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BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-6-241570306910.1186/1471-2105-6-24Research ArticleClustering the annotation space of proteins Kunin Victor [email protected] Christos A [email protected] Computational Genomics Group, EMBL-EBI, Cambridge, CB10 1SO, UK2005 9 2 2005 6 24 24 19 8 2004 9 2 2005 Copyright © 2005 Kunin and Ouzounis; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Current protein clustering methods rely on either sequence or functional similarities between proteins, thereby limiting inferences to one of these areas.
Results
Here we report a new approach, named CLAN, which clusters proteins according to both annotation and sequence similarity. This approach is extremely fast, clustering the complete SwissProt database within minutes. It is also accurate, recovering consistent protein families agreeing on average in more than 97% with sequence-based protein families from Pfam. Discrepancies between sequence- and annotation-based clusters were scrutinized and the reasons reported. We demonstrate examples for each of these cases, and thoroughly discuss an example of a propagated error in SwissProt: a vacuolar ATPase subunit M9.2 erroneously annotated as vacuolar ATP synthase subunit H. CLAN algorithm is available from the authors and the CLAN database is accessible at
Conclusions
CLAN creates refined function-and-sequence specific protein families that can be used for identification and annotation of unknown family members. It also allows easy identification of erroneous annotations by spotting inconsistencies between similarities on annotation and sequence levels.
==== Body
Background
To achieve high quality of annotation, curators are using direct evidence from additional experiments or infer functional roles by sequence similarity to experimentally characterized genes or proteins. However, erroneous annotations generated at early stages may propagate to new homologous sequences, ultimately leading to erroneous annotation of entire families. The number of database errors is known to grow with time, as the number of entries and errors tend to accumulate. A recent analysis suggested that as databases grow annotation errors may propagate at an exponential rate [1].
One of the most accurate and consistently annotated databases is SwissProt [2] – a manually curated protein sequence database which strives to provide a high level of annotations such as the description of the function of a protein, its domain structure, post-translational modifications, variants, etc. All entries in SwissProt are annotated by experts, thus reducing the amount of errors expected from fully automatic methods.
This impressive collection of textual information provides excellent opportunities for natural language processing in computational biology. Text mining of the free text in biomedical literature is well established (see [3] for a recent review). These methods use standard techniques such as the TF-IDF method, which considers background frequency of terms and the frequency of terms in the documents of interest. However, protein annotations are conceptually different from the free text of biomedical articles and abstracts upon which these methods are applied.
While articles normally contain thousands of words and abstracts contain a few hundred, annotations are limited to a few key terms, averaging 7 and rarely exceeding 20 words. Research articles are always unique, whereas orthologous proteins often have identical annotations. On the other hand, differences in annotations may or may not signal difference in function, depending on the context. For example, the addition of the word "precursor" to the annotation does not imply functional variation. Also, multiple occurrence of a term in an article or an abstract signals its higher relevance to the subject. In contrast, presence of a term in a protein annotation normally signals presence of a function, and its repetition is meaningless. All these differences require the development of a different method for clustering protein annotations.
This work describes a method called CLAN specially developed to assess the consistency of annotations. CLAN allows the rapid comparison of protein annotations, finding all proteins that are considered to have the same (or a closely related) function, according to the functional descriptions of the corresponding database entries. To group pairwise hits between these proteins, clustering of connected components was used, and sequence similarity within the clusters was considered. Using CLAN, an exemplar error was identified in the annotation of the M9.2 (M9.7) subunit of a vacuolar H+ ATPase (V-ATPase).
Results
General statistics
We used CLAN [see Methods] to cluster SwissProt entries based on annotation strings. We experimented with score cut-offs [see Figure 1 and Methods for derivation of scores] between 10-2 and 10-6. Depending on the threshold, the output contained between 2,070,221 (for e ≤ 10-6) and 3,980,763 (e ≤ 10-2) pairwise annotation similarities. The output of CLAN was subjected to clustering of connected components, resulting in 43,416 (e ≤ 10-2) up to 47,344 (e ≤ 10-4) clusters, including singletons. When a lower score threshold was used, the number of clusters in the output decreased, as more proteins were excluded from any clusters (see below).
A sample of few hundred of the annotation clusters was examined manually to assess whether they contained proteins annotated as functionally unrelated. At the low score cut-offs, we failed to find any such examples, thus suggesting a high degree of specificity. Also, hypothetical proteins were correctly not grouped together, which reflects their singularity in the protein function space. However, at these cut-off values, proteins annotated with a single word (i.e. Plastocyanin) failed to pass the threshold of meaningful annotation. This effect is due to the fact that no single term has a sufficiently low frequency to provide an score below the threshold. In total, 29,845 entries were not even defined as singletons (these include both characterized and hypothetical proteins) as their annotation strings are non-descriptive, resulting in high scores above the threshold.
On the other hand, at the highest score threshold the method succeeds in clustering together many single-word annotations. However, this increase in sensitivity is counter-balanced by a decrease in specificity. At the score of 10-2, the biggest cluster contains proteins annotated as "Hypothetical X kDa protein in Y intergenic region", where X is the calculated protein molecular weight and Y is the genomic location. These proteins are most probably unrelated, perform different functions or may not be expressed at all, and thus should not be clustered together. In fact, this cluster is eliminated at score cut-off of 10-3.
Comparison to Pfam database
To estimate how well annotation clusters correspond to the sequence-based protein families, we compared the CLAN clusters (e ≤ 10-2) to the Pfam-A database [4]. This database is a curated semi-automatic collection of protein domains, defined by experts as sharing sequence (and usually functional) similarity. To perform a meaningful comparison, we used only CLAN clusters containing more than 3 members. We found that on average 91% of CLAN cluster members belong to a single Pfam family.
However, the reverse relationship does not hold: only 51% of Pfam entries correspond to a single CLAN cluster, on average. This suggests that annotation-based clusters are about twice as small and contain only parts of sequence-based families. This is partially due to inconsistent annotation of protein families discussed below. On the other hand, it also reflects multifunctionality and diversification of some protein families. For example, a single Malate and Lactate dehydrogenase (MDH/LDH) family from Pfam is divided by CLAN to distinct annotation families, thus providing a finer distinction between family members that can be used for more accurate annotation of functional specificity [5].
As many of the CLAN clusters which correspond to several Pfam families contain multi-subunit complexes or multidomain proteins, a rigorous domain detection procedure is expected to drastically improve this result. For the results of the combination of CLAN annotation clusters with sequence-based clustering, see the section describing the CLAN database.
Though SwissProt annotation is not independent from Pfam assignments and often depend on them, a few clusters were found that did not correspond to Pfam families. Thus, CLAN system might facilitate identification and rapid incorporation of new families to curated protein family databases such as Pfam.
Analysis of individual clusters
We aimed to determine whether similarity of the annotation corresponds to the sequence similarity of the proteins. For this, we compared protein sequences within annotation clusters using BlastP program [6]. Overall, we found very consistent sequence similarity relationships within annotation clusters. The proteins participating in the annotation clusters usually formed tight protein families, with all (or most) proteins sharing detectable sequence similarity to the other cluster members. This pattern is expected, since protein annotation is usually based upon sequence similarity. We considered this pattern to be typical and examined any deviations from it.
Analysis of sequence similarity within annotation-based families identified the following exceptions to the consistent similarity patterns between all family members.
1. Fragments of proteins. The fragments often fail to produce significant Blast scores, thus appearing as singletons on the similarity graph.
2. Taxa – specific forms of enzymes that form separate protein families, such as Bacterial and Archaeal adenylate kinases, forming distinctive sequence-based families.
3. Non-orthologous gene displacements, such as malate dehydrogenases.
4. Protein complexes. Annotation – derived families often included multiple chains/subunits of protein complexes, such as Glutamyl-tRNA(Gln) amidotransferase subunits A, C, D and E.
5. False positives of CLAN. The only case found so far involves the 'hypothetical protein' cluster, appearing at the permissive score cut-off 0.01 (see previous section).
6. SwissProt annotation errors.
Fragments
SwissProt contains 7,232 entries annotated as "fragment". These are normally short fragments of larger protein molecules. An extreme example is LUXE_VIBFI (3 residues only), a fragment of Long-chain-fatty-acid-luciferin-component ligase. The complete sequence from a related Vibrio species documented in SwissProt (LUXE_VIBHA) contains 378 residues. There are at least 71 entries annotated as "fragment", whose sequences are less then 10 residues long. As their sequence often contains a subset from full-length close homologs, the information from these fragments is often redundant. Also, these short fragments fail to produce significant scores in any sequence similarity search procedure, and thus are usually irrelevant for sequence search engines. Moreover, fragments often create noise in sequence alignments and HMM analyses creating pseudo-conserved domains, and pseudo-gaps. In summary, we question the value of the presence of short protein fragments in a manually curated database, aiming for maximum precision rather then maximum coverage.
Taxa-specific forms of enzymes
In some taxa, proteins performing identical functions might diverge to a point where they are defined as separate sequence-based families, sharing marginal similarity. One such example is the bacterial and archaeal adenylate kinases. Though similar at the 3-dimensional level [7], the sequences of members of these families diverged beyond recognition by BlastP, and are thus considered as separate families.
Non-orthologous gene displacements
Enzymes belonging to different structural families may perform identical cellular function. In this case, annotation does not provide enough information about the type of the family a particular enzyme belongs to. One such case is represented by glucokinases, which belong to different families but perform identical function. For example, GNTK_BACSU and GNTK_ECOLI are both glucokinases and are annotated as such, were thus clustered together by CLAN but are unrelated to each other [8].
Protein complexes
Subunits in protein complexes often have almost identical annotation. For example, Glutamyl-tRNA(Gln) amidotransferase subunits A, C, D, and E differ only in the chain identifier. The annotation of the subunits is somewhat misleading, as subunits A, B and C form one form of a rather ubiquitous complex [9], and subunits D and E form an alternative archaeal-specific complex [10]. Each of the chains forms a tight sequence-similarity based family, with no similarity detected between chains. Entries describing chains A, C, D and E appear in a single CLAN cluster. Interestingly, a few organisms have the entire complexes annotated in SwissProt (Table 1), although in many cases the documentation of these protein complexes is incomplete. The CLAN approach can help to identify the organisms where the chains remain to be identified, while the phylogenetic pattern of the complex may serve as an additional evidence for further annotation.
Annotation errors
Finally, CLAN can serve as a powerful tool to identify annotation errors. Overall, we found SwissProt annotations very consistent and robust. Most of the annotation-based sets resulted in tight sequence-based similarity clusters. A few cases where no similarity was found between a protein and the rest of the cluster were normally due to the limits in sensitivity by BlastP, and the homology was clear when multiple alignments, profile- or HMM-based search methods were used. Nevertheless, there are cases where annotation is problematic. Below, we discuss in detail an example of inconsistency found in the annotation of a cluster containing vacuolar H-ATPase.
False annotation example: Vacuolar ATP synthase subunit M9.2
When analyzing the output of CLAN with score threshold 10-6, we found an interesting example of a propagated error. The protein VAOH_CAEEL (Q20591) from Caenorhabditis elegans has been annotated as "Probable vacuolar ATP synthase subunit H (EC 3.6.3.14) (V-ATPase H subunit) (Vacuolar proton pump H subunit)". This protein is clustered by CLAN with other vacuolar ATP synthase subunits. However, BlastP alignment failed to produce evidence of similarity between this protein and other members of the cluster annotated as vacuolar ATP synthase subunit H. Also, this protein is much shorter then other members of the annotation cluster, comprising only of 96 residues, while other members of the cluster have more then 400 residues. In fact, C. elegans was reported to contain another two V-ATPase H subunits, with length of 451 and 470 residues that share substantial sequence similarity with other members of the family. Multiple alignments of the family suggested that VA0H_CAEEL is a distant protein not related to the rest of the family. The SwissProt entry does not contain a link to a published work. A PSI-Blast search against the NCBI NR database allowed to identify other members of the family. The family was first described in the literature as "M9.2 V-ATPase subunit" in bovine [11]. Interestingly, an appropriate sequence entry exists in SwissProt with an adequate reference (VA0H_BOVIN). However, this protein is named in SwissProt as "vacuolar ATP synthase subunit H", and the name V-ATPase M9.2 subunit is suggested as a synonym. Another SwissProt entry (VA0H_HUMAN) contains a human homolog, with identical annotation. Interestingly, the bovine and human proteins were unified into a separate annotation cluster at score 10-6, which was unified with other members of "vacuolar ATP synthase subunit" cluster at lower score cut-offs.
The M9.2 V-ATPase subunits were identified in mammals [11,12], insects [11,13] and C. elegans [11]. The alternative names included "M9.7" or subunit 'e' in insects [14,15] and ATP6H in dog [12]. A potential homolog was identified in the plant Mesembryanthemum crystallinum (gi 26986112) [16]. Using PSI-BLAST searches we have identified homologs in several other species, including Arabidopsis thaliana, Anopheles gambiae and a few paralogs in Drosophila melanogaster. The multiple alignment of these proteins is provided as supplementary data.
The M9.2 protein was suggested to be a homolog [11] of yeast Vma21p protein (VM21_YEAST). The Vma21p protein is required for assembly of the V-ATPase, but is not found in the mature complex [17]. The orthology of the two proteins was suggested based on the weak sequence identity, association with V-ATPase complex, and a similar hydrophobicity profile [11]. However, the doubts concerning orthology of the two proteins were already expressed [14]. While Vma21p is localized in endoplasmic reticulum and is not a part of the mature V-ATPase [17], M9.2 is a subunit of mature V-ATPase located in the vacuolar membrane [11]. In addition, the topology of the yeast protein seems to be inverse to that of the mammalian and insect proteins: glycosylation at the C-terminus of the M. sexta protein indicates that the C-terminus is exposed to the extracellular surface, whereas the C-terminus of Vma21p appears to be localized on the cytosolic side of the membrane [14]. To identify any similarity between these two protein families, we used sequence-versus-sequence (BlastP), sequence to profile (PSI-BLAST) [6], and profile-profile (LAMA) [18] search methods. Each of these methods failed to detect any significant similarity between the two families. Our sequence analysis suggests that the initial reports of such homology between these families were not substantial, and the reported marginal similarity between the two sequences was due to the extent of hydrophobic regions in the two protein families. In summary, while the exact function of the M9.2 subunit of V-ATPase is still not known, it has a turbulent history of misinterpretation. It was confused with V-ATPase H chain in SwissProt, it was misaligned with yeast Vmi21p protein, it has several names in different organisms of which the name 'subunit e' is most confusing, as there is already a 'subunit E' in the complex. The current study used (i) CLAN to detect the discrepancies for protein annotation in SwissProt and (ii) sensitive sequence searches in order to demonstrate the independent status of this protein family.
The error in the annotation of M9.2 subunit of V-ATPase was reported to the SwissProt developers, and is likely to be corrected at the time of publication of this work.
False negative example
Another form of annotation error results in a false negative case for CLAN. It occurs when a protein clearly belongs to a certain family but is not annotated as such. This event can be detected when several CLAN families map to a single sequence similarity-based family. One example is SUCD_BACSU, annotated as " Succinyl-CoA synthetase alpha chain (EC 6.2.1.5) (SCS-alpha)". This protein belongs to the SUCD family of proteins, and should cluster with its other members. However, its annotation contains synonyms " (Vegetative protein 239) (VEG239)", which makes this protein virtually unique in annotation space. Another well-known example is the initial characterization of some general stress proteins in bacteria, later shown to be ribosomal proteins (e.g. S1, L25) [19] – the initial annotations have not been modified. This type of assignments are usually based on genetics experiments and do not reflect the precise biochemical function of these proteins. In fact, there are multiple examples of proteins having unique or phenotype-based identifiers in their names, whereas it is clear that these proteins belong to larger protein families of known biochemical function. In our opinion, these proteins are examples of conflicting annotation with other family members and their annotations should be amended.
The clan database
Based on the results described above, we designed a database using the CLAN output with score threshold of 10-2. The CLAN database contains two levels of clustering. The first level contains clusters based on comparison of protein annotations, as described in the Methods section. To ensure completeness and reliability of the results, any protein annotated as 'fragment' was excluded from further processing. The second level in the database is generated by applying sequence-similarity-based grouping to each of the clusters obtained in the first stage. All members within annotation clusters are aligned using BlastP. The output of BlastP is subjected to various clustering procedures. In order to distinguish between the annotation-based clusters of the first database level, and the sequence-based sub-clusters of the second level, we call these 'clusters' and 'sub-clusters' accordingly. When referring to both these categories simultaneously, we use the notation (sub)clusters.
To estimate the contribution from the second level of clustering, we repeated the comparison to Pfam database, using sub-clusters instead of clusters. We found that on average 97% of CLAN sub-clusters members belong to a single Pfam family, (compared to the 91% for clusters). The gain in selectivity was accompanied by gain in sensitivity, and 66% of Pfam family members corresponded to a single sub-cluster (compared to 51% for clusters). Compared to annotation-based clustering alone, the dramatic increase in the sensitivity reflects the fact that in the second level of clustering, non-homologous sequences are removed and form separate sub-clusters.
The CLAN database has a web-based interactive interface, located at . To facilitate database navigation, we introduced (sub)cluster names, computed as a minimal consensus string for all (sub)cluster members. The MySQL-based storage of data allows fast searches based on sequence or cluster name, SwissProt accession or cluster identifier. The sequence similarity within clusters can be examined visually using the BioLayout software [20]. To facilitate the annotation of protein complexes, phylogenetic distribution of clusters between sub-clusters (such as Table 1) can be automatically generated upon the user's request. For all sub-clusters full-length multiple alignments are pre-computed with the ClustalW program [21], and the alignments can be viewed via an interactive Java interface.
Discussion
We have presented CLAN, a rapid and powerful method to build consistent annotation-based families, and assessed the consistency of annotation in protein databases. We have also presented the CLAN database, as a collection of protein families grouped by function. We believe that these tools can be used to improve the quality of database curation and assist annotation of newly sequenced proteins.
The double-step clustering presented here has resulted in the creation of a reliable database of protein families, with include proteins similar on both annotation and sequence levels. One of the potential uses of this database is an accurate assignment of function to newly sequenced proteins by protein – to – profile alignments or HMMs. Our method has the potential to distinguish between subtle changes that might reflect diversification of protein function. For example, homologous Malate and Lactate dehydrogenases are found in different (sub)clusters, allowing their characterization as separate protein families. As most protein family databases group proteins by homology, as opposed to function, to the best of our knowledge, our approach is unique in its ability to build protein sequence families distinguishing between functions. In the future, other such comparisons could include other structural classifications such as CATH [22] or TRIBEs [23] and functional classifications such as EC or GO [24].
Methods
We used SwissProt release of Feb 13, 2003 that contained 121,744 protein sequences. We aimed to find the similarity between the database entries solely based on the annotation and without any reference to sequence similarity. The annotation corpus considered here consisted of description line (protein name) and synonyms from the SwissProt database. We interchangeably use the term 'function' or 'annotation' to denote the contents of this corpus, implying the use of the corresponding annotation strings from the database.
Definition of distance measure between annotation fields
To obtain a distance measure, we first calculated the frequency φ of each word as the number of database entries containing the word N divided by the total number of entries in the database D (Equation 1). The word frequencies served as weights, because the words describing specific functions (such as "plastocyanin") occur less frequently than non-specific words (such as "protein").
We aimed to devise a scoring scheme that would produce highly significant scores for proteins with related function, while being able to distinguish between proteins with different functions. The score for the common terms p between protein functions may be computed as the product of frequencies of n terms shared between the two annotations (Equation 2).
However, sharing only part of the total number of significant words is insufficient: proteins may use similar substrates for different reactions, or perform similar chemical reactions on different substrates. There may be three proteins with annotations AB, BC, and CD respectively, and clustering by similarity only could lead to clustering together AB and CD. For example, "lactate dehydrogenase" should be differentiated from "lactate permease" or "alcohol dehydrogenase".
To achieve this distinction, we computed the difference between the two annotations, as above, for the case of the similarity measure. The score for the unique terms q is the product of frequencies of m terms unique to any of the two annotations (Equation 3).
Finally, the distance e between the two annotations is defined as the fraction between the score for common terms p and the score for unique terms q for the two annotation strings (Equation 4). We refer to this scoring function as 'score' throughout the manuscript.
An example of calculation of the score is shown on Figure 1.
Implementation
The procedure calculating pairwise distances between protein annotations described above was implemented in a perl program. Its sole input consists of SwissProt identifiers and annotation lines, and the output contains pairs of proteins with the score of annotation similarity lower than a given threshold. Compared to sequence-similarity clustering, this procedure is very rapid: an all-against-all comparison of SwissProt database containing 121,744 entries took about 26 minutes on a single processor of a Sun-Fire-480R server with 2 GB of RAM.
The result of the pairwise comparisons between protein annotations were then subjected to clustering of connected components. The protein sequences of the clusters obtained were subsequently analyzed with the BlastP program [6] and the results of similarities were clustered using GeneRage algorithm [25]. The results were visualized using the BioLayout software [20] (and Goldovsky, Cases et al., submitted). To evaluate the ability of CLAN to detect genuine protein families on the basis of annotation alone, the output (e ≤ 10-2) was compared to the Pfam-A database release 9.0 [4]. For each CLAN cluster with more then 3 entries, we have identified the corresponding Pfam entry (if available), and counted the fraction of CLAN entries found in the same Pfam cluster. The reverse comparison was computed in a similar manner.
Acknowledgements
The authors thank Michael Lappe, Carolin Cosiol and members of the CGG group for the helpful discussions.
Figures and Tables
Figure 1 Score calculation. An example of calculation of the score by CLAN. A. In the pre-processing stage, a dictionary is constructed with occurrences of terms in the SwissProt. Multiple occurrence of a term in a single entry is counted once. Frequency is calculated by dividing the term occurrence to the number of entries in the database. Numbers are rounded to the third decimal digit. B. An example of calculation of score for an alignment of two actual annotations.
Table 1 Phylogenetic distribution of Glutamyl-tRNA(Gln) amidotransferase subunits as represented in SwissProt. Note that the table is not completely populated. Evidence of presence of part of the complex can assist annotation for absent members.
chain A chain B chain C chain D chain E
Aeropyrum pernix GATA_AERPE GATB_AERPE GATD_AERPE GATE_AERPE
Agrobacterium tumefaciens (strain C58 / ATCC 33970) GATB_AGRT5
Anabaena sp GATA_ANASP GATB_ANASP GATC_ANASP
Aquifex aeolicus GATA_AQUAE GATB_AQUAE GATC_AQUAE
Archaeoglobus fulgidus GATA_ARCFU GATB_ARCFU GATC_ARCFU GATD_ARCFU GATE_ARCFU
Bacillus halodurans GATA_BACHD GATB_BACHD GATC_BACHD
Bacillus stearothermophilus GATA_BACST GATB_BACST GATC_BACST
Bacillus subtilis GATA_BACSU GATB_BACSU GATC_BACSU
Borrelia burgdorferi GATA_BORBU GATB_BORBU GATC_BORBU
Brucella melitensis GATB_BRUME GATC_BRUME
Brucella suis GATB_BRUME GATC_BRUME
Campylobacter jejuni GATA_CAMJE GATB_CAMJE GATC_CAMJE
Caulobacter crescentus GATB_CAUCR GATC_CAUCR
Chlamydia muridarum GATA_CHLMU GATB_CHLMU GATC_CHLMU
Chlamydia pneumoniae GATA_CHLPN GATB_CHLPN GATC_CHLPN
Chlamydia trachomatis GATA_CHLTR GATB_CHLTR GATC_CHLTR
Chlorobium tepidum GATB_CHLTE
Clostridium acetobutylicum GAB1_CLOAB
GAB2_CLOAB GAC1_CLOAB
GAC2_CLOAB
Corynebacterium glutamicum GATB_CORGL
Deinococcus radiodurans GATB_DEIRA GATC_DEIRA
Fusobacterium nucleatum (subsp nucleatum) GATA_FUSNN GATB_FUSNN
Halobacterium sp GATB_HALN1 GATC_HALN1 GATD_HALN1 GATE_HALN1
Helicobacter pylori GATA_HELPY GATB_HELPY GATC_HELPY
Helicobacter pylori J99 GATA_HELPJ GATB_HELPJ GATC_HELPJ
Lactococcus lactis (subsp lactis) GATA_LACLA GATB_LACLA GATC_LACLA
Listeria innocua GATA_LISIN GATB_LISIN GATC_LISIN
Listeria monocytogenes GATA_LISMO GATB_LISMO GATC_LISMO
Methanobacterium thermoautotrophicum GATA_METTH GATB_METTH GATD_METTH GATE_METTH
Methanococcus jannaschii GATA_METJA GATB_METJA GATC_METJA GATD_METJA GATE_METJA
Methanopyrus kandleri GATB_METKA GATD_METKA GATE_METKA
Methanosarcina acetivorans GATA_METAC GATB_METAC GATD_METAC GATE_METAC
Methanosarcina mazei GATA_METMA GATB_METMA GATD_METMA GATE_METMA
Moraxella catarrhalis GATA_MORCA GATB_MORCA GATC_MORCA
Mycobacterium leprae GATA_MYCLE GATB_MYCLE GATC_MYCLE
Mycobacterium tuberculosis GATA_MYCTU GATB_MYCTU GATC_MYCTU
Mycoplasma genitalium GATA_MYCGE GATB_MYCGE
Mycoplasma pneumoniae GATA_MYCPN GATB_MYCPN
Mycoplasma pulmonis GATB_MYCPU
Neisseria meningitidis (serogroup A) GATA_NEIMA GATB_NEIMA GATC_NEIMA
Neisseria meningitidis (serogroup B) GATA_NEIMB GATB_NEIMB GATC_NEIMB
Pseudomonas aeruginosa GATA_PSEAE GATB_PSEAE GATC_PSEAE
Pyrobaculum aerophilum GATD_PYRAE GATE_PYRAE
Pyrococcus abyssi GATD_PYRAB GATE_PYRAB
Pyrococcus furiosus GATD_PYRFU GATE_PYRFU
Pyrococcus horikoshii GATD_PYRHO GATE_PYRHO
Ralstonia solanacearum GATB_RALSO GATC_RALSO
Rhizobium loti GATB_RHILO GATC_RHILO
Rhizobium meliloti GATB_RHIME GATC_RHIME
Rickettsia conorii GATA_RICCN GATB_RICCN GATC_RICCN
Rickettsia prowazekii GATA_RICPR GATB_RICPR GATC_RICPR
Saccharomyces cerevisiae GATH_YEAST
Schizosaccharomyces pombe GATH_SCHPO
Staphylococcus aureus GATA_STAAU GATB_STAAU GATC_STAAM
Staphylococcus sp GATB_STASP
Streptococcus pneumoniae GATA_STRPN GATB_STRPN GATC_STRPN
Streptococcus pyogenes GATA_STRPY GATB_STRPY GATC_STRPY
Streptomyces coelicolor GATA_STRCO GATB_STRCO GATC_STRCO
Sulfolobus solfataricus GATA_SULSO GATB_SULSO GATC_SULSO GATD_SULSO GATE_SULSO
Sulfolobus tokodaii GATA_SULTO GATB_SULTO GATC_SULTO GATD_SULTO GATE_SULTO
Synechocystis sp GATA_SYNY3 GATB_SYNY3 GATC_SYNY3
Thermoanaerobacter tengcongensis GATA_THETN GATB_THETN
Thermoplasma acidophilum GATD_THEAC GATE_THEAC
Thermoplasma volcanium GATD_THEVO GATE_THEVO
Thermotoga maritima GATA_THEMA GATB_THEMA GATC_THEMA
Thermus thermophilus GATA_THETH GATB_THETH GATC_THETH
Treponema pallidum GATA_TREPA GATB_TREPA GATC_TREPA
Ureaplasma parvum GATB_UREPA
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| 15703069 | PMC552314 | CC BY | 2021-01-04 16:02:51 | no | BMC Bioinformatics. 2005 Feb 9; 6:24 | utf-8 | BMC Bioinformatics | 2,005 | 10.1186/1471-2105-6-24 | oa_comm |
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BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-6-281570748610.1186/1471-2105-6-28Research ArticleAn adaptive method for cDNA microarray normalization Zhao Yingdong [email protected] Ming-Chung [email protected] Richard [email protected] Biometric Research Branch, National Cancer Institute, National Institutes of Health, Rockville, Maryland, USA2 The EMMES Corporation, Rockville, Maryland, USA2005 11 2 2005 6 28 28 21 10 2004 11 2 2005 Copyright © 2005 Zhao et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Normalization is a critical step in analysis of gene expression profiles. For dual-labeled arrays, global normalization assumes that the majority of the genes on the array are non-differentially expressed between the two channels and that the number of over-expressed genes approximately equals the number of under-expressed genes. These assumptions can be inappropriate for custom arrays or arrays in which the reference RNA is very different from the experimental samples.
Results
We propose a mixture model based normalization method that adaptively identifies non-differentially expressed genes and thereby substantially improves normalization for dual-labeled arrays in settings where the assumptions of global normalization are problematic. The new method is evaluated using both simulated and real data.
Conclusions
The new normalization method is effective for general microarray platforms when samples with very different expression profile are co-hybridized and for custom arrays where the majority of genes are likely to be differentially expressed.
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Background
Microarray technology provides simultaneous measurements of expression levels for thousands of genes. Each step from sample preparation to data analysis, however, contains potential sources of bias and variability. Proper normalization adjusts for differences which interfere with the comparison of intensities of different labels at a given probe and with the comparison of intensities of corresponding probes on different arrays. Proper data normalization should allow for the comparison of expression levels across different arrays. Subsequent data analysis results are heavily dependent on effective normalization.
Normalization issues differ for dual-labeled platforms compared to single labeled platforms such as the Affymetrix GeneChip arrays. In this paper we address normalization for dual-labeled arrays with either cDNA or oligonucleotide probes. The objective of normalization for dual-labeled arrays is to correct for differences in intensities for the two labels on the same array. These differences arise from factors such as differences in sample concentrations, differences in photomultiplier tube setting, and differences in the affinity of the two labels for DNA.
Median or mean based global normalization methods use a single normalization factor applied to all genes on the array to adjust for labeling bias [1,2]. Such methods are widely used because of their simplicity. Intensity-based and location-based methods take into account intensity and spatial dependence on dye bias normalization factors [3,4]. Both global and intensity/location based normalization methods assume that most of the genes are not differentially expressed between the two samples hybridized on the array, and that for the differentially expressed genes, the direction of the difference is symmetric between the two samples. In many important cases, however, these assumptions are not appropriate because: 1) more than half of the genes are differentially expressed on the array; 2) the numbers of over- and under-expressed genes on the array are unequal; 3) only genes of specific biological interest are selected to make a customized array, which are highly variable across the samples. In the above cases, the global normalization methods and intensity/location based normalization methods become less accurate and a more sophisticated method is needed [5,6].
There are some methods which attempt to adaptively identify the subset of 'housekeeping' genes [6-8]. These methods require multiple arrays in order to identify the 'housekeeping' gene set, which does not always exist.
Newton et al. proposed a Gamma-Gamma-Bernoulli model for identifying differentially expressed genes in dual labeled arrays [9]. We have generalized Newton's model and here propose an adaptive method based on three-component mixture model for normalization of dual labeled microarray data.
Results
As described in the Methods section, we have applied our adaptive method to both the simulated data and real data. We have also compared our method with the global method and the intensity-based lowess method.
Results of the simulation studies are shown as bar plots in Figure 1. Figure 1A shows the comparison of our adaptive method, the global method and the lowess method when no noise was added. When the majority of genes in the array were non-differentially expressed (Case 1), or the numbers of over- and under-expressed genes on the array were equal (Case 2), the root mean squared error (RMSE) of the adaptive method was comparable with the other two methods; all were very small. When the array contained unequal numbers of over- and under-expressed genes and when the majority of genes were differentially expressed (Cases 3–6), the RMSEs of the global normalization method and the lowess method were much larger than those of the adaptive method. The differences ranged from around a two fold difference (0.895 in log2 scale) when the number of under-, null, and over-expressed genes were 200, 100, and 100, to more than a three fold difference (1.617 in log2 scale) when the number of under-, null, and over-expressed genes were 200, 50, and 50. The RMSEs for the adaptive method ranged from 0.078 to 0.159 in log2 scale.
We compared the histogram of observed intensities to the fitted marginal density from the adaptive method as a simple check to see whether the proposed model and the estimation procedure are in line with available data. Figure 2 shows the histograms of log(ratio) and log intensities of red and green channels of the simulated data, and the curve in each plot is the estimated density obtained from the fitted model. It is seen the data fits to the model quite well.
Gaussian noise with SD of 0.25 and 0.50 were added so that the data was not generated from the same model used for analysis with the adaptive method. The RMSEs of the global normalization method and the lowess method remained large, while the RMSEs of the adaptive method remained small, ranging from 0.083 to 0.569 on the log2 scale (Figure 1B and 1C).
In the above simulation, no apparent groups could be seen in the histograms of log(ratio) (Figure 2A). Better results for the adaptive method were also obtained for a simulation case where the three groups (under-expressed, non-differentially expressed, and over-expressed) are apparent in the histogram of log(ratio). The results can be seen in Figure 4 and Figure 5 [see Additional files 3, 4].
Results comparing RMSEs for the adaptive method, the global method and the lowess method with real data are shown in Table 2. The RMSEs of the adaptive method on data generated from ten different arrays ranged from 0.128 to 0.529, in comparison with RMSEs of around 1.0 using the global normalization method. The average RMSE (0.607) of the lowess method is almost two times that of our adaptive method (0.328), although the lowess method performed better than the global method (average RMSE = 1.016). Figure 3 shows the histograms of log(ratio) and log intensities of red and green channels of the real data, and the curve in each plot is the estimated density from the adaptive method.
Discussion
In this paper, we propose a new method for normalization of dual-labeled arrays in cases where the number of differentially expressed genes is substantial and not necessarily symmetric in direction. The method performed effectively with both simulated and real data.
We started our model building initially by introducing an unknown constant c into Newton's Gamma-Gamma-Bernoulli model [9]. The mixture model consisted of two groups: non-differentially expressed genes (Equation 1A) and differentially expressed genes (Equation 1B).
log(cRk) ~ Gamma(a, sk)
log(Gk) ~ Gamma(a, sk) (1A)
sk ~ Gamma(a0, γ)
log(cRk) ~ Gamma(a, )
log(Gk) ~ Gamma(a, ) (1B)
~ Gamma(a0, γ)
~ Gamma(a0, γ)
We found that when the differential expression was symmetric between the two samples, the model worked well. However, the error increased significantly when the ratio of the numbers of under- to over-expressed genes shifted from 1.
In order to make the model more flexible, we modified the model by assigning different scale factors γR and γG for the red channel and green channel intensities. For this modified two-component mixture, the error still remained large. We then extended the model into a three-component mixture model listed as Equations 8A-8C in the additional material [see Additional file 2]. The model was then quite flexible but there were too many parameters that needed to be optimized. After we tested it with simulation data and real data, we found the estimated model was not stable and difficult to optimize. We finally simplified the model to our final model given by Equations 2A-2C (see Methods section). When applying it to real data or simulated data, the estimates converged well close to globe optima. When different start points were used, the optimizations remained relatively robust.
Evaluation of normalization methods can be difficult since the true normalization factors are unknown with real data for custom arrays. We avoided this problem by synthesizing customized arrays based on real data for standard arrays containing thousands of genes. In order to make the distribution of each component group look smoother, we allowed certain range of overlap between the adjacent groups. Additional sampling method was tried to divide the whole distribution range into many non-overlapping intervals. In each interval the number of genes sampled increased when the absolute value of log2(ratio) became larger (Table 3 [see Additional file 6]). The model fitting results using data generated by this sampling method are listed in Table 4 [see Additional file 7] and Figure 6 [see Additional file 5].
We compared our adaptive method with the global method and the intensity-based lowess method. The lowess method assumes that in each intensity interval either the majority of genes are non-differentially expressed or the numbers of up- and down-regulated genes are equal. The global median normalization makes these assumptions only over the array as a whole. It is not surprising that our method performed much better that the above two methods, because the global median method only works well when the assumptions are valid while the intensity-based lowess method is only effective when there are intensity-dependent biases.
Correlation structure is complicated for the thousands of genes on a microarray. In our model, the intensity of each channel is conditionally independent given the scale parameter, but not marginally independent.Therefore, we are not assuming the intensities in two channels are independent. Although we did not generate correlated genes in our simulated data sets, correlations of genes do exist in the real data sets we tested. Spatial correlations are also possible but our method is not designed for that purpose. Yang et al. proposed using the lowess normalization separately within each grid on the array [3]. Our algorithm could be similarly applied within each grid to control for spatial effects.
Limited simulations were performed in this study. We also tried to use real data to test our method. Since an appropriate data set with known normalization factor was not available, we synthesized such data sets by sub-setting large arrays in which the true normalization factor could be accurately estimated. In the process of synthesizing such small arrays we had to choose an empirical threshold to stratify the differentially expressed genes and non-differentially expressed genes. Although we do not believe that the superiority shown for our algorithm depends critically on the threshold chosen nor on details of the synthesis, it would be preferable to evaluate the algorithm on real data sets with know normalization factors.
Although our method is designed for dual-labeled cDNA array, it can be extended to single channel Affymetrix chip data. The most popular normalization method for the Affymetrix chip compares each array to a single base line array for probe set summaries. The assumptions behind the normalization method are that the majority of the genes are non-differentially expressed and the numbers of over- and under-expressed genes are roughly equal; the same assumptions as those for dual-labeled cDNA arrays. We could treat the base line array as the 'reference channel' and the other array as the 'test' channel and apply our algorithm to probe set summaries. For Affymetrix chip data, there are multiple base pairs in a probe set and each probe has an intensity measurement. Several alternative normalization methods of Affymetrix arrays utilize the probe level information. For example, method based on an 'invariant set' proposed by Li and Wong assumes that a probe of a non-differentially expressed genes in two arrays to have similar ranks and uses an iterative procedure to identify the invariant set which presumably consists of points from non-differentially expressed genes [10].
Conclusions
Our new normalization method does not require that the majority of genes be non-differentially expressed, and doesn't require multiple array replicates, dye swaps, spiked controls, or housekeeping genes. It appears much more effective than standard methods when the numbers of over- and under-expressed genes are unequal, and the majority of the genes are differentially expressed. It can be very useful for general microarray platforms when samples with very different expression profile are co-hybridized and for custom arrays where the majority of genes are likely to be differentially expressed. In both of these settings, standard normalization methods are problematic.
Methods
Model
We define true intensities for a specific gene k in two channels as Rk (red) and Gk (green). Let c be a positive constant which is related to the normalization constant. The observed intensities for gene k in two channels are cRk and Gk. We assume the logarithm of intensity in each channel has a Gamma distribution. The genes on the array belong to three different groups: 1) non-differentially expressed; 2) under-expressed; and 3) over-expressed. The overall data will be fitted into a mixture model listed below.
For a non-differentially expressed gene k,
log(cRk) ~ Gamma(a, sk)
log(Gk) ~ Gamma(a, sk) (2A)
sk ~ Gamma(a0, γ).
For an under-expressed gene k,
log(cRk) ~ Gamma(a, )
log(Gk) ~ Gamma(a, ) (2B)
~ Gamma(a0, γ1)
~ Gamma(a0, γ2).
For an over-expressed gene k,
log(cRk) ~ Gamma(a, )
log(Gk) ~ Gamma(a, ) (2C)
~ Gamma(a0, γ2)
~ Gamma(a0, γ1).
In the above Gamma distributions, the parameters a and a0 are shape factors, and the parameters sk, γ, γ1,γ2, , are scale factors. The parameters a, a0, γ, γ1, γ2 will be estimated from the data.
Let pu(Rk, Gk), po(Rk, Gk) and pn(Rk, Gk) be the densities of (Rk, Gk) for under-expressed, over-expressed and non-differentially expressed genes, respectively. The joint distributions of (Rk, Gk) in three groups can be derived as follows [details see Additional file 1]:
Let θ denote the unknown parameter vector (a, a0, γ, γ1, γ2, c), which can be estimated by maximizing the likelihood function of observed data. We used the EM algorithm [11] for this maximization. Let p1 be the proportion of under-expressed genes and p2 be the proportion of over-expressed genes. We define indicator binary variable zk1 to be 1 if the kth gene is under expressed, 0 otherwise; and zk2 to be 1 if the kth gene is over expressed, 0 otherwise. The complete-data loglikelihood for all spots can be derived as follows,
In the M-step, we first take derivative on Equation (4) with respect to p1 and p2. This yields
where K is the total number of genes on the array.
To maximize Equation (4), we only need to maximize Equation (6) because the left out terms do not depend on the parameter θ.
In the E-step, we compute the conditional expectations of zk1 and zk2 given the other parameters from the M-step.
Once the constant c was obtained, the normalization constant for log intensity ratio data can be calculated as log(1/c).
Model evaluation
Simulation studies were performed by generating two channel intensities from the mixture model with c = 1.5, a = 118, a0 = 410, γ = 31, γ1 = 23, and γ2 = 29. Six scenarios were included using different proportions of non-differentially expressed genes and different ratios of under- to over- expressed genes, as listed in Table 1.
One hundred data sets were generated for each scenario and the RMSE between the estimated log2(1/) and the true log2(1/c) was calculated. The global method takes the median log2(ratio) of all genes in each data set as the normalization factor. The lowess method performs robust locally linear fits of M-A plot and corrects the biases that are dependent on spot intensity [3]. The RMSE between the normalized log2(ratio) using the lowess method and the normalized log2(ratio) using the global method with the true normalization factor (log2(1/c)) for all genes was also calculated for the same 100 data sets.
Gaussian white noise was also added when generating the simulated data. We used standard deviation of 0.25 in log2 scale to reflect the experimental noise in inbred strains of mice or cell line data and 0.5 in log2 scale to reflect a larger experimental noise in human tissue data [12].
In-silico studies were performed on real data. We tested the method on ten arrays from publicly available breast cancer data [13]. Each array consists of 9216 genes. The common reference sample was a pool of RNA isolated from 11 different cultured cell lines (green channel, labeled with Cy3). RNA from tissues of breast cancer patients were used in the test channel (red channel, labeled with Cy5). The array was first normalized by the global normalization method. The median log2(ratio) of all genes was considered as the true normalization factor c. The genes were then divided into three groups: over-expressed genes (log2(ratio)>1), non-differentially expressed genes(-1.5<log2(ratio)<1.5), and under-expressed genes (log2(ratio)<-1). We randomly sampled a specified number of genes from each group (100 non-differentially expressed genes, 200 under-expressed and 100 over-expressed genes) and then combined them into an in-silico array. We constructed 100 datasets for each of the 10 arrays in this way and the RMSE between the estimated log2(1/ ) and the true log2(1/c) was calculated.
Supplementary Material
Additional File 3
Figure 4: Bar plots show comparison of RMSE by using the adaptive method (black bar) and global method (grey bar) with simulated data generated from a mixture model with c = 1.5, a = 90, a0 = 120, γ = 8, γ1 = 6, and γ2 = 10 at three different noise levels (A) SD = 0; (B) SD = 0.25; and (C) SD = 0.50.
Click here for file
Additional File 4
Figure 5: Histograms and the estimated densities of log(ratio) and log(intensity) for a simulated data of a mixture model with c = 1.5, a = 90, a0 = 120, γ = 8, γ1 = 6, and γ2 = 10. The superimposed curve on each plot is generated from the fitted model.
Click here for file
Additional File 2
Equations 8A-8C: a three-component mixture model.
Click here for file
Additional File 5
Figure 6: Histograms and the estimated densities of log(ratio) and log(intensity) for a set of real data generated from array svcc109. The superimposed curve on each plot is generated from the fitted model. The procedure to generate the data was described in the paper and the sampling rate was shown in Table 3 [see Additional file 6].
Click here for file
Additional File 6
Table 3: Different number of genes sampled in each interval.
Click here for file
Additional File 7
Table 4: Comparison of RMSE by using the adaptive method and global method with real data by a different sampling method. The procedure to generate the data was described in the paper and the sampling rate was shown in Table 3 [see Additional file 6].
Click here for file
Additional File 1
Derivation of joint distributions of (R, G) for pn, pu, and po
Click here for file
Acknowledgement
We thank Dr. George Wright for reading our manuscript and helpful discussions, and the editor and reviewers who provided valuable suggestions.
Figures and Tables
Figure 1 Bar plots show comparison of RMSEs by using the global method (black bar), the lowess method (grey bar), and the adaptive method (white bar) for normalization with simulated data generated from a mixture model with c = 1.5, a = 118, a0 = 410, γ = 31, γ1 = 23, and γ2 = 29 at three different noise levels (A) SD = 0; (B) SD = 0.25; and (C) SD = 0.50.
Figure 2 Histograms and the estimated densities of log(ratio) and log(intensity) for a set of simulated data generated from a mixture model with c = 1.5, a = 118, a0 = 410, γ = 31, γ1 = 23, and γ2 = 29. The superimposed curve on each plot is generated from the fitted model.
Figure 3 Histograms and the estimated densities of log(ratio) and log(intensity) for a set of real data generated from array svcc109. The superimposed curve on each plot is generated from the fitted model.
Table 1 Six scenarios using different proportions of non-differentially expressed genes and different ratios of under- to over-expressed genes with simulated data.
Number of genes
Case Under-expressed Non-differentially expressed Over-expressed
1 100 500 100
2 100 100 100
3 200 100 100
4 200 100 50
5 200 50 100
6 200 50 50
Table 2 Comparison of RMSEs by using the global method, the lowess method, and the adaptive method for normalization with real data.
Case Array ID Global Lowess Adaptive
1 svcc134 1.082 0.600 0.315
2 svcc104 1.023 0.601 0.508
3 svcc120 1.005 0.552 0.435
4 svcc64 0.999 0.593 0.516
5 svcc106 0.967 0.704 0.128
6 svcc89 1.018 0.593 0.284
7 svcc109 1.014 0.577 0.264
8 svcc103 1.011 0.653 0.138
9 svcc98 1.022 0.631 0.159
10 svcc82 1.017 0.567 0.529
Average RMSE 1.016 0.607 0.328
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| 15707486 | PMC552315 | CC BY | 2021-01-04 16:02:49 | no | BMC Bioinformatics. 2005 Feb 11; 6:28 | utf-8 | BMC Bioinformatics | 2,005 | 10.1186/1471-2105-6-28 | oa_comm |
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BMC Palliat CareBMC Palliative Care1472-684XBioMed Central London 1472-684X-4-21571792310.1186/1472-684X-4-2Research ArticleThe uses of provincial administrative health databases for research on palliative care: Insights from British Columbia, Canada Allan Diane E [email protected] Kelli I [email protected] R Colin [email protected] Centre on Aging, University of Victoria, Sedgewick A104, PO Box 1700, STN CSC, Victoria, BC, V8W 2Y2, Canada2 Centre on Aging and School of Nursing, University of Victoria, Sedgewick A104, PO Box 1700, STN CSC, Victoria, BC, V8W 2Y2, Canada3 Centre for Population and Health Services Research, Okanagan University College, 3333 University Way, Kelowna, BC, V1V 1V7, Canada2005 17 2 2005 4 2 2 22 7 2004 17 2 2005 Copyright © 2005 Allan et al; licensee BioMed Central Ltd.2005Allan et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Research indicating that people increasingly prefer to die at home suggests that palliative care is likely to play a more prominent role in the future of Canada's health care system. Unfortunately, at a time when research evidence should be informing policy and service delivery, little is known about health service utilization by Canadians at the end of life. One existing mechanism that can help address this gap is provincial administrative health data. The purpose of this study was to explore the potential of administrative health data to identify characteristics of palliative care users, patterns of formal service utilization and predictors of palliative care use.
Methods
Bivariate and multivariate analyses were used to examine data from the Capital Health Region, British Columbia Linked Health Databases for the period 1992/93 to 1998/99. The databases examined include continuing care, physician claims, hospital separations, and vital statistics. As the name implies, these databases can be linked at the individual level using unique identifiers so that health services utilization can be tracked across sectors.
Results
General patterns of service use among palliative care patients suggest that general practitioner and medical specialist visits have decreased over time and the utilization of hospital beds has increased. Utilization of community-based services (i.e. home support and home nursing care) shows an overall pattern of decline. However, when compared to non-palliative care patients, palliative care patients spent fewer nights in hospital, used fewer hours of home support, and had a greater number of home nursing care visits.
Conclusions
Administrative health databases can provide valuable information for examining service utilization patterns over time. However, given that decisions surrounding the designation of palliative care include factors beyond the scope of administrative databases (such as quality of life, personal preferences, social support), these databases should only be seen as one source of information to inform service delivery and policy decision making.
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Background
Despite the fact that we all die, the philosophy and practice of palliative and end of life care is relatively new [1]. Certainly, in the broader context of the Canadian health care system, and within most western cultures, palliative and end of life care has played a secondary role due in part to the dominance of the biomedical model and its inherent focus on cure [2,3]. Care at the end of life is likely to play a more prominent role, however, in the future of Canada's health care system [4-6]. This expectation is based on the projected increase in numbers of older persons and the associated heightened risk of developing age-related chronic diseases such as cancer and cardiopulmonary disease. Conjoint with this expectation, the focus of care is shifting from institutional to community care. Thus, as governments seek to contain health care costs and caregivers search for improved quality of life for those nearing the ends of their lives, effective palliative and end of life care will become a more prominent research and policy issue.
Quality palliative and end of life care has emerged as a core value of Canada's health system [7]. Indeed, over the past few years, the Canadian government, policymakers and health care professionals have responded to increasing concerns about the quality of care for the dying. In 2001, the Canadian government appointed a Minister with Special Responsibility for palliative care and a national palliative care Secretariat was established within Health Canada [8]. A national action plan on palliative and end of life care was subsequently developed and several national subcommittees and working groups have formed to deal specifically with issues related to health care services at the end of life. One of these issues is the lack of research and data to inform health service delivery and policy decision-making in palliative and end of life care [7].
At a time when research evidence should be informing policy and health service delivery, little is known about health service utilization by Canadians at the end of life. Although countries such as Australia and the United Kingdom have established national palliative care (PC) surveillance data and extensive work has been done in the United States to identify key data elements pertaining to the end of life, Canada has been slower to develop in this area. Many Canadian PC programs have developed regional data systems [9], and a core data set for national surveillance is currently being developed [10]. National data standards and a surveillance system, however, are yet to be fully implemented in Canada. Therefore, researchers are examining existing sources of data to assist in informing health service and policy decisions. One existing mechanism that may assist in informing such decisions is provincial administrative health databases.
The primary purpose of this paper is to explore the usefulness of provincial administrative health databases as a source of information to inform health care decision making and policy development in palliative and end of life care. Data from the Capital Regional District in British Columbia, Canada are examined for the period 1992/93 to 1998/99. Specifically, our analyses identify characteristics of PC patients, patterns of formal service utilization over time, and predictors of PC service utilization. The implications of our findings for both research and policy, along with recommendations for future data collection initiatives are addressed.
Palliative care: Patient characteristics and service utilization
Research on dying persons has grown in the past decade due, in part, to a rapidly aging population, policy concerns about increasing health expenditures, and a perception that the quality of care at the end of life is inadequate [11,12]. Several patterns have emerged describing patients who receive specialized PC. In Canada and the United Kingdom, for example, over 90 percent of palliative service users have a primary diagnosis of cancer [13]; 70–90 percent of palliative service users in Australia and more than 60 percent in the United States have likewise been diagnosed with cancer [13,14].
Levels of PC services utilization tend to be similar for both males and females [13,15,16,18] although there are exceptions. For example, Grande and associates (1998) reported that more males than females used PC services, while a later study by Grande and colleagues (2002) found more females used PC services. Generally, the relationship between age and PC service use is consistent across studies with services typically provided to adults in the younger older age range (i.e. 65–74 years) [13,15,17-21]. Results of studies investigating the relationship between socioeconomic status and PC service use have not been consistent with some studies reporting a positive relationship between income and referral to PC services [17,22,21,23] while others report no significant relationships [15,18,24].
Some research has examined the patterns of formal service utilization of PC patients. The more frequently cited measures of service utilization include scope of services used, along with length of stay (on a particular PC service) and location of death [4,13,24,25]. Yet, even these have received limited research attention. In Canada, population-based studies examining PC patients commonly use vital statistics and/or cancer registry data [4,22,25]. While providing important information, both data sources are limited in scope. Mortality data typically include underlying cause of death and date of death but little information on demographic characteristics or service utilization. Cancer registries contain demographic and service utilization variables, but are restricted to capturing only data on cancer patients and therefore exclude patients dying from other diseases [26].
Administrative health databases
While administrative health databases have been used for research purposes for decades in Canada, it was the establishment of the Manitoba Centre for Health Policy and Evaluation and the subsequent development of their population health information system, POPULIS [27], which brought this data source into the spotlight. Similar developments in other Canadian provinces since that time now offer health researchers access to provincial administrative health data that can be linked across services. In British Columbia, the Centre for Health Services and Policy Research maintains and links provincial health data and is responsible for extracting and providing data to health researchers upon request from and approval by the Ministry of Health [28]. Like mortality and cancer registry data, however, there are limitations to administrative health data. These databases, for example, were originally constructed to serve a billing role; service providers submit claims in order to be reimbursed for services provided. The data contained in these datasets were thus not originally intended for research purposes. Therefore, while the databases are rich in information for select utilization, supply, and cost issues, their usefulness for addressing other factors that may influence the health of populations is limited. In the context of palliative and end of life care, it is this final point that bears further examination. Specifically, do these administrative databases identify persons receiving PC, and if so, what information is available to researchers interested in studying PC patients and their health service utilization? In addressing these questions, we are primarily concerned with providing examples of what can be done with these data rather than what the data are actually telling us about PC patients and their health care utilization.
Methods
Data for this paper came from the British Columbia Linked Health Database. Housed at the University of British Columbia (BC), this database contains all provincial administrative health data collected by the BC Ministry of Health including physician claims, hospital separations, long-term care data, pharmacare data and vital statistics. Each of these components can be analyzed separately, as a stand-alone database, or linked through unique identification numbers and examined in combination or as a whole. The data were originally collected as part of a larger project examining the impact of regionalization in BC from 1990/91 to 1998/99 [29]. However, as the purpose of this particular paper fell outside the objectives of the larger study, a separate request to the BC Ministry of Health for data access was submitted and approved. Ethics approval from the academic institution was also received.
Given that PC patients could not be identified in the data prior to 1992/93, for the purposes of this paper, data from fiscal years 1992/93 to 1998/99 for all databases (with the exception of vital statistics where only 1998/99 figures were available) were included. Results are based on a 100 percent sample of the population aged 50 and over residing in the Capital Regional District, on Vancouver Island, British Columbia, Canada for each of the years. Both univariate and multivariate analyses were used to determine the usefulness of administrative databases for PC research in British Columbia.
The starting point for these analyses was the identification of all patients designated as being in need of PC (it should be noted that not all persons who may require PC are identified as needing it in the databases, nor are all people who use PC services included in the databases). This was made possible through the 'service type' variable found in the direct care services database, one of many databases that make up the continuing care component of the administrative databases. Next, using the unique identification numbers that are consistent across the different databases, it is possible to link these different datasets thereby allowing for the examination of individual service utilization across the various segments of the health care system (specifically: physician claims, hospital separations, and the home support and home nursing care components of continuing care).
The continuing care database is a suite of databases, however for the purpose of this paper only the direct care services and home support databases were accessed. The direct care services databases were used in this paper for 2 purposes. First, and most importantly, it allowed us to identify those designated palliative through an individual variable. It should be noted that those never entering the continuing care system but who receive palliative services in another sector (i.e., entered the hospital, received palliative services and died in hospital) could not be identified as such and thus are not designated palliative for these analyses. Second, the direct care services database also contains information on home nursing care. For this paper, the number of home nursing visits received by each individual in a given year was calculated. Home support utilization is tracked on a monthly basis. From these files it was possible to calculate the number of hours of home support received by each individual in a given year.
All claims made by physicians are tracked in the Medical Services Plan database. It is therefore possible to calculate the number of visits to a physician made by individuals over the course of a year. The hospital separations database is so named because a hospital patient is entered into the system only when they leave the hospital (this includes deaths). Since admission and separation dates are included as variables, it is possible to calculate the number of nights each individual spends in the hospital (no overnight stay is assigned a 0). Total nights in a given year for each individual can then be summed. The vital statistics data that was accessed included underlying cause of death. This variable is comprised of ICD-9 codes that can be classified into disease categories. This procedure allowed for the identification of cancer versus non-cancer deaths used in the multivariate analysis.
Each of these health databases includes a small number of demographic variables such as age and gender, and geographic identifiers such as health authority and census tract. Marital status is included in one of the continuing care databases. This lack of what are typically referred to as 'control variables' is one of the biggest limitations of administrative databases. To compensate, it is possible to link Census variables at an aggregate level to the administrative data. Of course this procedure only works for variables that lend themselves to averages. For example, it is possible to calculate the average household income for an area but it is not possible to calculate the average gender for an area. Using this premise, average household income was linked to the administrative data at the enumeration area level (the smallest geographic unit released by Statistics Canada and the BC Ministry of Health). In brief, the average household income for an enumeration area is assigned to an individual residing anywhere within the enumeration area. Thus, income is an aggregate measure while the remainder of the variables used in this paper are individual measures.
Finally, as mentioned earlier, each of these databases is linkable. The term linkable is used to describe the assignment of the same unique identification number to an individual regardless of database. In other words, Person A will be assigned the same number in the hospital database and the continuing care database. As such, linkable databases allow researchers to examine health service utilization and health status of the same individual across sectors. Without the ability to link, it would be impossible, for example, to examine underlying cause of death (vital statistics database) in relation to the designation of palliative care (continuing care database).
As described in the preceding paragraphs, 5 health service utilization measures were calculated: number of general practitioner visits per year; number of medical specialist visits per year; number of nights spent in hospital per year; number of hours of home support received per month; and number of home nursing care visits received per month. Next, using these variables, three different analyses were conducted:
1. Descriptive statistics (distribution, mean, median) were used to examine PC patient characteristics and health service utilization by gender. All those identified as being in need of PC for each fiscal year were examined by age, gender, income, and the 5 health service utilization measures. The sample size of PC patients ranged from a low of 74 in 1992/93 to a high of 568 in 1997/98.
2. Because service use is likely to differ by diagnosis, we compared patients designated as being in need of PC with patients who were not designated as in need of PC in order to control for diagnosis. Previous research indicates that the majority of persons receiving specialized palliative and end of life care have a cancer diagnosis [13]. While it is acknowledged that different types of cancer place different demands on the health care system, for the purposes of this study, all cancers were examined in combination. Using the underlying cause of death code from vital statistics available for 1998/99, all those who died of cancer in 1998/99 (n = 2,734) were identified. Next, comparisons involving age, gender, income, and the 5 health service utilization measures were then made between cancer patients designated palliative (n = 119) and cancer patients not designated palliative (n = 2,615).
3. To examine the influence of a PC designation on health service utilization, five multiple linear regression models were estimated using the 1998/99 data from the palliative and non-palliative sample (n = 2,734) described above. The following five dependent variables were regressed on age, gender, average household income, and designation of palliative/non-palliative: (1) number of general practitioner visits; (2) number of medical specialist visits; (3) number of nights spent in the hospital; (4) number of hours of home support; and (5) number of home nursing care visits. Prior to conducting the multivariate analyses, assumptions of normality, linearity, and collinearity were tested and adjustments were made where necessary.
Results
1. Palliative care patient and health service utilization characteristics
The linked administrative database allowed for a description of patient characteristics including age, gender, and income. Table 1 presents characteristics of PC patients by gender from 1992/93 to 1998/99. Results indicate that almost 40 percent of male and female PC patients are between the ages of 70 and 79 years and this trend is consistent across years for males, and varies from 34% to 45% in females. Median values for annual household income range from $46,757 to $53,377 for males and $41,923 to $48,753 for females over the study period. With the exception of 1992/93 and 1997/98 where the median income for males is substantially higher than that of females, the income differences between males and females are slight.
Table 1 Palliative Care Patient Characteristics by Gender: 1992/93–1995/96
1992/93 1993/94 1994/95 1995/96
M (n = 42) F (n = 32) M (n = 212) F (n = 178) M (n = 225) F (n = 194) M (n = 207) F (n = 216)
Age (%)
50–59 9.6 3.1 13.2 9.5 6.7 13.9 10.1 6.5
60–69 21.4 18.8 21.7 32.0 25.7 25.8 23.7 21.7
70–79 47.6 46.9 39.7 34.3 42.7 39.1 38.7 39.4
80+ 21.4 31.2 25.4 24.2 24.9 21.2 27.5 32.4
Median income 53377 41923 47682 46997 47272 48753 47238 47272
Palliative Care Patient Characteristics by Gender: 1996/97–1998/99
1996/97 1997/98 1998/99
M (n = 209) F (n = 189) M (n = 245) M (n = 209) F (n = 189) F (n = 194)
Age (%)
50–59 8.6 9.4 6.6 8.6 9.4 13.9
60–69 27.2 14.9 14.6 27.2 14.9 25.8
70–79 39.2 45.5 40.4 39.2 45.5 39.1
80+ 25.0 30.2 38.4 25.0 30.2 21.2
Median income 46757 45896 49984 46757 45896 48753
The means for the use of the five health services are presented in Table 2. T-test results suggest that there are few health service utilization differences between male and female PC patients. General patterns suggest that female PC patients spend more nights in hospital and receive a greater number of monthly home support hours and home nursing care visits than do their male counterparts, however, few of these gender differences reach statistical significance. One exception is number of home support hours where the results suggest that in three of the seven years examined, females receive a greater number of home support hours than males.
Table 2 Mean Palliative Care Patient Service Utilization by Gender: 1992/93 – 1998/99
Annual GP visits Annual specialist visits Nights spent in hospital annually Monthly hours of home support Monthly home nursing care visits
M F M F M F M F M F
1992/93 19.2 21.3 14.3 19.4 99.9 106.7 15.3 26.0 23.0 29.8
1993/94 15.0 15.7 13.8 13.3 102.9 185.5 21.9** 31.0 25.7** 26.7
1994/95 15.1 13.9 12.9 13.6 136.6 172.3 24.6 30.9 19.7 22.0
1995/96 15.3 14.9 12.7 12.4 139.1 176.0 24.9 26.9 21.5 22.5
1996/97 12.8 13.9 11.9* 14.6 138.1 211.4 26.2* 30.5 19.5 24.0
1997/98 6.8 7.3 6.8 6.8 205.9 313.4 31.8** 28.2 23.1 25.3
1998/99 3.0 3.0 1.3 1.3 117.9 150.1 18.9 27.4 17.3 21.7
*p < .05; **p < .01 (t-test results comparing males and females)
Health service utilization trends observed across the years show that the number of annual general practitioner and specialist visits is fairly steady until 1997/98 when there is a dramatic drop. In contrast, the number of nights spent in hospital steadily increases over the same period until 1998/99 when a sharp reduction in hospital bed utilization is observed. Patterns of home support and home nursing care utilization over time are more variable. Home support hours per month exhibit a pattern similar to number of hospital nights, although the increases and decreases are not as large. Number of home nursing care visits has fluctuated over the years and shows a general pattern of decline from 1992/93 to 1998/99.
2. Palliative care vs. non-palliative care cancer patients and health service utilization characteristics
Results comparing cancer patients designated as being in need of PC and cancer patients who were not designated as in need of PC are presented in Table 3. Findings suggest that there are no differences between palliative and non-palliative cancer patients in terms of gender, average household income, and number of general practitioner and medical specialist visits. Differences are observed between the two groups for age, number of nights spent in hospital, number of hours of home support, and number of home nursing care visits. Specifically, PC cancer patients spend fewer nights in the hospital (p < .001), use fewer hours of home support (p < .05), and have a greater number of home nursing care visits (p < .001) than do non-PC cancer patients.
Table 3 Cancer Patient Characteristics and Service Utilization by Designation of Palliative: 1998/99
Palliative Non-palliative
Age group (%)***
50–59 7.7 8.5
60–69 26.6 17.0
70–79 35.3 37.8
80+ 30.4 36.7
Gender (%)
Male 47.6 47.4
Female 52.4 52.6
Income () 51,718 49,804
GP visits () 3.23 3.21
Specialist visits () 1.21 1.34
Nights in hospital ()*** 20.97 24.19
Home support hours ()* 105.48 172.60
Home nursing care visits ()*** 21.66 6.84
* p < .05; ** p < .01; *** p < .001 (X2 and t-test results comparing palliative and non-palliative)
3. Predictors of health service utilization
Regression results for the five health service utilization regression models are presented in Table 4. Results suggest that age, gender, income, and designation of PC are not strongly predictive of health service utilization. There is a significant relationship between age and number of nights spent in hospital (p < .001), suggesting that the likelihood of spending a greater amount of time in the hospital increases with age. Being female (p < .01) and being older (p < .01) increases the amount of home support hours received, while being younger (p < .05) and being designated as being in need of PC (p < .001) increases the number of home nursing care visits received.
Table 4 Regression estimates and standard errors for five regressions
GP visits Specialist Visits Nights in hospital Home support hours Home nursing care visits
B SE B SE B SE B SE B SE
Age .00 .00 -.00 .00 .14*** .01 .14** .02 -.11* .01
Gender -.02 .01 -.01 .01 .05 .04 .12** .06 .03 .05
Income .01 .00 .00 .00 .00 .00 .04 .00 0.04 .00
Palliative .02 .03 -.02 .02 -.05 .07 -.05 .10 .29*** .05
Adj. R2 -.001 -.001 .02 .03 .10
* p < .05; **p < .01; ***p < .001
Discussion
The primary purpose of this study was to explore the usefulness of provincial administrative health databases as a source of information to inform health care decision making and policy development in palliative and end of life care. Administrative databases represent an existing source of longitudinal information on health system users. The need to track individuals' health service utilization over time has long been recognized by researchers, and this is a key advantage of such databases. At the same time, however, the breadth of information collected is limited. For example, no direct measurements of quality of life, or for that matter, quality of death are compiled. The researcher who makes use of the databases must therefore work within these confines. The findings from the present study provide insights into the precise manner in which these databases can be of value to health services researchers, within these limits. More specifically, the analyses conducted for this paper exhibit that a sample of palliative patients can be identified in one of the many provincial administrative health databases. Since individuals can be linked across sectors, it is possible to examine a number of health status and utilization indicators of these individuals. Finally, linking the health databases with the vital statistics variables gives the researcher insight into the underlying cause of death.
The limitations of administrative databases arise directly from their intended purpose. They were created to serve a billing role, and although they are of value as a research tool, their limitations should be noted. First, socioeconomic variables are limited to age, gender and income. The importance of these variables is unquestioned; however, a wider range of such variables is normally a requirement for social science based health services research. Second, the precise date within a year that patients are designated palliative is not available. Since health service resource utilization has been shown to be much greater during the final few months and weeks of life, information on date designated palliative would be informative. Third, the data that would allow for an examination of either quality of care provided or quality of dying – both of which are central to any study seeking to improve service delivery and associated outcomes – is not collected. Finally, given the complexities of data collection within the health care system, these databases are unlikely to capture all individuals in need of PC. For example, given the fluctuation in the number of palliative patients across years, with special emphasis on the precipitous decline observed between 1997/98 and 1998/99, it appears that the definition and coding of the variable designating an individual palliative may be subject to change over time. If these data are to be used for research purposes, this indicates the need for careful monitoring for continuity of both definition and coding procedures.
While the primary objective of this paper was not to focus on the actual trends and findings, the results hold potential for future studies. For example, the observed decrease in both GP and specialist visits between 1992/93 and 1998/99 for males and females designated as being in need of PC may be indicative of changing trends. First, persons designated palliative are increasingly signaling their desire to die at home. Second, perhaps GPs are increasingly visiting dying patients at home, with the result that these visits are not captured in the health databases since physicians cannot bill for such visits. As GPs become more aware of the need for effective PC, and as they acquire the requisite skills to do so, the territory once thought to be the exclusive realm of the specialist may be experiencing erosion. In the larger picture, this may be indicative of a shift away from a curative medical model approach to end of life care, to a more appropriate social model.
At the same time that GP and specialist visits among persons designated as being in need of PC have declined, mean number of nights spent in hospital annually have shown a general increase. Ostensibly, the increase in hospital nights until the 1997/98 period is a puzzling trend: especially given findings on the general population that show a decrease in nights spent in hospital throughout the 1990's [30]. A potential explanation is that although the rhetoric surrounding the issue trumpets the need to orchestrate a "closer to home" health care delivery system for palliative persons, the reality is that the resources have not yet been committed. Consistent with this explanation, the sharp decline in hospital visits by palliative persons observed for the 1998/99 period may reflect an increased flow of resources towards effective community care. However, prior to making any conclusions about a shift in direction of resources and subsequent utilization patterns, it is necessary to examine the figures beyond 1998/99. In other words, does the decline in hospital nights continue or was this year simply an anomaly?
Although it is not monotonic, the general decline in monthly home nursing visits between 1992/93 and 1998/99 is also somewhat puzzling on the surface, given the calls to enhance home nursing services for the dying. A comparison of home nursing visits between palliative and non-palliative cancer patients, however, reveals that those designated palliative receive considerably more home nursing visits. This latter observation is consistent with the increased care requirements at the end of life. In trying to decipher the differences between these two groups it would be important to examine the time of the visits in relation to death. For instance, do more visits occur in the last months or weeks leading up to death?
In the multivariate analyses, it is of particular interest that being designated palliative is related only to number of home nursing care visits. In other words, being designated palliative has no bearing on general practitioner visits, medical specialist visits, number of hospital nights and number of hours of home support. This, of course, is contrary to bivariate findings presented in Table 3 that reveal significant differences between palliative and non-palliative patients in number of nights spent in hospital and number of hours of home support received. Thus, it appears that when age, gender and income are controlled for, the initial differences found between palliative and non-palliative individuals disappear.
Conclusions
These data provide another piece of the overall picture of service utilization at the end of life. Research into palliative and end of life care is in its infancy at a time when effective and immediate information based on sound scientific research is urgently required. To the extent that administrative databases can bridge this gap, they are of immediate use. Their primary usefulness, however, is probably as a means of identifying and sketching a larger picture, and from that picture, facilitating the generation of key questions that may serve as platforms for launching more in-depth studies. For example, the decline over time in GP and specialist visits, combined with the general decline in home nursing visits for palliative persons raises the question of whether resources have met rhetoric, and if not, why not? Given that health care and policy decisions must include information on factors beyond the scope of administrative databases (such as quality of life, personal preferences, social support), these databases should only be seen as one piece of information to inform service delivery and policy decision making for patients and families at the end of life.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
All authors participated in the conceptualization and design of the study. DEA drafted the initial background, methods and results sections. KIS and DEA conducted all the analyses. KIS and RCR drafted the discussion section and provided additional edits to all sections. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The authors wish to thank Dr. Margaret J. Penning for use of the data collected as part of a larger study entitled Health Care Restructuring and Community-Based Care: A Longitudinal Study, supported by a grant (LOI 1997-054) from the Canadian Health Services Research Foundation with contributions from the Capital Health Region and the Ministry of Health/Ministry Responsible for Seniors in British Columbia, the Manitoba Centre for Health Policy and Evaluation, and the South Eastman and Interlake Regional Health Authorities in Manitoba. Dr. Stajduhar was supported by a Post Doctoral Fellowship from the Canadian Institutes of Health Research and the Michael Smith Foundation for Health Research.
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| 15717923 | PMC552316 | CC BY | 2021-01-04 16:30:52 | no | BMC Palliat Care. 2005 Feb 17; 4:2 | utf-8 | BMC Palliat Care | 2,005 | 10.1186/1472-684X-4-2 | oa_comm |
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BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-6-161566765810.1186/1471-2105-6-16SoftwareEfficient decoding algorithms for generalized hidden Markov model gene finders Majoros William H [email protected] Mihaela [email protected] Arthur L [email protected] Steven L [email protected] Bioinformatics Department, The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD, USA2005 24 1 2005 6 16 16 15 11 2004 24 1 2005 Copyright © 2005 Majoros et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The Generalized Hidden Markov Model (GHMM) has proven a useful framework for the task of computational gene prediction in eukaryotic genomes, due to its flexibility and probabilistic underpinnings. As the focus of the gene finding community shifts toward the use of homology information to improve prediction accuracy, extensions to the basic GHMM model are being explored as possible ways to integrate this homology information into the prediction process. Particularly prominent among these extensions are those techniques which call for the simultaneous prediction of genes in two or more genomes at once, thereby increasing significantly the computational cost of prediction and highlighting the importance of speed and memory efficiency in the implementation of the underlying GHMM algorithms. Unfortunately, the task of implementing an efficient GHMM-based gene finder is already a nontrivial one, and it can be expected that this task will only grow more onerous as our models increase in complexity.
Results
As a first step toward addressing the implementation challenges of these next-generation systems, we describe in detail two software architectures for GHMM-based gene finders, one comprising the common array-based approach, and the other a highly optimized algorithm which requires significantly less memory while achieving virtually identical speed. We then show how both of these architectures can be accelerated by a factor of two by optimizing their content sensors. We finish with a brief illustration of the impact these optimizations have had on the feasibility of our new homology-based gene finder, TWAIN.
Conclusions
In describing a number of optimizations for GHMM-based gene finders and making available two complete open-source software systems embodying these methods, it is our hope that others will be more enabled to explore promising extensions to the GHMM framework, thereby improving the state-of-the-art in gene prediction techniques.
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Background
Generalized Hidden Markov Models have seen wide use in recent years in the field of computational gene prediction. A number of ab initio gene-finding programs are now available which utilize this mathematical framework internally for the modeling and evaluation of gene structure [1-6], and newer systems are now emerging which expand this framework by simultaneously modeling two genomes at once, in order to harness the mutually informative signals present in homologous gene structures from recently diverged species. As greater numbers of such genomes become available, it is tempting to consider the possibility of integrating all this information into increasingly complex models of gene structure and evolution.
Notwithstanding our eagerness to utilize this expected flood of genomic data, methods have yet to be demonstrated which can perform such large-scale parallel analyses without requiring inordinate computational resources. In the case of Generalized Pair HMMs (GPHMMs), for example, the only systems in existence of which we are familiar make a number of relatively restrictive assumptions in order to reduce the computational complexity of the problem to a more tolerable level [7,8,15]. Yet, even these systems are currently capable of handling no more than two genomes at once. If larger numbers of genomes are to be simultaneously integrated into the gene prediction process in a truly useful manner, then it is reasonable to suggest that new methods will be needed for efficient modeling of parallel gene structures and their evolution. Assuming for now that these methods are likely to continue to build on the basic GHMM framework, we feel it is important that efficient methods of GHMM implementation be properly disseminated for the benefit of those who are to work on this next generation of eukaryotic gene finders.
Modeling genes with a GHMM
A Hidden Markov Model (HMM) is a state-based generative model which transitions stochastically from state to state, emitting a single symbol from each state. A GHMM (or semi-Markov model) generalizes this scenario by allowing individual states to emit strings of symbols rather than one symbol at a time [9,10]. A GHMM is parameterized by its transition probabilities, its state duration (i.e., feature length) probabilities, and its state emission probabilities. These probabilities influence the behavior of the model in terms of which sequences are most likely to be emitted and which series of states are most likely to be visited by the model as it generates its output.
Eukaryotic gene prediction entails the parsing of a DNA sequence into a set of putative CDSs (coding segments, hereafter referred to informally as "genes") and their corresponding exon-intron structures [11]. Thus, the problem of eukaryotic gene prediction can be approximately stated as one of parsing sequences over the nucleotide alphabet Σ = {A,C,G,T} according to the regular expression:
Σ*(ATGΣ*(GTΣ*AG)*Σ*Γ)*Σ*, (1)
where the signals (start and stop codons, donors, and acceptors) have been underlined for clarity, and where Γ = {TAG,TGA,TAA} represents a stop codon. (The actual nucleotides comprising these signals may differ between organisms; we have given the most common ones). An additional constraint not explicitly represented in Formula 1 is that the number of non-intron nucleotides between the start and stop codons of a single gene must be a multiple of three, and furthermore, if these nucleotides are aggregated into a discrete number of nonoverlapping triples, or codons, then none of these codons must be a stop codon, other than the stop codon which terminates the gene. Note that the Σ* terms in Formula 1 permit the occurrence of pseudo-signals – e.g., an ATG triple which does not comprise a true start codon. Gene prediction with a GHMM thus entails parsing with an ambiguous stochastic regular grammar; the challenge is to find the most probable parse of an input sequence, given the GHMM parameters and the input sequence.
In the case of simple Hidden Markov Models, this optimal parsing (or decoding) problem can be solved with the well-known Viterbi algorithm, a dynamic programming algorithm with run time linear in the sequence length (for a fixed number of states) [12]. A modified Viterbi algorithm is required in the case of GHMMs, since each state can now emit more than one symbol at a time [2], resulting in the following optimization problem:
where φ is a parse of the sequence consisting of a series of states qi and state durations di, 0≤i≤n, with each state qi emitting subsequence Si of length di, so that the concatenation of all S0S1...Sn produces the complete output sequence S (but note that states q0 and qn are silent, producing no output). Pe(Si|qi,di) denotes the probability that state qi emits subsequence Si, given duration di; Pt(qi|qi-1) is the probability that the GHMM transitions from state qi-1 to state qi; and Pd(di|qi) is the probability that state qi has duration di. The argmax is over all parses of the DNA sequence into well-formed exon-intron structures; hence, the problem is one of finding the parse which maximizes the product in Equation 2.
Implementation
The PSA decoding algorithm
The approach commonly used in GHMM gene finders for evaluating Equation 2 is to allocate several arrays, one per variable-length feature state, and to evaluate the arrays left-to-right along the length of the input sequence according to a dynamic programming algorithm, which we will detail below. We refer to this approach as the Prefix Sum Arrays (PSA) approach, since the values in the aforementioned arrays represent cumulative scores for prefixes of the sequence.
Without loss of generality, let us consider the GHMM structure depicted in Figure 1. Although individual GHMMs will differ from this particular structure on specific points, the model in Figure 1 is general enough to serve as a concrete example as we illustrate the operation of the algorithm.
The diamonds denote the states for fixed length features (ATG = start codon, TAG = stop codon, GT = donor, AG = acceptor) and the circles denote states for variable length features (N = intergenic, I = intron, Esng = single exon, Einit = initial exon, Eint = internal exon, Efin = final exon). This model generates genes only on the forward strand of the DNA; to obtain a double-stranded model one can simply mirror the structure and link the forward and reverse models through a single merged intergenic state.
Associated with each diamond state is a signal sensor such as a weight matrix (WMM) or some other fixed-length model (e.g., a WAM, WWAM, MDD tree, etc.) [13], and with each circular state is associated a variable-length content sensor, such as a Markov chain (MC) or an Interpolated Markov Model (IMM) [14].
For the purposes of illustration, we will consider only the simplest of each model type, since the more complex model types commonly in use can in general be handled generically within the GHMM framework. The simplest fixed-length model is the WMM:
where xh..xh+n denotes the subsequence currently within a sliding (n + 1)-element window, called the context window, and P(x|θ[i]) denotes the probability of nucleotide x occurring at position i within the window, for model θ. In practice, all of the probabilities described in all of these models are represented in log space (to reduce the incidence of numerical underflow on the computer), so that products of probabilities can be replaced with sums of their logs.
The simplest variable-length model used in practice is the Markov chain. An nth-order Markov chain M for state qi would evaluate the probability P(Si|qi,di) of a putative feature Si according to:
where xj is the jth nucleotide in the sequence of the putative feature, di is the length of that feature, and PM(xj|xj-n..xj-1) is the probability of nucleotide xj conditional on the identities of its n predecessor nucleotides, according to content model M. As with the fixed-length model described above, this computation is typically done in log space.
In scoring the signals and content regions of a putative gene parse, it will be important for us to carefully differentiate between the nucleotides which are scored by a signal sensor and those which are scored by a content sensor in a putative parse. As shown in Figure 2, the content and signal regions must partition the sequence into non-overlapping segments; allowing overlaps would result in double-counting of nucleotide probabilities, which can lead to undesirable biases in the decoding algorithm.
The first step of the PSA algorithm is to compute a prefix sum array for each content sensor. For noncoding states (introns and intergenic) this can be formalized as shown in Figure 3.
In the case of exon states, it is important to capture the different statistical properties present in the three codon positions, referred to as phase 0, phase 1, and phase 2. We employ three Markov chains, M0, M1, and M2, corresponding to these three phases. Together, these three chains constitute a three-periodic Markov chain, M{0,1,2}. Exon states then require three arrays, each of which can be initialized using the procedure shown in Figure 4.
In this way, we can initialize the three arrays αi, 0, αi, 1, and αi, 2 for an exon state qi as follows:
for ω ← 0 to 2 do
init_phased(αi,ω, S, M{0, 1, 2},ω) ;
The individual chains M0, M1, and M2 comprising M{0,1,2} are applied in periodic fashion within the procedure init_phased() to compute conditional probabilities of successive nucleotides along the length of the array. The three arrays are phase-shifted by one from each other, with each element in the array storing the cumulative score of the prefix up to the current nucleotide. The first nucleotide is taken to be in phase ω for array αi,ω. Initializing the arrays for reverse-strand states can be achieved by simply reverse-complementing the DNA sequence and then reversing the order of the resulting arrays (keeping in mind later that the reverse-strand arrays tabulate their sums from the right, rather than the left, and that ω is the phase of the last array entry rather than the first).
Once the prefix sum arrays have been initialized for all variable-duration states, we make another left-to-right pass over the input sequence to look for all possible matches to the fixed-length states, via the signal sensors. In general, a signal sensor θ models the statistical biases of nucleotides at fixed positions surrounding a signal of a given type, such as a start codon. Whenever an appropriate consensus is encountered (such as ATG for the start codon sensor), the signal sensor's fixed-length window is superimposed around the putative signal (i.e., with a margin of zero or more nucleotides on either side of the signal consensus) and evaluated to produce a logarithmic signal score RS = log P(xh..xh+n-1|θ), where h is the position of the beginning of the window and n is the window length. If signal thresholding is desired, RS can be compared to a pre-specified threshold and those locations scoring below the threshold can be eliminated from consideration as putative signals.
The remaining candidates for signals of each type are then inserted into a type-specific signal queue for consideration later as possible predecessors of subsequent signals in a putative gene model. As each new signal is encountered, the optimal predecessors for the signal are selected from among the current contents of the signal queues, using a scoring function described below. In the example (forward strand) GHMM depicted in Figure 1, the possible (predecessor→successor) patterns are:
ATG→TAG
ATG→GT
GT→AG
AG→GT
AG→TAG
TAG→ATG
Associated with each of these patterns is a transition probability, Pt(qi|qi-1), which is included in the scoring of a possible predecessor; this probability can be accessed quickly by indexing into a two-dimensional array. The logarithmic transition score will be denoted RT(qi-1,qi) = log Pt(qi|qi-1).
The distance from a prospective predecessor to the current signal is also included in the evaluation in the form of Pd(di|qi) for distance (=duration) di and signal type (=state) qi. This probability can usually be obtained relatively quickly, depending on the representation of the duration distributions. If the distributions have been fitted to a curve with a simple algebraic formula, then evaluation of the formula is typically a constant-time operation. If a histogram is instead maintained, then a binary search is typically required to find the histogram interval containing the given distance. We denote the logarithmic duration score RD(qi,qj) = log Pd(di|qi:j) where di is the length of the content region delimited by signals qi and qj, and qi:j is the variable-length state corresponding to that content region.
Following Equation 2, the final component of the scoring function is the emission probability Pe(Si|qi,di). For a fixed-length state, this is simply the score produced by the signal sensor. For a variable-length state qi, Pe can be evaluated very quickly by indexing into the prefix sum array αi,γ for state qi and phase γ at the appropriate indices for the two signals and simply performing subtraction:
RC(spred,scur,ω) ← αi,γ [wpos(scur) - 1] - αi,γ [wpos(spred) + wlen(spred) - 1], (5)
where wpos(s) is the 0-based position (within the full input sequence) of the first nucleotide in the context window for signal s, wlen(s) is the length of the context window for signal s, and spred and scur are the predecessor and current signals, respectively. In the case of coding features, γ is the phase of the array and ω = (γ + pos(scur))mod3 is the phase of scur, for pos(scur) the position of the leftmost consensus base of scur. For reverse-strand features, since the prefix sum arrays tabulate their sums from the right instead of the left, the subtraction must be reversed:
RC(spred,scur,ω) ← αi,γ[wpos(spred) + wlen(spred)] - αi,γ[wpos(scur)], (6)
and ω = (γ +L - pos(scur) - 1)mod3, for L the sequence length. For noncoding features, the phases can be ignored when computing RC, since there is only one array per noncoding state.
The resulting optimization function is:
for current signal sj and predecessor signal si; RI(si,γi) denotes the logarithmic inductive score for signal si in phase γi. For forward-strand coding features, the phases γi and γj are related by:
γi = (γj - Δ)mod3, (8)
for Δ the putative exon length, or, equivalently,
γj = (γi + Δ)mod3. (9)
These relations can be converted to the reverse strand by swapping + and -. For introns, γi = γj. For intergenic features, the phase will always be 0 for a forward strand signal and 2 for a reverse strand signal (since on the reverse strand the leftmost base of a 3-base signal would be in phase 2).
The result of Equation 7 is the optimal predecessor for signal sj. This scoring function is evaluated for all appropriate predecessor signals, which are readily available in one or more queues, as mentioned above. A pointer called a trellis link is then created, pointing from the current signal to its optimal predecessor. In the case of those signals that can terminate an exon or an intron, three optimal predecessors must be retained, one for each phase. The inductive score RI(sj,γj) of the new signal sj is then initialized from the selected predecessor si as follows:
RI(sj, γj) ← RI(si, γj) + RT(si, sj) + RD(si, sj) + RC(si, sj, γj) + RS(sj), (10)
where RS(sj) is the logarithmic score produced by the signal sensor for signal sj.
A final step to be performed at each position along the input sequence is to drop from each queue any signal that has been rendered unreachable from all subsequent positions due to intervening stop codons. Except for the final stop codon of a gene, in-phase (i.e., in phase 0) stop codons are generally not permitted in coding exons; for this reason, any potential stop codon (regardless of its signal score) will eclipse any preceding start codon or acceptor site (or, on the reverse strand, stop codon or donor site) in the corresponding phase. The algorithm shown in Figure 5 addresses this issue by dropping any fully eclipsed signal (i.e., eclipsed in all three phases) from its queue.
For the reverse strand, line 3 of eclipse() should be changed to:
ω ← (p-pos(s)-len(s) - 1) mod3;
where len(s) is the length of the consensus sequence for signal s (e.g., 3 for ATG). Note that by xmod3 we mean the positive remainder after division of x by 3; in some programming languages (such as C/C++), a negative remainder may be returned, in which case 3 should be added to the result.
A special case of eclipsing which is not handled by eclipse() is that which occurs when a stop codon straddles an intron; this can be handled fairly simply by checking for such when considering each donor signal as a prospective predecessor for an acceptor signal (or vice-versa on the reverse strand). As each predecessor is evaluated, the bases immediately before the donor and immediately following the acceptor are examined, and if a stop codon is formed, the predecessor is no longer considered eligible for selection in the corresponding phase.
As shown in Figure 5, when a signal has been eclipsed in all three phases it can be removed from its queue. In this way, as a signal falls further and further behind the current position in the sequence, the signal becomes more and more likely to be eclipsed in all three phases as randomly formed stop codons are encountered in the sequence, so that coding queues (e.g., those holding forward strand start codons and acceptors, or reverse strand donors and stop codons) tend not to grow without bound, but to be limited on average to some maximal load determined by the nucleotide composition statistics of the sequence. Because of this effect, the expected number of signals which must be considered during predecessor evaluation can be considered effectively constant in practice.
In the case of noncoding queues (e.g., those holding forward strand donors or stop codons, etc.), the assumption that noncoding features follow a geometric (i.e., exponentially decreasing) distribution allows us to limit these queues to a single element (per phase), because once a noncoding predecessor has been selected in a given phase, no other noncoding predecessor which has already been compared to the selected predecessor can ever become more attractive by virtue of its transition probability (since they are the same for signals of the same type, of which all the signals in a single queue are), its duration probability (since the geometric distribution ensures that their respective duration probabilities decrease at the same rate), nor its sequence probability (since any nucleotides encountered after seeing the two potential predecessors will affect their sequence scores identically).
Because the coding and noncoding queues are effectively limited to a constant load (as argued above), the expected processing time at each nucleotide is O(1) in practice and therefore the entire algorithm up to this point requires time O(L) for an input sequence of length L and a GHMM with a fixed number of states. It will be seen that the traceback procedure described below also requires time O(L), and so this is the time complexity of the PSA decoding algorithm for normal eukaryotic genomes (i.e., those not especially lacking in random stop codons).
Once the end of the sequence is reached, the optimal parse φ can be reconstructed by tracing back through the trellis links. In order for this to be done, a set of virtual, anchor signals (one of each type) must be instantiated at either terminus of the sequence (each having signal score RS = 0). Those at the left terminus will have been entered into the appropriate queues at the very start of the algorithm as prospective targets for the first trellis links (and having inductive scores RI = 0), and those at the right terminus are the last signals to be evaluated and linked into the trellis. The highest scoring of these right terminal anchor signals is selected (in its highest-scoring phase) as the starting point for the traceback procedure. Traceback consists merely of following the trellis links backward while adjusting for phase changes across exons, as shown in Figure 6.
Modifications to Figure 6 for features on the reverse-strand include changing the AG on line 8 to GT, changing the subtraction on line 9 to addition, and changing the 0 on line 7 to 2.
It should be clear from the foregoing that the space requirements of the PSA decoding algorithm are O(L|Q|) for sequence length L and variable-duration state set Q. If, for example, array elements are 8-byte double-precision floating point numbers, then the GHMM depicted in Figure 1 would require 14 prefix sum arrays (4 exon states × 3 phases + 1 intergenic state + 1 intron state), resulting in a memory requirement of at least 112 bytes per nucleotide. Generalizing this GHMM to handle both DNA strands would increase this to 216 bytes per nucleotide, so that processing of a 1 Mb sequence would require at least 216 Mb of RAM just for the arrays. Adding states for 5' and 3' untranslated regions would increase this to 248 Mb of RAM for a 1 Mb sequence, or over 1 Gb of RAM for a 5 Mb sequence. For the purposes of comparative gene finding on multiple organisms with large genes, these requirements seem less than ideal, especially when one considers the possibility of adding yet other states.
The memory requirements can be reduced in several ways. First, Markov chains can be shared by similar states. For example, the intron and intergenic states can share a single Markov chain trained on pooled noncoding DNA, and all the exon states can use the same three-periodic Markov chain trained on pooled coding DNA. To our knowledge, the extent to which this optimization affects the accuracy of the resulting gene finder has not been systematically investigated, though it is commonly used in practice. Second, the models for exons can be modified so as to utilize likelihood ratios instead of probabilities. If the models for exons are re-parameterized to compute:
and the noncoding models are modified to compute:
then the latter can be seen to be unnecessary, since it will always evaluate to 1. Such a modification is valid and will have no effect on the mathematical structure of the optimization problem given in Equation 2 as long as the denominator is evaluated using a Markov chain or other multiplicative model, since the effect of the denominator on inductive scores will then be constant across all possible predecessors for any given signal. Using such ratios allows us to skip the evaluation of all noncoding states, so that the number of prefix sum arrays required for a double-stranded version of the GHMM in Figure 1 would be only 6 (assuming the previous optimization is applied as well), corresponding to the three exon phases on two strands. Furthermore, to the extent that these likelihood ratios are expected to have a relatively limited numerical range, lower-precision floating point numbers can be used, or the ratios could instead be multiplied by an appropriate scaling factor and then stored as 2-byte integers [2]. This is a significant reduction, though asymptotically the complexity is still O(L|Q|). An additional consideration is that the log-likelihood strategy makes unavailable (or at least inseparable) the raw coding and noncoding scores, which might be desired later for some unforeseen application.
A third method of reducing the memory requirements is to eliminate the prefix sum arrays altogether, resulting in what we call the Dynamic Score Propagation (DSP) algorithm.
The DSP decoding algorithm
Informally, the DSP algorithm is similar to the PSA algorithm except that rather than storing all nucleotide scores for all content sensors in a set of prefix sum arrays, we instead store only the specific elements of those arrays that are needed for assessing prospective predecessors during the trellis formation. Associated with each signal is a "propagator" variable which represents the log probability of the highest-scoring partial parse up to and including this signal. As processing proceeds left-to-right along the sequence, these propagators are updated so as to extend these partial parses up to the current position. In this way, the inductive score of each signal is incrementally propagated up to each potential successor signal that is encountered during processing; when a signal is eclipsed in all phases by stop codons (i.e., removed from its respective queue), propagation of that signal's inductive score halts, since further updates would be useless beyond that point. Because no prefix sum arrays are allocated, and because the signal queues are effectively limited in size (as argued previously), the expected memory requirements of DSP will be seen to be O(L+|Q|), where the constant factor associated with the L term is small, reflecting only the number of signals per nucleotide emitted by the signal sensors, as well as the memory required to store the sequence itself.
Let us introduce some notation. We define a propagator π to be a 3-element array, indexed using the notation π[i] for 0≤i≤2; when dealing with multiple propagators, πj[i] will denote element i of the jth propagator.
Each signal si will now have associated with it a propagator, denoted πi. For signals which can be members of multiple queues (such as start codons, which can be members of both the initial exon queue and the single exon queue), the signal will have one propagator per queue, but it will be clear from the context to which propagator we refer. Each queue will also have a propagator associated with it, though for the sake of reducing ambiguity we will refer to these as accumulators and represent them with the symbol α. The purpose of the accumulators is to reduce the number of updates to individual signal propagators; otherwise, every signal propagator in every queue would need to be updated at every position in the input sequence. The accumulator for a given queue will accumulate additions to be made to the propagators of the signals currently in the queue. The update of signal propagators from their queue's accumulator is delayed as long as possible, as described below. Accumulator scores are initialized to zero, as are the propagator scores for the left terminus anchor signals; the general case of propagator initialization will be described shortly.
Updating of a propagator π from an accumulator α is simple in the case of a noncoding queue:
∀0≤ω≤2 π[ω] ← π[ω] + α[0]. (13)
For coding queues, the update must take into account the location of the signal s associated with the propagator π, in order to synchronize the periodic association between phase and array index:
∀0≤ω≤2 π[ω] ← π[ω] + α[(ω - pos(s) - len(s))mod3], (14)
or, on the reverse strand:
∀0≤ω≤2 π[ω] ← π[ω] + α[(ω + pos(s) + len(s))mod3]. (15)
Given a content sensor M, a coding accumulator can be updated according to the rule:
∀0≤ω≤2 α[ω] ← α[ω] + log PM[(ω+f)mod3](xf), (16)
or, on the reverse strand:
∀0≤ω≤2 α[ω] ← α[ω] + log PW[(ω-f)mod3](xf), (17)
where f is the position of the current nucleotide xf, PM[ω](xf) is the probability assigned to xf by the content sensor M in phase ω, and W is the reverse-complementary model to M which computes the probability of its parameter on the opposite strand and taking contexts from the right rather than from the left. This update occurs once at each position along the input sequence. Use of f provides an absolute frame of reference when updating the accumulator. This is necessary because the accumulator for a queue has no intrinsic notion of phase: unlike an individual signal, a queue is not rooted at any particular location relative to the sequence.
For noncoding queues, only the 0th element of the accumulator must be updated:
α[0] ← α[0] + log PM(xf). (18)
All that remains is to specify the rule for selecting an optimal predecessor and using it to initialize a new signal's propagator. We first consider new signals which terminate a putative exon. Let si denote the predecessor under consideration and sj the new signal. Denote by Δ the length of the putative exon. Then on the forward strand, we can compare predecessors with respect to phase ω via the scoring function RCI + RD + RT, where RD and RT are the duration and transition scores described earlier and RCI includes the content score and the inductive score from the previous signal:
∀0≤ω≤2 RCI(si,ω) ← πi[(ω - Δ)mod3]. (19)
On the reverse strand we have:
∀0≤ω≤2 RCI(si,ω) ← πi [(ω + Δ)mod3]. (20)
For introns it is still necessary to separate the three phase-specific scores to avoid greedy behavior, though the phase does not change across an intron, so no Δ term is necessary:
∀0≤ω≤2 RCI(si,ω) ← πi[ω]. (21)
When the preceding feature is intergenic we need only refer to phase zero of the preceding stop codon:
RCI(si,ω) ← πi[0], (22)
or, on the reverse strand, phase 2 of the preceding start codon (since the leftmost base of the reverse-strand start codon will reside in phase 2).
Once an optimal predecessor with score RCI + RD + RT is selected with respect to a given phase ω, the appropriate element of the new signal's propagator can be initialized directly:
πj[ω] ← RCI(si,ω) + RD(si,sj) + RT(si,sj) + RS(sj), (23)
where RS(sj) = P(context(sj)|θj) is the score assigned to the context window of the new signal sj by the appropriate signal sensor θj. An exception to Equation 23 occurs when ω is not a valid phase for signal sj (e.g., phase 1 for a start codon), in which case we instead set πj[ω] to -∞.
One final complication arises from the fact that the algorithm, as we have presented it, does not permit adjacent signals in a prospective parse to have overlapping signal sensor windows; to allow such would be to permit double-counting of nucleotide probabilities, thereby biasing the probabilistic scoring function. It is a simple matter to reformulate the algorithm so that signal sensors score only the two or three consensus nucleotides of the signals under consideration; this would allow adjacent signals in a prospective parse to be as close as possible without actually overlapping (i.e., a single exon consisting of the sequence ATGTAG would be permitted, even if the start codon and stop codon context windows overlapped). However, doing so might be expected to decrease gene finder accuracy, for two reasons: (1) statistical biases occurring at fixed positions relative to signals of a given type can in general be better exploited by a signal sensor specifically trained on such positions than by a content sensor trained on data pooled from many positions at variable distances from the signal, and (2) in the case of Markov chains and Interpolated Markov Models, probability estimates for nucleotides immediately following a signal can be inadvertently conditioned on the few trailing nucleotides of the preceding feature (assuming the chain has a sufficiently high order), even though the models are typically not trained accordingly. For these reasons, we prefer to use signal sensors which impose a moderate margin around their respective signals, both to detect any biologically relevant biases which might exist within those margins, and to ensure that content sensors condition their probabilities only on nucleotides within the same feature.
Given the foregoing, it is necessary to utilize a separate "holding queue" for signals which have recently been detected by their signal sensors but which have context windows still overlapping the current position in the DSP algorithm. The reason for this is that propagator updates via Equations 13–15 must not be applied to signals having context windows overlapping any nucleotides already accounted for in the accumulator scores, since to do so would be to double-count probabilities. It is therefore necessary to observe the following discipline.
Associated with each signal queue Gi there must be a separate holding queue, Hi. When a signal is instantiated by a signal sensor it is added to the appropriate Hi rather than to Gi. As the algorithm advances along the sequence, at each new position we must examine the contents of each holding queue Hi to identify any signal having a context window which has now passed completely to the left of the current position. If one or more such signals are identified, then we first update the propagators of all the signals in the main queue Gi using Equations 13–15, then zero-out the values of the accumulator αi for that queue, and then allow the recently passed signals to graduate from Hi to Gi. Observe that at this point all the signals in Gi have in their propagators scores which have effectively been propagated up to the same point in the sequence, and that point is immediately left of the current position; this invariant is necessary for the proper operation of the algorithm. All content sensors are then evaluated at the current position and their resulting single-nucleotide scores are used to update the accumulators for their respective queues. Finally, whenever it becomes necessary to evaluate the signals in some queue Gi as possible predecessors of a new signal, we must first update the propagators of all the elements of Gi as described above, so that the comparison will be based on fully propagated scores.
Equivalence of DSP and PSA
We now give a proof that DSP is mathematically equivalent to PSA, since it may not be entirely obvious from the foregoing description. We will consider only the forward strand cases; the proof for the reverse strand cases can be derived by a series of trivial substitutions in the proof below.
To begin, we show by induction that the signal propagator πj[ω] for signal sj is initialized to the PSA inductive score RI(sj,ω). For the basis step, recall that the left terminus anchor signals were initialized to have zero scores in both PSA and DSP, regardless of whether a given signal began a coding or noncoding feature. In the case of coding features, substituting Equation 19 into Equation 23 yields:
πj[ω] ← πi[(ω - Δ)mod3] + RD(si,sj) + RT(si,sj) + RS(sj). (24)
According to Equation 10, this initialization will result in πj[ω] = RI(sj,ω) only if:
πi[(ω - Δ)mod3] = RI(si,γi) + RC(si,sj,ω), (25)
where γi = (ω - Δ)mod3 according to Equation 8. At the time that signal sj is instantiated by its signal sensor, πi has been propagated up to e = wpos(sj) - 1, the nucleotide just before the leftmost position of the context window for sj. By the inductive hypothesis, πi[γi] was initialized to RI(si,γi). This initialization occurred at the time when the current DSP position was at the beginning of the predecessor's context window. Note, however, that πi effectively began receiving updates at position b = wpos(si) + wlen(si), the position immediately following the end of the signal's context window, at which point si graduated from its holding queue. Thus, πi[γi] will have accumulated content scores for positions b through e, inclusive. In order to establish Equation 25, we need to show that these accumulations sum to precisely RC(si,sj,ω).
Substituting Equation 16 into Equation 14 we get the following formula describing propagator updates as if they came directly from content sensor M:
∀0≤ω≤2 π[ω] ← π[ω] + log PM[(ω+Δ)mod3](xf), (26)
where Δ = f-(pos(si) + len(si)) is the distance between the rightmost end of signal si and the current position f in the DSP algorithm. Let us introduce the notation:
F(i,j,ω) = ∑k = i..jlog PM[(ω+k)mod3](xk). (27)
Using this notation, πi[γi] has since its initialization accumulated F(b,e,γi - pos(si) - len(si)); this can be verified by expanding this expression via Equation 27 and observing that the result equals a summation of the log term in Equation 26 over f = b to e. Looking at init_phased(), it should be obvious that the effect of lines 5 and 8 will be that:
αi,γ [h] = ∑k = 0..hlog PM [(k+γ)mod3](xk) = F(0,h,γ). (28)
According to Equation 5, showing that πi[γi] has accumulated RC(si,sj,ω) is therefore equivalent to:
F(b,e,ψ) = F(0,wpos(sj) - 1,γ) - F(0,wpos(si) + wlen(si) - 1,γ), (29)
where ψ = γi - pos(si) - len(si) and γ = ω - pos(sj). Equivalently:
F(b,e,ψ) = F(0,e,γ) - F(0,b - 1,γ). (30)
To see that ψ ≡ γ(mod3), observe that pos(sj) - (pos(si) + len(si)) = Δ, the length of the putative exon (possibly shortened by three bases, in the case where si is a start codon), and further that γi - ω ≡ -Δ(mod3) according to Equation 8, so that ψ - γ ≡ Δ-Δ ≡ 0(mod3). Thus, Equation 30 is equivalent to:
F(b,e,γ) = F(0,e,γ) - F(0,b - 1,γ), (31)
which can be established as a tautology by simple algebra after expansion with Equation 27. This shows that the signal propagator for signal sj is initialized to the PSA inductive score RI(sj,ω), and thus establishes the inductive step of the proof in the case of coding features.
To see that the above arguments also hold for noncoding features, note that Equation 21 simplifies Equation 25 to:
πi[ω] = RI(si,ω) + RC(si,sj), (32)
that Equations 13 and 18 combine to simplify Equation 26 to:
∀0≤ω≤2 π[ω] ← π[ω] + log PM(xf), (33)
and that lines 4 and 6 of init_nonphased() cause:
αi[h] = ∑k = 0..hlog PM(xk) = FNC(0,h), (34)
for FNC(i,j) = ∑k = i..jlog PM(xk). We can thus reformulate Equation 29 as:
FNC(b,e) = FNC(0,wpos(scur) - 1) - FNC(0,wpos(spred) + wlen(spred) - 1), (35)
or, equivalently:
FNC(b,e) = FNC(0,e) - FNC(0,b - 1), (36)
which is again a tautology. In the interests of brevity, we leave it up to the reader to verify that the above arguments still apply when the noncoding features are intergenic, thereby invoking Equation 22 rather than Equation 21 in formulating Equation 31.
To see that the selection of optimal predecessors is also performed identically in the two algorithms, note that the PSA criterion given in Equation 7 is equivalent to the argmax(RCI + RD + RT) criterion of DSP as long as RCI(si,ω) = RC(si,sj,ω) + RI(si,γi) at the time the optimal predecessor is selected, which we have in fact already shown by establishing Equation 25.
Thus, DSP and PSA build identical trellises; application of the same traceback() procedure should therefore produce identical gene predictions.
Fast decoding of Markov chains
Markov chains are typically implemented in GHMM-based gene finders using hash tables, due to the simplicity of such an implementation. Thus, for a given Markov chain M we may utilize a hash table which associates the probability PM(xj|xj-n..xj-1) with the sequence xj-n..xj. Although hash tables provide a relatively efficient solution for this task, they are wasteful in the sense that as we evaluate the chain on successive nucleotides in a sequence, we repeatedly manipulate preceding nucleotides in forming successive substrings to be indexed into the hash table.
A much faster (and much more elegant) solution is to employ a Finite State Machine (FSM) in which states exist for all possible sequences of length n+1 or less, and where the state having label xj-n..xj emits the probability PM(xj|xj-n..xj-1), for nth-order Markov chain M. In this way, the transition probabilities of the Markov chain become the state emissions of the FSM. During a single left-to-right scan of a sequence, each base requires only a single two-dimensional array indexing operation to access the desired probability, and a single integer value store operation to remember the identity of the new state. When compared to the typical regime of arithmetic and bit-shift operations over an (n+1)-element string that would be required for a typical hash function, the difference can be significant.
Implementing this optimization is fairly straightforward, both for conventional Markov chains and for Interpolated Markov Models, whether homogeneous or three-periodic. Central to the method is a means of mapping between state labels and integer state identifiers for use in indexing into the transition table. The base-4 number system can be utilized for this purpose, assuming a nucleotide mapping such as ∇ = {A↔0, C↔1, G↔2, T↔3}. To account for lower-order states, define:
which gives the total number of strings of length less than L. Converting a string S = x0..xL-1 to base-4 can be accomplished as follows:
Now a string S can be mapped to a state index using:
state(S) = B(|S|) + λ(S), (39)
where |S| denotes the length of S.
Given this integer↔label mapping and an nth-order Markov chain in hash table format, the FSM state emissions can be initialized by indexing state labels into the hash table to obtain the Markov chain transition probabilities. The transition table can be initialized fairly simply by noting that the successor of state x0..xL-1 upon seeing symbol s is x1..xL-1s if L = n + 1, or x0..xL-1s for L <n + 1. A model for the reverse strand can be handled by applying this scheme in reverse, so that the state with label xj-n..xj emits the probability PM(xj-n|xj-n+1..xj), and the lower-order states are reserved for the end of the sequence rather than the beginning.
Results
Table 1 shows the memory and time requirements for two GHMM gene finders, one using the PSA algorithm and the other the DSP algorithm, on a 922 Kb sequence. Note that the DSP gene finder has 31 states, while the PSA gene finder explicitly evaluates only 6 states, so that they both give a ratio of 2.8 seconds per state on this sequence, while the ratio of memory per state is 14 Mb for the PSA gene finder and 0.95 Mb for the DSP gene finder. Thus, the DSP and PSA algorithms appear to consume the same amount of time per state, while DSP requires only a fraction of the memory (per state) as PSA.
Table 2 shows the results of applying the FSM optimization to a DSP gene finder to accelerate its content sensors. As can be seen from the table, the FSM approach reduces execution time by more than half (as compared to a hash table implementation), while also reducing total RAM usage. The DSP/FSM configuration reported here utilized both conventional Markov chains as well as Interpolated Markov Models, both represented using FSMs. Note that the hashing software used for comparison was a very efficient implementation which used native C character arrays; in particular, we did not use the C++ Standard Template Library (STL) implementations of string and hash, due to efficiency concerns regarding the re-copying of string arguments to the hash function. Our custom string hashing implementation was found to be much faster than the STL implementation (data not shown). Accordingly, one can expect an FSM implementation to show even greater gains as compared to an STL-based hashing implementation.
We utilized our DSP-based gene finder TIGRscan [5] in the construction of our syntenic gene finder TWAIN, a Generalized Pair HMM which performs gene prediction in two genomes simultaneously. TWAIN operates by invoking a modified version of TIGRscan to build a directed acyclic graph of all high-scoring parses of each of the two input sequences. Early experiments indicated that these parse graphs could be quite large in practice and may therefore require a significant portion of available RAM for their storage. In addition, the dynamic programming matrix used by TWAIN promised to be large as well. It was in anticipation of this problem that we were prompted to develop TIGRscan using the DSP architecture, to minimize the memory requirements of the underlying GHMM, thereby freeing the remaining available memory for use by the rest of the machinery within TWAIN.
As a result of these and other optimizations (such as our use of a sparse matrix representation for TWAIN's dynamic programming algorithm) we were able to apply TWAIN's gene prediction component to a pair of fungal genomes (Aspergillus fumigatus and A. nidulans) while consuming under 50 Mb of RAM, whereas an earlier prototype of this system applied to the same input data routinely exhausted all available memory on a computer with 1 Gb of RAM. We are hopeful that through the use of optimizations such as those described here we will be able to apply TWAIN to other pairs of genomes with longer genes, and possibly extend the program to handle more than two species simultaneously.
Conclusions
In describing a number of optimizations for GHMM-based gene finders and making available two complete open-source software systems embodying these methods, it is our hope that others will be more enabled to explore promising extensions to the GHMM framework, thereby improving the state-of-the-art in gene prediction techniques.
Availability and requirements
* Project name: TIGRscan, GlimmerHMM
* Project home page:
* Operating system(s): Linux/UNIX
* Programming language: C/C++
* Other requirements: compiled using gcc 3.3.3
* License: Artistic License, see
* Any restrictions to use by non-academics: terms of Artistic License
Authors' contributions
The DSP algorithm was devised by WHM, who also performed the computational experiments and wrote the manuscript. The PSA gene finder GlimmerHMM was implemented by MP. MP, ALD, and SLS provided detailed insights into the PSA architecture and provided valuable comments on the manuscript.
Acknowledgements
This work was supported in part by NIH grants R01-LM06845 and R01-LM007938.
Figures and Tables
Figure 1 An example GHMM topology. Diamonds represent signal states (for fixed-length features) and circles represent content states (for variable-length features). Allowable transitions are shown with arrows. ATG = start codon, TAG = stop codon, GT = donor splice site, AG = acceptor splice site, N = intergenic region, I = intron, Einit = initial exon, Eint = internal exon, Efin = final exon, Esng = single exon gene. The denoted machine operates by transitioning stochastically from state to state, emitting a gene feature of a particular type upon entering a given state.
Figure 2 Non-overlapping of content and signal sensors. Fixed-length features such as start codons and donor sites are detected by signal sensors, which are used to score an entire context window surrounding the signal. To avoid double-counting, content sensors score only the nucleotides strictly between two signal sensors. In this example, the CTA at the end of the start codon sensor window and the CGA at the beginning of the donor site sensor window are not scored by the exon content sensor, even though they are part of the putative exon, since those bases are already scored by the signal sensors.
Figure 3 The init_nonphased() algorithm. Initialization of a noncoding array α, given a sequence S = x0..xL-1 and nth-order Markov chain M. Note that all parameters are assumed passed by reference. The procedure initializes each array element to the log probability of the nucleotide at the corresponding position in the sequence, conditional on some number of preceding bases.
Figure 4 The init_phased() algorithm. Initialization of a single exon array σ, given a sequence S = x0..xL-1, a set of three Markov chains P{0,1,2}, and initial phase (i.e., phase of the first array element) ω. All parameters are assumed to be passed by reference. This procedure is similar to init_nonphased(), except that the conditional probabilities are computed in a phase-specific manner by the appropriate member of the three-periodic Markov chain.
Figure 5 The eclipse() algorithm. Eclipsing signals in queue G when a stop codon has been encountered at position p. All parameters are assumed to be passed by reference. pos(s) is the position of the first base of the signal's consensus sequence (e.g., the A in ATG). len(s) is the length of the signal's consensus sequence (e.g., 3 for ATG). The procedure operates by computing the phase ω in which each signal is eclipsed by the stop codon, and then identifies those signals which are now eclipsed in all three phases. Any signal eclipsed in all three phases is then dropped from the queue, since any exon starting at that signal and extending up to the current position in the sequence would have an in-frame stop codon.
Figure 6 The traceback() algorithm. Reconstruction of the optimal parse by tracing back through trellis links. Parameters are the selected right-terminus signal s and its chosen phase ω. Returns a stack of signals constituting the optimal parse, with the top signal at the beginning of the parse and the bottom signal at the end. exon_length(p, s) denotes the number of coding nucleotides between p and s. The procedure operates by iteratively following the highest-scoring predecessor link from the current signal, adjusting the current phase as necessary when a trellis link corresponding to a coding feature is traversed.
Table 1 Space and time requirements for two gene finders Two gene finders, the 31-state DSP gene finder TIGRscan, and the 6-state PSA gene finder GlimmerHMM, were run on a 922 Kb sequence. The DSP gene finder used raw probabilities and the PSA gene finder used log-likelihood ratios. The DSP implementation required less memory, both in total and per state, than the PSA implementation. Although the PSA implementation required less total time, the DSP implementation required the same amount of time per state, so that for a given gene finder with a fixed number of states, DSP decoding can be expected to be fully as fast as PSA decoding.
RAM (Mb) RAM/state (Mb) Time, min:sec seconds/state
31-state DSP 29 0.95 1:28 2.8
6-state PSA 84 14 0:17 2.8
Table 2 Efficiency of Markov chain implementations Execution time for a 31-state GHMM gene finder utilizing hash tables or FSMs for its content sensors, applied to a 1.8 Mb sequence. The FSM implementation was over twice as fast as the hash table implementation, and required significantly less memory.
time (min:sec) total RAM
DSP/Hash 1:15 53 Mb
DSP/FSM 0:34 44 Mb
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| 15667658 | PMC552317 | CC BY | 2021-01-04 16:02:49 | no | BMC Bioinformatics. 2005 Jan 24; 6:16 | utf-8 | BMC Bioinformatics | 2,005 | 10.1186/1471-2105-6-16 | oa_comm |
==== Front
BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-6-161566765810.1186/1471-2105-6-16SoftwareEfficient decoding algorithms for generalized hidden Markov model gene finders Majoros William H [email protected] Mihaela [email protected] Arthur L [email protected] Steven L [email protected] Bioinformatics Department, The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD, USA2005 24 1 2005 6 16 16 15 11 2004 24 1 2005 Copyright © 2005 Majoros et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The Generalized Hidden Markov Model (GHMM) has proven a useful framework for the task of computational gene prediction in eukaryotic genomes, due to its flexibility and probabilistic underpinnings. As the focus of the gene finding community shifts toward the use of homology information to improve prediction accuracy, extensions to the basic GHMM model are being explored as possible ways to integrate this homology information into the prediction process. Particularly prominent among these extensions are those techniques which call for the simultaneous prediction of genes in two or more genomes at once, thereby increasing significantly the computational cost of prediction and highlighting the importance of speed and memory efficiency in the implementation of the underlying GHMM algorithms. Unfortunately, the task of implementing an efficient GHMM-based gene finder is already a nontrivial one, and it can be expected that this task will only grow more onerous as our models increase in complexity.
Results
As a first step toward addressing the implementation challenges of these next-generation systems, we describe in detail two software architectures for GHMM-based gene finders, one comprising the common array-based approach, and the other a highly optimized algorithm which requires significantly less memory while achieving virtually identical speed. We then show how both of these architectures can be accelerated by a factor of two by optimizing their content sensors. We finish with a brief illustration of the impact these optimizations have had on the feasibility of our new homology-based gene finder, TWAIN.
Conclusions
In describing a number of optimizations for GHMM-based gene finders and making available two complete open-source software systems embodying these methods, it is our hope that others will be more enabled to explore promising extensions to the GHMM framework, thereby improving the state-of-the-art in gene prediction techniques.
==== Body
Background
Generalized Hidden Markov Models have seen wide use in recent years in the field of computational gene prediction. A number of ab initio gene-finding programs are now available which utilize this mathematical framework internally for the modeling and evaluation of gene structure [1-6], and newer systems are now emerging which expand this framework by simultaneously modeling two genomes at once, in order to harness the mutually informative signals present in homologous gene structures from recently diverged species. As greater numbers of such genomes become available, it is tempting to consider the possibility of integrating all this information into increasingly complex models of gene structure and evolution.
Notwithstanding our eagerness to utilize this expected flood of genomic data, methods have yet to be demonstrated which can perform such large-scale parallel analyses without requiring inordinate computational resources. In the case of Generalized Pair HMMs (GPHMMs), for example, the only systems in existence of which we are familiar make a number of relatively restrictive assumptions in order to reduce the computational complexity of the problem to a more tolerable level [7,8,15]. Yet, even these systems are currently capable of handling no more than two genomes at once. If larger numbers of genomes are to be simultaneously integrated into the gene prediction process in a truly useful manner, then it is reasonable to suggest that new methods will be needed for efficient modeling of parallel gene structures and their evolution. Assuming for now that these methods are likely to continue to build on the basic GHMM framework, we feel it is important that efficient methods of GHMM implementation be properly disseminated for the benefit of those who are to work on this next generation of eukaryotic gene finders.
Modeling genes with a GHMM
A Hidden Markov Model (HMM) is a state-based generative model which transitions stochastically from state to state, emitting a single symbol from each state. A GHMM (or semi-Markov model) generalizes this scenario by allowing individual states to emit strings of symbols rather than one symbol at a time [9,10]. A GHMM is parameterized by its transition probabilities, its state duration (i.e., feature length) probabilities, and its state emission probabilities. These probabilities influence the behavior of the model in terms of which sequences are most likely to be emitted and which series of states are most likely to be visited by the model as it generates its output.
Eukaryotic gene prediction entails the parsing of a DNA sequence into a set of putative CDSs (coding segments, hereafter referred to informally as "genes") and their corresponding exon-intron structures [11]. Thus, the problem of eukaryotic gene prediction can be approximately stated as one of parsing sequences over the nucleotide alphabet Σ = {A,C,G,T} according to the regular expression:
Σ*(ATGΣ*(GTΣ*AG)*Σ*Γ)*Σ*, (1)
where the signals (start and stop codons, donors, and acceptors) have been underlined for clarity, and where Γ = {TAG,TGA,TAA} represents a stop codon. (The actual nucleotides comprising these signals may differ between organisms; we have given the most common ones). An additional constraint not explicitly represented in Formula 1 is that the number of non-intron nucleotides between the start and stop codons of a single gene must be a multiple of three, and furthermore, if these nucleotides are aggregated into a discrete number of nonoverlapping triples, or codons, then none of these codons must be a stop codon, other than the stop codon which terminates the gene. Note that the Σ* terms in Formula 1 permit the occurrence of pseudo-signals – e.g., an ATG triple which does not comprise a true start codon. Gene prediction with a GHMM thus entails parsing with an ambiguous stochastic regular grammar; the challenge is to find the most probable parse of an input sequence, given the GHMM parameters and the input sequence.
In the case of simple Hidden Markov Models, this optimal parsing (or decoding) problem can be solved with the well-known Viterbi algorithm, a dynamic programming algorithm with run time linear in the sequence length (for a fixed number of states) [12]. A modified Viterbi algorithm is required in the case of GHMMs, since each state can now emit more than one symbol at a time [2], resulting in the following optimization problem:
where φ is a parse of the sequence consisting of a series of states qi and state durations di, 0≤i≤n, with each state qi emitting subsequence Si of length di, so that the concatenation of all S0S1...Sn produces the complete output sequence S (but note that states q0 and qn are silent, producing no output). Pe(Si|qi,di) denotes the probability that state qi emits subsequence Si, given duration di; Pt(qi|qi-1) is the probability that the GHMM transitions from state qi-1 to state qi; and Pd(di|qi) is the probability that state qi has duration di. The argmax is over all parses of the DNA sequence into well-formed exon-intron structures; hence, the problem is one of finding the parse which maximizes the product in Equation 2.
Implementation
The PSA decoding algorithm
The approach commonly used in GHMM gene finders for evaluating Equation 2 is to allocate several arrays, one per variable-length feature state, and to evaluate the arrays left-to-right along the length of the input sequence according to a dynamic programming algorithm, which we will detail below. We refer to this approach as the Prefix Sum Arrays (PSA) approach, since the values in the aforementioned arrays represent cumulative scores for prefixes of the sequence.
Without loss of generality, let us consider the GHMM structure depicted in Figure 1. Although individual GHMMs will differ from this particular structure on specific points, the model in Figure 1 is general enough to serve as a concrete example as we illustrate the operation of the algorithm.
The diamonds denote the states for fixed length features (ATG = start codon, TAG = stop codon, GT = donor, AG = acceptor) and the circles denote states for variable length features (N = intergenic, I = intron, Esng = single exon, Einit = initial exon, Eint = internal exon, Efin = final exon). This model generates genes only on the forward strand of the DNA; to obtain a double-stranded model one can simply mirror the structure and link the forward and reverse models through a single merged intergenic state.
Associated with each diamond state is a signal sensor such as a weight matrix (WMM) or some other fixed-length model (e.g., a WAM, WWAM, MDD tree, etc.) [13], and with each circular state is associated a variable-length content sensor, such as a Markov chain (MC) or an Interpolated Markov Model (IMM) [14].
For the purposes of illustration, we will consider only the simplest of each model type, since the more complex model types commonly in use can in general be handled generically within the GHMM framework. The simplest fixed-length model is the WMM:
where xh..xh+n denotes the subsequence currently within a sliding (n + 1)-element window, called the context window, and P(x|θ[i]) denotes the probability of nucleotide x occurring at position i within the window, for model θ. In practice, all of the probabilities described in all of these models are represented in log space (to reduce the incidence of numerical underflow on the computer), so that products of probabilities can be replaced with sums of their logs.
The simplest variable-length model used in practice is the Markov chain. An nth-order Markov chain M for state qi would evaluate the probability P(Si|qi,di) of a putative feature Si according to:
where xj is the jth nucleotide in the sequence of the putative feature, di is the length of that feature, and PM(xj|xj-n..xj-1) is the probability of nucleotide xj conditional on the identities of its n predecessor nucleotides, according to content model M. As with the fixed-length model described above, this computation is typically done in log space.
In scoring the signals and content regions of a putative gene parse, it will be important for us to carefully differentiate between the nucleotides which are scored by a signal sensor and those which are scored by a content sensor in a putative parse. As shown in Figure 2, the content and signal regions must partition the sequence into non-overlapping segments; allowing overlaps would result in double-counting of nucleotide probabilities, which can lead to undesirable biases in the decoding algorithm.
The first step of the PSA algorithm is to compute a prefix sum array for each content sensor. For noncoding states (introns and intergenic) this can be formalized as shown in Figure 3.
In the case of exon states, it is important to capture the different statistical properties present in the three codon positions, referred to as phase 0, phase 1, and phase 2. We employ three Markov chains, M0, M1, and M2, corresponding to these three phases. Together, these three chains constitute a three-periodic Markov chain, M{0,1,2}. Exon states then require three arrays, each of which can be initialized using the procedure shown in Figure 4.
In this way, we can initialize the three arrays αi, 0, αi, 1, and αi, 2 for an exon state qi as follows:
for ω ← 0 to 2 do
init_phased(αi,ω, S, M{0, 1, 2},ω) ;
The individual chains M0, M1, and M2 comprising M{0,1,2} are applied in periodic fashion within the procedure init_phased() to compute conditional probabilities of successive nucleotides along the length of the array. The three arrays are phase-shifted by one from each other, with each element in the array storing the cumulative score of the prefix up to the current nucleotide. The first nucleotide is taken to be in phase ω for array αi,ω. Initializing the arrays for reverse-strand states can be achieved by simply reverse-complementing the DNA sequence and then reversing the order of the resulting arrays (keeping in mind later that the reverse-strand arrays tabulate their sums from the right, rather than the left, and that ω is the phase of the last array entry rather than the first).
Once the prefix sum arrays have been initialized for all variable-duration states, we make another left-to-right pass over the input sequence to look for all possible matches to the fixed-length states, via the signal sensors. In general, a signal sensor θ models the statistical biases of nucleotides at fixed positions surrounding a signal of a given type, such as a start codon. Whenever an appropriate consensus is encountered (such as ATG for the start codon sensor), the signal sensor's fixed-length window is superimposed around the putative signal (i.e., with a margin of zero or more nucleotides on either side of the signal consensus) and evaluated to produce a logarithmic signal score RS = log P(xh..xh+n-1|θ), where h is the position of the beginning of the window and n is the window length. If signal thresholding is desired, RS can be compared to a pre-specified threshold and those locations scoring below the threshold can be eliminated from consideration as putative signals.
The remaining candidates for signals of each type are then inserted into a type-specific signal queue for consideration later as possible predecessors of subsequent signals in a putative gene model. As each new signal is encountered, the optimal predecessors for the signal are selected from among the current contents of the signal queues, using a scoring function described below. In the example (forward strand) GHMM depicted in Figure 1, the possible (predecessor→successor) patterns are:
ATG→TAG
ATG→GT
GT→AG
AG→GT
AG→TAG
TAG→ATG
Associated with each of these patterns is a transition probability, Pt(qi|qi-1), which is included in the scoring of a possible predecessor; this probability can be accessed quickly by indexing into a two-dimensional array. The logarithmic transition score will be denoted RT(qi-1,qi) = log Pt(qi|qi-1).
The distance from a prospective predecessor to the current signal is also included in the evaluation in the form of Pd(di|qi) for distance (=duration) di and signal type (=state) qi. This probability can usually be obtained relatively quickly, depending on the representation of the duration distributions. If the distributions have been fitted to a curve with a simple algebraic formula, then evaluation of the formula is typically a constant-time operation. If a histogram is instead maintained, then a binary search is typically required to find the histogram interval containing the given distance. We denote the logarithmic duration score RD(qi,qj) = log Pd(di|qi:j) where di is the length of the content region delimited by signals qi and qj, and qi:j is the variable-length state corresponding to that content region.
Following Equation 2, the final component of the scoring function is the emission probability Pe(Si|qi,di). For a fixed-length state, this is simply the score produced by the signal sensor. For a variable-length state qi, Pe can be evaluated very quickly by indexing into the prefix sum array αi,γ for state qi and phase γ at the appropriate indices for the two signals and simply performing subtraction:
RC(spred,scur,ω) ← αi,γ [wpos(scur) - 1] - αi,γ [wpos(spred) + wlen(spred) - 1], (5)
where wpos(s) is the 0-based position (within the full input sequence) of the first nucleotide in the context window for signal s, wlen(s) is the length of the context window for signal s, and spred and scur are the predecessor and current signals, respectively. In the case of coding features, γ is the phase of the array and ω = (γ + pos(scur))mod3 is the phase of scur, for pos(scur) the position of the leftmost consensus base of scur. For reverse-strand features, since the prefix sum arrays tabulate their sums from the right instead of the left, the subtraction must be reversed:
RC(spred,scur,ω) ← αi,γ[wpos(spred) + wlen(spred)] - αi,γ[wpos(scur)], (6)
and ω = (γ +L - pos(scur) - 1)mod3, for L the sequence length. For noncoding features, the phases can be ignored when computing RC, since there is only one array per noncoding state.
The resulting optimization function is:
for current signal sj and predecessor signal si; RI(si,γi) denotes the logarithmic inductive score for signal si in phase γi. For forward-strand coding features, the phases γi and γj are related by:
γi = (γj - Δ)mod3, (8)
for Δ the putative exon length, or, equivalently,
γj = (γi + Δ)mod3. (9)
These relations can be converted to the reverse strand by swapping + and -. For introns, γi = γj. For intergenic features, the phase will always be 0 for a forward strand signal and 2 for a reverse strand signal (since on the reverse strand the leftmost base of a 3-base signal would be in phase 2).
The result of Equation 7 is the optimal predecessor for signal sj. This scoring function is evaluated for all appropriate predecessor signals, which are readily available in one or more queues, as mentioned above. A pointer called a trellis link is then created, pointing from the current signal to its optimal predecessor. In the case of those signals that can terminate an exon or an intron, three optimal predecessors must be retained, one for each phase. The inductive score RI(sj,γj) of the new signal sj is then initialized from the selected predecessor si as follows:
RI(sj, γj) ← RI(si, γj) + RT(si, sj) + RD(si, sj) + RC(si, sj, γj) + RS(sj), (10)
where RS(sj) is the logarithmic score produced by the signal sensor for signal sj.
A final step to be performed at each position along the input sequence is to drop from each queue any signal that has been rendered unreachable from all subsequent positions due to intervening stop codons. Except for the final stop codon of a gene, in-phase (i.e., in phase 0) stop codons are generally not permitted in coding exons; for this reason, any potential stop codon (regardless of its signal score) will eclipse any preceding start codon or acceptor site (or, on the reverse strand, stop codon or donor site) in the corresponding phase. The algorithm shown in Figure 5 addresses this issue by dropping any fully eclipsed signal (i.e., eclipsed in all three phases) from its queue.
For the reverse strand, line 3 of eclipse() should be changed to:
ω ← (p-pos(s)-len(s) - 1) mod3;
where len(s) is the length of the consensus sequence for signal s (e.g., 3 for ATG). Note that by xmod3 we mean the positive remainder after division of x by 3; in some programming languages (such as C/C++), a negative remainder may be returned, in which case 3 should be added to the result.
A special case of eclipsing which is not handled by eclipse() is that which occurs when a stop codon straddles an intron; this can be handled fairly simply by checking for such when considering each donor signal as a prospective predecessor for an acceptor signal (or vice-versa on the reverse strand). As each predecessor is evaluated, the bases immediately before the donor and immediately following the acceptor are examined, and if a stop codon is formed, the predecessor is no longer considered eligible for selection in the corresponding phase.
As shown in Figure 5, when a signal has been eclipsed in all three phases it can be removed from its queue. In this way, as a signal falls further and further behind the current position in the sequence, the signal becomes more and more likely to be eclipsed in all three phases as randomly formed stop codons are encountered in the sequence, so that coding queues (e.g., those holding forward strand start codons and acceptors, or reverse strand donors and stop codons) tend not to grow without bound, but to be limited on average to some maximal load determined by the nucleotide composition statistics of the sequence. Because of this effect, the expected number of signals which must be considered during predecessor evaluation can be considered effectively constant in practice.
In the case of noncoding queues (e.g., those holding forward strand donors or stop codons, etc.), the assumption that noncoding features follow a geometric (i.e., exponentially decreasing) distribution allows us to limit these queues to a single element (per phase), because once a noncoding predecessor has been selected in a given phase, no other noncoding predecessor which has already been compared to the selected predecessor can ever become more attractive by virtue of its transition probability (since they are the same for signals of the same type, of which all the signals in a single queue are), its duration probability (since the geometric distribution ensures that their respective duration probabilities decrease at the same rate), nor its sequence probability (since any nucleotides encountered after seeing the two potential predecessors will affect their sequence scores identically).
Because the coding and noncoding queues are effectively limited to a constant load (as argued above), the expected processing time at each nucleotide is O(1) in practice and therefore the entire algorithm up to this point requires time O(L) for an input sequence of length L and a GHMM with a fixed number of states. It will be seen that the traceback procedure described below also requires time O(L), and so this is the time complexity of the PSA decoding algorithm for normal eukaryotic genomes (i.e., those not especially lacking in random stop codons).
Once the end of the sequence is reached, the optimal parse φ can be reconstructed by tracing back through the trellis links. In order for this to be done, a set of virtual, anchor signals (one of each type) must be instantiated at either terminus of the sequence (each having signal score RS = 0). Those at the left terminus will have been entered into the appropriate queues at the very start of the algorithm as prospective targets for the first trellis links (and having inductive scores RI = 0), and those at the right terminus are the last signals to be evaluated and linked into the trellis. The highest scoring of these right terminal anchor signals is selected (in its highest-scoring phase) as the starting point for the traceback procedure. Traceback consists merely of following the trellis links backward while adjusting for phase changes across exons, as shown in Figure 6.
Modifications to Figure 6 for features on the reverse-strand include changing the AG on line 8 to GT, changing the subtraction on line 9 to addition, and changing the 0 on line 7 to 2.
It should be clear from the foregoing that the space requirements of the PSA decoding algorithm are O(L|Q|) for sequence length L and variable-duration state set Q. If, for example, array elements are 8-byte double-precision floating point numbers, then the GHMM depicted in Figure 1 would require 14 prefix sum arrays (4 exon states × 3 phases + 1 intergenic state + 1 intron state), resulting in a memory requirement of at least 112 bytes per nucleotide. Generalizing this GHMM to handle both DNA strands would increase this to 216 bytes per nucleotide, so that processing of a 1 Mb sequence would require at least 216 Mb of RAM just for the arrays. Adding states for 5' and 3' untranslated regions would increase this to 248 Mb of RAM for a 1 Mb sequence, or over 1 Gb of RAM for a 5 Mb sequence. For the purposes of comparative gene finding on multiple organisms with large genes, these requirements seem less than ideal, especially when one considers the possibility of adding yet other states.
The memory requirements can be reduced in several ways. First, Markov chains can be shared by similar states. For example, the intron and intergenic states can share a single Markov chain trained on pooled noncoding DNA, and all the exon states can use the same three-periodic Markov chain trained on pooled coding DNA. To our knowledge, the extent to which this optimization affects the accuracy of the resulting gene finder has not been systematically investigated, though it is commonly used in practice. Second, the models for exons can be modified so as to utilize likelihood ratios instead of probabilities. If the models for exons are re-parameterized to compute:
and the noncoding models are modified to compute:
then the latter can be seen to be unnecessary, since it will always evaluate to 1. Such a modification is valid and will have no effect on the mathematical structure of the optimization problem given in Equation 2 as long as the denominator is evaluated using a Markov chain or other multiplicative model, since the effect of the denominator on inductive scores will then be constant across all possible predecessors for any given signal. Using such ratios allows us to skip the evaluation of all noncoding states, so that the number of prefix sum arrays required for a double-stranded version of the GHMM in Figure 1 would be only 6 (assuming the previous optimization is applied as well), corresponding to the three exon phases on two strands. Furthermore, to the extent that these likelihood ratios are expected to have a relatively limited numerical range, lower-precision floating point numbers can be used, or the ratios could instead be multiplied by an appropriate scaling factor and then stored as 2-byte integers [2]. This is a significant reduction, though asymptotically the complexity is still O(L|Q|). An additional consideration is that the log-likelihood strategy makes unavailable (or at least inseparable) the raw coding and noncoding scores, which might be desired later for some unforeseen application.
A third method of reducing the memory requirements is to eliminate the prefix sum arrays altogether, resulting in what we call the Dynamic Score Propagation (DSP) algorithm.
The DSP decoding algorithm
Informally, the DSP algorithm is similar to the PSA algorithm except that rather than storing all nucleotide scores for all content sensors in a set of prefix sum arrays, we instead store only the specific elements of those arrays that are needed for assessing prospective predecessors during the trellis formation. Associated with each signal is a "propagator" variable which represents the log probability of the highest-scoring partial parse up to and including this signal. As processing proceeds left-to-right along the sequence, these propagators are updated so as to extend these partial parses up to the current position. In this way, the inductive score of each signal is incrementally propagated up to each potential successor signal that is encountered during processing; when a signal is eclipsed in all phases by stop codons (i.e., removed from its respective queue), propagation of that signal's inductive score halts, since further updates would be useless beyond that point. Because no prefix sum arrays are allocated, and because the signal queues are effectively limited in size (as argued previously), the expected memory requirements of DSP will be seen to be O(L+|Q|), where the constant factor associated with the L term is small, reflecting only the number of signals per nucleotide emitted by the signal sensors, as well as the memory required to store the sequence itself.
Let us introduce some notation. We define a propagator π to be a 3-element array, indexed using the notation π[i] for 0≤i≤2; when dealing with multiple propagators, πj[i] will denote element i of the jth propagator.
Each signal si will now have associated with it a propagator, denoted πi. For signals which can be members of multiple queues (such as start codons, which can be members of both the initial exon queue and the single exon queue), the signal will have one propagator per queue, but it will be clear from the context to which propagator we refer. Each queue will also have a propagator associated with it, though for the sake of reducing ambiguity we will refer to these as accumulators and represent them with the symbol α. The purpose of the accumulators is to reduce the number of updates to individual signal propagators; otherwise, every signal propagator in every queue would need to be updated at every position in the input sequence. The accumulator for a given queue will accumulate additions to be made to the propagators of the signals currently in the queue. The update of signal propagators from their queue's accumulator is delayed as long as possible, as described below. Accumulator scores are initialized to zero, as are the propagator scores for the left terminus anchor signals; the general case of propagator initialization will be described shortly.
Updating of a propagator π from an accumulator α is simple in the case of a noncoding queue:
∀0≤ω≤2 π[ω] ← π[ω] + α[0]. (13)
For coding queues, the update must take into account the location of the signal s associated with the propagator π, in order to synchronize the periodic association between phase and array index:
∀0≤ω≤2 π[ω] ← π[ω] + α[(ω - pos(s) - len(s))mod3], (14)
or, on the reverse strand:
∀0≤ω≤2 π[ω] ← π[ω] + α[(ω + pos(s) + len(s))mod3]. (15)
Given a content sensor M, a coding accumulator can be updated according to the rule:
∀0≤ω≤2 α[ω] ← α[ω] + log PM[(ω+f)mod3](xf), (16)
or, on the reverse strand:
∀0≤ω≤2 α[ω] ← α[ω] + log PW[(ω-f)mod3](xf), (17)
where f is the position of the current nucleotide xf, PM[ω](xf) is the probability assigned to xf by the content sensor M in phase ω, and W is the reverse-complementary model to M which computes the probability of its parameter on the opposite strand and taking contexts from the right rather than from the left. This update occurs once at each position along the input sequence. Use of f provides an absolute frame of reference when updating the accumulator. This is necessary because the accumulator for a queue has no intrinsic notion of phase: unlike an individual signal, a queue is not rooted at any particular location relative to the sequence.
For noncoding queues, only the 0th element of the accumulator must be updated:
α[0] ← α[0] + log PM(xf). (18)
All that remains is to specify the rule for selecting an optimal predecessor and using it to initialize a new signal's propagator. We first consider new signals which terminate a putative exon. Let si denote the predecessor under consideration and sj the new signal. Denote by Δ the length of the putative exon. Then on the forward strand, we can compare predecessors with respect to phase ω via the scoring function RCI + RD + RT, where RD and RT are the duration and transition scores described earlier and RCI includes the content score and the inductive score from the previous signal:
∀0≤ω≤2 RCI(si,ω) ← πi[(ω - Δ)mod3]. (19)
On the reverse strand we have:
∀0≤ω≤2 RCI(si,ω) ← πi [(ω + Δ)mod3]. (20)
For introns it is still necessary to separate the three phase-specific scores to avoid greedy behavior, though the phase does not change across an intron, so no Δ term is necessary:
∀0≤ω≤2 RCI(si,ω) ← πi[ω]. (21)
When the preceding feature is intergenic we need only refer to phase zero of the preceding stop codon:
RCI(si,ω) ← πi[0], (22)
or, on the reverse strand, phase 2 of the preceding start codon (since the leftmost base of the reverse-strand start codon will reside in phase 2).
Once an optimal predecessor with score RCI + RD + RT is selected with respect to a given phase ω, the appropriate element of the new signal's propagator can be initialized directly:
πj[ω] ← RCI(si,ω) + RD(si,sj) + RT(si,sj) + RS(sj), (23)
where RS(sj) = P(context(sj)|θj) is the score assigned to the context window of the new signal sj by the appropriate signal sensor θj. An exception to Equation 23 occurs when ω is not a valid phase for signal sj (e.g., phase 1 for a start codon), in which case we instead set πj[ω] to -∞.
One final complication arises from the fact that the algorithm, as we have presented it, does not permit adjacent signals in a prospective parse to have overlapping signal sensor windows; to allow such would be to permit double-counting of nucleotide probabilities, thereby biasing the probabilistic scoring function. It is a simple matter to reformulate the algorithm so that signal sensors score only the two or three consensus nucleotides of the signals under consideration; this would allow adjacent signals in a prospective parse to be as close as possible without actually overlapping (i.e., a single exon consisting of the sequence ATGTAG would be permitted, even if the start codon and stop codon context windows overlapped). However, doing so might be expected to decrease gene finder accuracy, for two reasons: (1) statistical biases occurring at fixed positions relative to signals of a given type can in general be better exploited by a signal sensor specifically trained on such positions than by a content sensor trained on data pooled from many positions at variable distances from the signal, and (2) in the case of Markov chains and Interpolated Markov Models, probability estimates for nucleotides immediately following a signal can be inadvertently conditioned on the few trailing nucleotides of the preceding feature (assuming the chain has a sufficiently high order), even though the models are typically not trained accordingly. For these reasons, we prefer to use signal sensors which impose a moderate margin around their respective signals, both to detect any biologically relevant biases which might exist within those margins, and to ensure that content sensors condition their probabilities only on nucleotides within the same feature.
Given the foregoing, it is necessary to utilize a separate "holding queue" for signals which have recently been detected by their signal sensors but which have context windows still overlapping the current position in the DSP algorithm. The reason for this is that propagator updates via Equations 13–15 must not be applied to signals having context windows overlapping any nucleotides already accounted for in the accumulator scores, since to do so would be to double-count probabilities. It is therefore necessary to observe the following discipline.
Associated with each signal queue Gi there must be a separate holding queue, Hi. When a signal is instantiated by a signal sensor it is added to the appropriate Hi rather than to Gi. As the algorithm advances along the sequence, at each new position we must examine the contents of each holding queue Hi to identify any signal having a context window which has now passed completely to the left of the current position. If one or more such signals are identified, then we first update the propagators of all the signals in the main queue Gi using Equations 13–15, then zero-out the values of the accumulator αi for that queue, and then allow the recently passed signals to graduate from Hi to Gi. Observe that at this point all the signals in Gi have in their propagators scores which have effectively been propagated up to the same point in the sequence, and that point is immediately left of the current position; this invariant is necessary for the proper operation of the algorithm. All content sensors are then evaluated at the current position and their resulting single-nucleotide scores are used to update the accumulators for their respective queues. Finally, whenever it becomes necessary to evaluate the signals in some queue Gi as possible predecessors of a new signal, we must first update the propagators of all the elements of Gi as described above, so that the comparison will be based on fully propagated scores.
Equivalence of DSP and PSA
We now give a proof that DSP is mathematically equivalent to PSA, since it may not be entirely obvious from the foregoing description. We will consider only the forward strand cases; the proof for the reverse strand cases can be derived by a series of trivial substitutions in the proof below.
To begin, we show by induction that the signal propagator πj[ω] for signal sj is initialized to the PSA inductive score RI(sj,ω). For the basis step, recall that the left terminus anchor signals were initialized to have zero scores in both PSA and DSP, regardless of whether a given signal began a coding or noncoding feature. In the case of coding features, substituting Equation 19 into Equation 23 yields:
πj[ω] ← πi[(ω - Δ)mod3] + RD(si,sj) + RT(si,sj) + RS(sj). (24)
According to Equation 10, this initialization will result in πj[ω] = RI(sj,ω) only if:
πi[(ω - Δ)mod3] = RI(si,γi) + RC(si,sj,ω), (25)
where γi = (ω - Δ)mod3 according to Equation 8. At the time that signal sj is instantiated by its signal sensor, πi has been propagated up to e = wpos(sj) - 1, the nucleotide just before the leftmost position of the context window for sj. By the inductive hypothesis, πi[γi] was initialized to RI(si,γi). This initialization occurred at the time when the current DSP position was at the beginning of the predecessor's context window. Note, however, that πi effectively began receiving updates at position b = wpos(si) + wlen(si), the position immediately following the end of the signal's context window, at which point si graduated from its holding queue. Thus, πi[γi] will have accumulated content scores for positions b through e, inclusive. In order to establish Equation 25, we need to show that these accumulations sum to precisely RC(si,sj,ω).
Substituting Equation 16 into Equation 14 we get the following formula describing propagator updates as if they came directly from content sensor M:
∀0≤ω≤2 π[ω] ← π[ω] + log PM[(ω+Δ)mod3](xf), (26)
where Δ = f-(pos(si) + len(si)) is the distance between the rightmost end of signal si and the current position f in the DSP algorithm. Let us introduce the notation:
F(i,j,ω) = ∑k = i..jlog PM[(ω+k)mod3](xk). (27)
Using this notation, πi[γi] has since its initialization accumulated F(b,e,γi - pos(si) - len(si)); this can be verified by expanding this expression via Equation 27 and observing that the result equals a summation of the log term in Equation 26 over f = b to e. Looking at init_phased(), it should be obvious that the effect of lines 5 and 8 will be that:
αi,γ [h] = ∑k = 0..hlog PM [(k+γ)mod3](xk) = F(0,h,γ). (28)
According to Equation 5, showing that πi[γi] has accumulated RC(si,sj,ω) is therefore equivalent to:
F(b,e,ψ) = F(0,wpos(sj) - 1,γ) - F(0,wpos(si) + wlen(si) - 1,γ), (29)
where ψ = γi - pos(si) - len(si) and γ = ω - pos(sj). Equivalently:
F(b,e,ψ) = F(0,e,γ) - F(0,b - 1,γ). (30)
To see that ψ ≡ γ(mod3), observe that pos(sj) - (pos(si) + len(si)) = Δ, the length of the putative exon (possibly shortened by three bases, in the case where si is a start codon), and further that γi - ω ≡ -Δ(mod3) according to Equation 8, so that ψ - γ ≡ Δ-Δ ≡ 0(mod3). Thus, Equation 30 is equivalent to:
F(b,e,γ) = F(0,e,γ) - F(0,b - 1,γ), (31)
which can be established as a tautology by simple algebra after expansion with Equation 27. This shows that the signal propagator for signal sj is initialized to the PSA inductive score RI(sj,ω), and thus establishes the inductive step of the proof in the case of coding features.
To see that the above arguments also hold for noncoding features, note that Equation 21 simplifies Equation 25 to:
πi[ω] = RI(si,ω) + RC(si,sj), (32)
that Equations 13 and 18 combine to simplify Equation 26 to:
∀0≤ω≤2 π[ω] ← π[ω] + log PM(xf), (33)
and that lines 4 and 6 of init_nonphased() cause:
αi[h] = ∑k = 0..hlog PM(xk) = FNC(0,h), (34)
for FNC(i,j) = ∑k = i..jlog PM(xk). We can thus reformulate Equation 29 as:
FNC(b,e) = FNC(0,wpos(scur) - 1) - FNC(0,wpos(spred) + wlen(spred) - 1), (35)
or, equivalently:
FNC(b,e) = FNC(0,e) - FNC(0,b - 1), (36)
which is again a tautology. In the interests of brevity, we leave it up to the reader to verify that the above arguments still apply when the noncoding features are intergenic, thereby invoking Equation 22 rather than Equation 21 in formulating Equation 31.
To see that the selection of optimal predecessors is also performed identically in the two algorithms, note that the PSA criterion given in Equation 7 is equivalent to the argmax(RCI + RD + RT) criterion of DSP as long as RCI(si,ω) = RC(si,sj,ω) + RI(si,γi) at the time the optimal predecessor is selected, which we have in fact already shown by establishing Equation 25.
Thus, DSP and PSA build identical trellises; application of the same traceback() procedure should therefore produce identical gene predictions.
Fast decoding of Markov chains
Markov chains are typically implemented in GHMM-based gene finders using hash tables, due to the simplicity of such an implementation. Thus, for a given Markov chain M we may utilize a hash table which associates the probability PM(xj|xj-n..xj-1) with the sequence xj-n..xj. Although hash tables provide a relatively efficient solution for this task, they are wasteful in the sense that as we evaluate the chain on successive nucleotides in a sequence, we repeatedly manipulate preceding nucleotides in forming successive substrings to be indexed into the hash table.
A much faster (and much more elegant) solution is to employ a Finite State Machine (FSM) in which states exist for all possible sequences of length n+1 or less, and where the state having label xj-n..xj emits the probability PM(xj|xj-n..xj-1), for nth-order Markov chain M. In this way, the transition probabilities of the Markov chain become the state emissions of the FSM. During a single left-to-right scan of a sequence, each base requires only a single two-dimensional array indexing operation to access the desired probability, and a single integer value store operation to remember the identity of the new state. When compared to the typical regime of arithmetic and bit-shift operations over an (n+1)-element string that would be required for a typical hash function, the difference can be significant.
Implementing this optimization is fairly straightforward, both for conventional Markov chains and for Interpolated Markov Models, whether homogeneous or three-periodic. Central to the method is a means of mapping between state labels and integer state identifiers for use in indexing into the transition table. The base-4 number system can be utilized for this purpose, assuming a nucleotide mapping such as ∇ = {A↔0, C↔1, G↔2, T↔3}. To account for lower-order states, define:
which gives the total number of strings of length less than L. Converting a string S = x0..xL-1 to base-4 can be accomplished as follows:
Now a string S can be mapped to a state index using:
state(S) = B(|S|) + λ(S), (39)
where |S| denotes the length of S.
Given this integer↔label mapping and an nth-order Markov chain in hash table format, the FSM state emissions can be initialized by indexing state labels into the hash table to obtain the Markov chain transition probabilities. The transition table can be initialized fairly simply by noting that the successor of state x0..xL-1 upon seeing symbol s is x1..xL-1s if L = n + 1, or x0..xL-1s for L <n + 1. A model for the reverse strand can be handled by applying this scheme in reverse, so that the state with label xj-n..xj emits the probability PM(xj-n|xj-n+1..xj), and the lower-order states are reserved for the end of the sequence rather than the beginning.
Results
Table 1 shows the memory and time requirements for two GHMM gene finders, one using the PSA algorithm and the other the DSP algorithm, on a 922 Kb sequence. Note that the DSP gene finder has 31 states, while the PSA gene finder explicitly evaluates only 6 states, so that they both give a ratio of 2.8 seconds per state on this sequence, while the ratio of memory per state is 14 Mb for the PSA gene finder and 0.95 Mb for the DSP gene finder. Thus, the DSP and PSA algorithms appear to consume the same amount of time per state, while DSP requires only a fraction of the memory (per state) as PSA.
Table 2 shows the results of applying the FSM optimization to a DSP gene finder to accelerate its content sensors. As can be seen from the table, the FSM approach reduces execution time by more than half (as compared to a hash table implementation), while also reducing total RAM usage. The DSP/FSM configuration reported here utilized both conventional Markov chains as well as Interpolated Markov Models, both represented using FSMs. Note that the hashing software used for comparison was a very efficient implementation which used native C character arrays; in particular, we did not use the C++ Standard Template Library (STL) implementations of string and hash, due to efficiency concerns regarding the re-copying of string arguments to the hash function. Our custom string hashing implementation was found to be much faster than the STL implementation (data not shown). Accordingly, one can expect an FSM implementation to show even greater gains as compared to an STL-based hashing implementation.
We utilized our DSP-based gene finder TIGRscan [5] in the construction of our syntenic gene finder TWAIN, a Generalized Pair HMM which performs gene prediction in two genomes simultaneously. TWAIN operates by invoking a modified version of TIGRscan to build a directed acyclic graph of all high-scoring parses of each of the two input sequences. Early experiments indicated that these parse graphs could be quite large in practice and may therefore require a significant portion of available RAM for their storage. In addition, the dynamic programming matrix used by TWAIN promised to be large as well. It was in anticipation of this problem that we were prompted to develop TIGRscan using the DSP architecture, to minimize the memory requirements of the underlying GHMM, thereby freeing the remaining available memory for use by the rest of the machinery within TWAIN.
As a result of these and other optimizations (such as our use of a sparse matrix representation for TWAIN's dynamic programming algorithm) we were able to apply TWAIN's gene prediction component to a pair of fungal genomes (Aspergillus fumigatus and A. nidulans) while consuming under 50 Mb of RAM, whereas an earlier prototype of this system applied to the same input data routinely exhausted all available memory on a computer with 1 Gb of RAM. We are hopeful that through the use of optimizations such as those described here we will be able to apply TWAIN to other pairs of genomes with longer genes, and possibly extend the program to handle more than two species simultaneously.
Conclusions
In describing a number of optimizations for GHMM-based gene finders and making available two complete open-source software systems embodying these methods, it is our hope that others will be more enabled to explore promising extensions to the GHMM framework, thereby improving the state-of-the-art in gene prediction techniques.
Availability and requirements
* Project name: TIGRscan, GlimmerHMM
* Project home page:
* Operating system(s): Linux/UNIX
* Programming language: C/C++
* Other requirements: compiled using gcc 3.3.3
* License: Artistic License, see
* Any restrictions to use by non-academics: terms of Artistic License
Authors' contributions
The DSP algorithm was devised by WHM, who also performed the computational experiments and wrote the manuscript. The PSA gene finder GlimmerHMM was implemented by MP. MP, ALD, and SLS provided detailed insights into the PSA architecture and provided valuable comments on the manuscript.
Acknowledgements
This work was supported in part by NIH grants R01-LM06845 and R01-LM007938.
Figures and Tables
Figure 1 An example GHMM topology. Diamonds represent signal states (for fixed-length features) and circles represent content states (for variable-length features). Allowable transitions are shown with arrows. ATG = start codon, TAG = stop codon, GT = donor splice site, AG = acceptor splice site, N = intergenic region, I = intron, Einit = initial exon, Eint = internal exon, Efin = final exon, Esng = single exon gene. The denoted machine operates by transitioning stochastically from state to state, emitting a gene feature of a particular type upon entering a given state.
Figure 2 Non-overlapping of content and signal sensors. Fixed-length features such as start codons and donor sites are detected by signal sensors, which are used to score an entire context window surrounding the signal. To avoid double-counting, content sensors score only the nucleotides strictly between two signal sensors. In this example, the CTA at the end of the start codon sensor window and the CGA at the beginning of the donor site sensor window are not scored by the exon content sensor, even though they are part of the putative exon, since those bases are already scored by the signal sensors.
Figure 3 The init_nonphased() algorithm. Initialization of a noncoding array α, given a sequence S = x0..xL-1 and nth-order Markov chain M. Note that all parameters are assumed passed by reference. The procedure initializes each array element to the log probability of the nucleotide at the corresponding position in the sequence, conditional on some number of preceding bases.
Figure 4 The init_phased() algorithm. Initialization of a single exon array σ, given a sequence S = x0..xL-1, a set of three Markov chains P{0,1,2}, and initial phase (i.e., phase of the first array element) ω. All parameters are assumed to be passed by reference. This procedure is similar to init_nonphased(), except that the conditional probabilities are computed in a phase-specific manner by the appropriate member of the three-periodic Markov chain.
Figure 5 The eclipse() algorithm. Eclipsing signals in queue G when a stop codon has been encountered at position p. All parameters are assumed to be passed by reference. pos(s) is the position of the first base of the signal's consensus sequence (e.g., the A in ATG). len(s) is the length of the signal's consensus sequence (e.g., 3 for ATG). The procedure operates by computing the phase ω in which each signal is eclipsed by the stop codon, and then identifies those signals which are now eclipsed in all three phases. Any signal eclipsed in all three phases is then dropped from the queue, since any exon starting at that signal and extending up to the current position in the sequence would have an in-frame stop codon.
Figure 6 The traceback() algorithm. Reconstruction of the optimal parse by tracing back through trellis links. Parameters are the selected right-terminus signal s and its chosen phase ω. Returns a stack of signals constituting the optimal parse, with the top signal at the beginning of the parse and the bottom signal at the end. exon_length(p, s) denotes the number of coding nucleotides between p and s. The procedure operates by iteratively following the highest-scoring predecessor link from the current signal, adjusting the current phase as necessary when a trellis link corresponding to a coding feature is traversed.
Table 1 Space and time requirements for two gene finders Two gene finders, the 31-state DSP gene finder TIGRscan, and the 6-state PSA gene finder GlimmerHMM, were run on a 922 Kb sequence. The DSP gene finder used raw probabilities and the PSA gene finder used log-likelihood ratios. The DSP implementation required less memory, both in total and per state, than the PSA implementation. Although the PSA implementation required less total time, the DSP implementation required the same amount of time per state, so that for a given gene finder with a fixed number of states, DSP decoding can be expected to be fully as fast as PSA decoding.
RAM (Mb) RAM/state (Mb) Time, min:sec seconds/state
31-state DSP 29 0.95 1:28 2.8
6-state PSA 84 14 0:17 2.8
Table 2 Efficiency of Markov chain implementations Execution time for a 31-state GHMM gene finder utilizing hash tables or FSMs for its content sensors, applied to a 1.8 Mb sequence. The FSM implementation was over twice as fast as the hash table implementation, and required significantly less memory.
time (min:sec) total RAM
DSP/Hash 1:15 53 Mb
DSP/FSM 0:34 44 Mb
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| 15717925 | PMC552318 | CC BY | 2021-01-04 16:40:17 | no | BMC Dev Biol. 2005 Feb 17; 5:4 | latin-1 | BMC Dev Biol | 2,005 | 10.1186/1471-213X-5-4 | oa_comm |
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Microb Cell FactMicrobial Cell Factories1475-2859BioMed Central London 1475-2859-4-61570748810.1186/1475-2859-4-6ResearchThe small heat-shock proteins IbpA and IbpB reduce the stress load of recombinant Escherichia coli and delay degradation of inclusion bodies LeThanh Ha [email protected] Peter [email protected] Frank [email protected] Institute for Biotechnology, Department of Biochemistry/Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle, Germany2 Bioprocess Engineering Laboratory, P.O. Box 4300, Department of Process and Environmental Engineering, Biocenter Oulu, University of Oulu, FIN-90014 Oulu, Finland2005 11 2 2005 4 6 6 16 12 2004 11 2 2005 Copyright © 2005 LeThanh et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The permanently impaired protein folding during recombinant protein production resembles the stress encountered at extreme temperatures, under which condition the putative holding chaperones, IbpA/IbpB, play an important role. We evaluated the impact of ibpAB deletion or overexpression on stress responses and the inclusion body metabolism during production of yeast α-glucosidase in Escherichia coli.
Results
Deletion of ibpAB, which is innocuous under physiological conditions, impaired culture growth during α-glucosidase production. At higher temperatures, accumulation of stress proteins including disaggregation chaperones (DnaK and ClpB) and components of the RNA degradosome, enolase and PNP, was intensified. Overexpression of ibpAB, conversely, suppressed the heat-shock response under these conditions. Inclusion bodies of α-glucosidase started to disaggregate after arrest of protein synthesis in a ClpB and DnaK dependent manner, followed by degradation or reactivation. IbpA/IbpB decelerated disaggregation and degradation at higher temperatures, but did hardly influence the disaggregation kinetics at 15°C. Overexpression of ibpAB concomitant to production at 42°C increased the yield of α-glucosidase activity during reactivation.
Conclusions
IbpA/IbpB attenuate the accumulation of stress proteins, and – at high temperatures – save disaggregated proteins from degradation, at the cost, however, of delayed removal of aggregates. Without ibpAB, inclusion body removal is faster, but cells encounter more intense stress and growth impairment. IbpA/IbpB thus exert a major function in cell protection during stressful situations.
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Background
Production of recombinant proteins as inclusion bodies in Escherichia coli can induce several stress responses [1-3] and may interfere with primary metabolism and cellular protein synthesis [4-6]. The metabolic burden of the synthesis of recombinant and stress proteins [7,8] may result in stop of the synthesis of components of the protein synthesising machinery, and ribosomes may be actively degraded [4,9,10]. Most commonly, the heat-shock response is induced, as the recombinant proteins – unable to fold properly – overload the chaperone network [11,12]. While appropriate execution of stress responses is of vital importance for the host Escherichia coli [13], the high metabolic load of strong foreign protein production can prevent proper adaptation [14,15]. The stress situation can become permanent, and only few cellular proteins except heat-shock proteins are synthesised.
Analogously, incubation at extreme temperatures is characterised by near exclusive heat-shock protein synthesis as a consequence of extensive aggregation of cellular proteins [18,19]. To deal with this challenge, the chaperone DnaK changes its function: Under physiological conditions, DnaK promotes folding of kinetically trapped intermediates by ATP-dependent partial unfolding, but at high temperatures it becomes a holding chaperone and binds its client proteins permanently, unable to release them in a folding-competent conformation [20].
The small heat-shock proteins (sHsps) are intrinsic holding chaperones of multimeric structure. The multimers dissociate at high temperatures into smaller entities for exposure of client binding sites. After binding of misfolded client proteins, the sHsps form large globular assemblies [21]. Thereby, they prevent irreversible aggregation, and under permissive conditions transfer the client proteins to ATP-dependent chaperones for refolding or degradation [22,23]. Also the E. coli sHsps, IbpA and IbpB, reversibly dissociate upon prolonged incubation at 50°C from multimers of 2 – 3 MDa to monomers in the case of IbpA and to oligomers of 650 – 700 kDa in the case of IbpB, exposing hydrophobic sites [24,25]. IbpA and IbpB are highly homologous [26], but show distinct features. While IbpB is soluble when overexpressed alone, prevents inactivation or aggregation of proteins upon heat and oxidative stress in vitro and facilitates their subsequent refolding by DnaK [23-25], a role for IbpA in mediating the transfer of IbpB and other proteins into the insoluble cell fraction has been proposed [19,27]. IbpA and IbpB have been found associated with thermally aggregated cellular proteins and with inclusion bodies [26,28]. The resolubilisation of protein aggregates, which in vitro and in vivo is mediated by ClpB together with DnaK [28-31], can be retarded both in strains overexpressing ibpAB as well as in some cases in strains lacking ibpAB [19,32,33]. IbpA and IbpB might control the reversible transition between soluble and aggregated forms that account for the plasticity of inclusion bodies [27,34,35].
The role of IbpA/IbpB in cell protection is less clear. Overexpression of ibpAB confers resistance to heat and other stresses [37], but deletion of the ibpAB operon does not affect growth even at high temperatures. Only recovery from prolonged exposure to extreme temperatures such as 50°C are impaired in an ibpAB mutant, and growth of ibpAB dnaK double mutants at high temperatures [19,36]. In this study we exploit the similarities between incubation at extreme temperature and production of aggregation-protein proteins to examine the functions of the small heat-shock proteins IbpA/IbpB in inclusion body metabolism and stress relief during α-glucosidase production. The enzyme α-glucosidase from Saccharomyces cerevisiae produced in recombinant E. coli is a suitable model system. It accumulates as inclusion bodies and induces the heat-shock response including prolonged expression of the ibpAB operon [17]. It is, however, also able to fold into its native conformation in E. coli if produced at low temperature [38], enabling the study of in-vivo-reactivation kinetics.
Results
IbpA/IbpB levels during α-glucosidase production with and without IbpAB coexpression
The concentrations of IbpA and IbpB were estimated from the proteomes of cells producing α-glucosidase for two hours. The ibpAB operon was induced already during α-glucosidase production at 30°C, with moderately higher levels at elevated temperatures (Table 1), whereas no IbpA/IbpB was detected in the plasmid-free strain. The ibpAB operon including its own promoter was inserted immediately downstream of the α-glucosidase gene into the plasmid pKK177-3/GLUCP1, expecting overexpression of ibpAB from its heat-shock promoter and as a tricistronic operon together with α-glucosidase. This plasmid gave ten and four times higher concentrations of IbpA and IbpB, respectively, at 30°C, and the sHsp level increased further in proportion to temperature (Table 1). With ibpAB overexpression, the IbpA accumulation was intensified more strongly and the ratio of IbpA to IbpB rose from 2–3 to 6–8. As the two sHsp may exert different functions, this change need to be accounted for. Depending on the temperature, overexpression increased the concentrations of IbpA and IbpB relative to their concentrations without overexpression four to more than 25 times (Table 1). This wide range of concentrations served to investigate the impact of IbpA/IbpB on α-glucosidase production and in-vivo-reactivation.
Elevated levels of IbpA/IbpB increase α-glucosidase accumulation
α-Glucosidase was produced in E. coli MC4100 with and without ibpAB overexpression and in an otherwise isogenic ibpAB deletion mutant. α-Glucosidase accumulation was enhanced at elevated IbpA/IbpB level and reduced in the deletion strain in a temperature dependent manner; the effect was strongest at high temperature (Table 2). At temperatures below 30°C, the IbpA/IbpB levels did not influence the amount of α-glucosidase, which accumulated mainly in the soluble cell fraction in active form (Table 2).
Post-induction growth of the ibpAB deletion mutant was retarded at higher temperatures (Fig. 1), although there was no impact of the ibpAB deletion on growth without induction of α-glucosidase synthesis (not shown) or concomitant to production at lower temperature.
Thus, the sHsp are effective only under conditions that favour inclusion body formation.
IbpA/IbpB attenuate accumulation of stress proteins during α-glucosidase production
To study the impact of IbpA/IbpB on cellular reactions during α-glucosidase production, the proteomes of cultures producing α-glucosidase for two hours at various temperatures were separated by two-dimensional gel electrophoresis (shown for 42°C in Fig. 2). While chaperones assisting de-novo protein folding such as GroEL and GroES were not significantly influenced by IbpA/IbpB level (Fig. 2), the DnaK concentration showed a negative correlation with IbpA/IbpB availability at 42°C (Fig. 3). The disaggregating chaperone ClpB reached higher concentrations in the ibpAB deletion mutant at all temperatures, in addition to the induction at elevated temperatures (Fig. 3). ClpB accumulation was repressed, however, in the wildtype strain at 30°C, and with overexpression of ibpAB both at 30 and 37°C. As the overall level of IbpA/IbpB was low at these temperatures, and other cellular proteins were not affected, this repression is unlikely to be a passive effect of the metabolic burden of ibpAB expression.
A similar pattern was found for enolase, which showed higher levels at elevated temperatures only in the deletion mutant (Fig. 3). As enolase is a glycolytic enzyme as well as a putative structural component of the RNA degradosome [39], we examined another metabolic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and polynucleotide phosphorylase (PNP), another RNA degradosome member. The positions on the 2D gels of further degradosome components, RNAse E and RhlB, are not listed in the database. While PNP accumulated to higher concentrations at elevated temperatures and reached highest levels in the deletion mutant (Fig. 3), GAPDH concentration was not significantly affected by temperature and had a slightly lower concentration in the ibpAB deletion mutant at 42°C (Fig. 3). Thus, enolase seems to be induced as a part of a stress response rather than as a metabolic enzyme.
A typical house-keeping protein, the translational elongation factor EF-Tu, had a two to three times higher level at 42°C than at lower temperatures, but the concentrations were not effected by IbpA/IbpB level (Fig. 3).
Thus, while housekeeping proteins are not influenced by IbpA/IbpB levels, the accumulation of ClpB and components of the RNA degradosome was intensified in the ibpAB deletion mutant and attenuated with ibpAB overexpression. This indicates that IbpA/IbpB can relieve the cells of the stress encountered during α-glucosidase production.
Impact of chaperones on in-vivo-reactivation and degradation of α-glucosidase from inclusion bodies
The impact of chaperones on the metabolism of α-glucosidase inclusion bodies was examined in wildtype E. coli MC4100 with or without overexpression of ibpAB and in isogenic strains carrying deletions of ibpAB, clpB, or dnaK. After production of α-glucosidase at 37°C for two hours, the cultures were treated with tetracycline to arrest protein synthesis and transferred to 30°C to initiate disaggregation of inclusion bodies as reported for other proteins [34]. In the wildtype and ibpAB strains, disintegration of inclusion bodies, increase of α-glucosidase activity, and higher concentrations of α-glucosidase in the soluble cell fraction could be detected already within 20 min; the disaggregation was accompanied by degradation (cf. below). While the concentration of α-glucosidase was 10–25% higher in dnaK and clpB deletion mutants than in the wildtype strain, the specific activities obtained during the production phase were less than 0.1 Umg-1, compared to about 0.3 Umg-1 in the other strains (data not shown). Importantly, neither disaggregation, nor reactivation nor degradation took place in these mutants within two hours, and the concentration of α-glucosidase inclusion bodies remained unchanged.
The kinetics of disaggregation, reactivation and degradation of inclusion bodies at modified levels of IbpA/IbpB, i.e. with deletion or overexpression of ibpAB, were investigated as a function of temperature. After production of α-glucosidase at 37°C, the cultures were transferred to various temperatures. As protein synthesis was arrested before the transfer, the initial IbpA/IbpB level did not depend on the disaggregation temperature. The α-glucosidase concentration in the insoluble cell fractions of all cultures decreased with time, but with different kinetics. In the ibpAB deletion strain, the disaggregation kinetics were not influenced by the incubation temperature (Fig. 4A–D). With overexpression of ibpAB, the removal of α-glucosidase from the inclusion bodies was fastest at 15°C (Fig. 4A), proceeding with similar rates as in the other strains. The disaggregation was, however, progressively slower at 30 and 37°C (Fig. 4C,D). At 37°C, α-glucosidase was largely preserved in the insoluble cell fraction of the ibpAB overexpressing strain (Fig. 4D). Thus, disaggregation was retarded at higher temperatures in an IbpA/IbpB dependent manner.
Disaggregation of α-glucosidase inclusion bodies was accompanied by an increase of the specific α-glucosidase activities in the cell extracts. The reactivation showed a clear temperature optimum in the range of 24 to 30°C, most evident with wildtype levels of IbpA/IbpB (Fig. 4F,G). The specific α-glucosidase activities increased sevenfold up to 2 Umg-1 within 3 h, whereas at both higher and lower temperatures of 37°C and 15°C, only 0.5–0.75 Umg-1 were obtained (Fig. 4E,H). Both, ibpAB overexpression and deletion, did not improve the maximum reactivation yields. The resolubilisation, measured as an increase of the α-glucosidase concentrations in the soluble cell fraction, showed very similar kinetics as the reactivation (data not shown).
A large part of the disaggregated α-glucosidase was subjected to proteolysis: within three hours after arrest of protein synthesis, 30 – 40% of α-glucosidase were lost due to degradation at 15°C in all three strains (Fig. 4I), and in the ibpAB deletion mutant at all temperatures (Fig. 4I–L). Similar to the effect on disaggregation, higher ibpAB expression levels or elevated temperatures synergistically retarded degradation, reducing the loss to 10% with ibpAB overexpression at 37°C (Fig. 4L). IbpA/IbpB thus influenced disaggregation, resolubilisation, reactivation and degradation in a temperature-dependent manner.
IbpA/IbpB favour reactivation of α-glucosidase produced at high temperature
As IbpA/IbpB are designed for action at extreme temperature, α-glucosidase inclusion bodies were produced at 42°C, followed by arrest of protein synthesis and incubation at 30°C. The production temperature did not influence the initial disaggregation kinetics (data not shown). About one third of the α-glucosidase produced concomitant to ibpAB overexpression was resistant to disaggregation after incubation over night, while it was eliminated apart from traces in the other strains (Fig. 5A). Also the removal of other components of the inclusion bodies, which appear as smear in the SDS PAGE of the insoluble cell fractions, were impaired in the overexpressing strain (Fig. 5A), resembling the core inclusion bodies that remain with other proteins [34], whereas the inclusion bodies produced at low IbpA/IbpB levels were completely dissolved after prolonged incubation. Only outer membrane proteins (indicated by arrows) that result from coprecipitation of cell debris during inclusion body preparation [40] remain at constant level in the insoluble cell fraction (Fig. 5A). Thus, the delay of disaggregation by IbpA/IbpB is not specific to α-glucosidase, but effects also cellular proteins in a similar way.
Production at 37°C resulted in degradation of 20% of α-glucosidase during incubation at 30°C for 3 h in the wildtype strain (Fig. 4K). After production at 42°C, however, there was less than 10% of α-glucosidase lost during reactivation at 30°C with or without ibpAB overexpression (Fig. 6). Thus, degradation of α-glucosidase after arrest of protein synthesis was predetermined by the preceding production temperature. The protection was dependent on IbpA/IbpB availability, as in the deletion mutant the loss of α-glucosidase was substantial regardless of the production temperature (Fig. 4K, 6).
Consequently, disaggregated α-glucosidase was quantitatively transferred to the soluble cell fraction after production at 42°C concomitant to ibpAB overexpression, and became the most prominent band in the gel. The band intensity increased from 2 to 7% of the total cellular protein (Fig. 5B). In contrast, significantly less α-glucosidase could be detected in the soluble fraction of the deletion mutant even after prolonged incubation, finally corresponding to only 4% of cellular protein (Fig. 5B).
The α-glucosidase activity increased during the initial reactivation phase with similar slopes with and without ibpAB overexpression (Fig. 7B). Stationary level of activity was reached earlier, however, in the ibpAB deletion mutant and in the wildtype strain without overexpression. Hence the final activities in these strains were independent of the production temperature (Fig. 7A,B). In contrast, the activity continued to increase for about 12 h after arrest of protein synthesis subsequent to α-glucosidase production at 42°C with ibpAB overexpression (Fig. 7B), resulting in higher activity yields than obtained in the wild type strain. The final α-glucosidase activities after production with overexpression of ibpAB at 42°C were two times higher than after production at 37°C (Fig. 7A,B). IbpA/IbpB can thus ameliorate reactivation of inclusion bodies produced at high temperature.
Discussion
Intensified stress response and growth impairment in ibpAB deletion strain
Impairment of cell growth by ibpAB deletion is observed only under conditions characterised by depletion of free DnaK, such as under extremely high temperature or artificially controlled low level [19,33,36,37]. Accumulation of α-glucosidase as inclusion bodies mimics the aspect of permanently impaired protein folding, and growth impairment during α-glucosidase production was more intense in the ibpAB mutant. Moreover, DnaK controls the expression of heat-shock genes. Consequently, chromosomal ibpAB and other heat-shock genes were induced during α-glucosidase production. Especially in the deletion strain, lack of IbpA/IbpB was compensated by enhanced accumulation of the disaggregating chaperone ClpB. Also the levels of two components of the RNA degradosome, enolase and PNP, were elevated in the ibpAB deletion strain at 42°C. Enolase homologues have been reported to be induced by heat shock in a variety of species, including Bacillus subtilis [41], but there was no indication that the E. coli eno gene can be induced by heat-shock or high temperature [42].
Both ClpB and DnaK are essential for disaggregation and de novo protein folding
ClpB and DnaK accumulated to higher levels in the ibpAB deletion strain. These chaperones cooperate with IbpA/IbpB in a "disaggregation triad" [33]. Each chaperone, ClpB and DnaK, was essential for reactivation as well as degradation of α-glucosidase, indicating that both processes operate after disaggregation of the inclusion bodies. With other protein aggregates, varying dependencies on DnaK and ClpB for degradation or reactivation were observed [29,30,32,43-45]. To the aggregate-specific factors influencing the chaperone demand for disaggregation in vitro belong their sizes and accessible surface areas [31,46,47]. Also the substrate specificity of chaperones may influence the protein-specific requirements.
The impaired disaggregation in clpB and dnaK mutants reduced the accumulation of active α-glucosidase already during production, and a negative impact of the clpB deletion on de novo folding has been observed also with a number of other proteins [48]. Resolubilisation from aggregates might thus be a general route to active protein, in accordance with a quantitative model of inclusion body formation kinetics [16]. This constitutes a major difference to the irreversible aggregation at extreme temperatures, when the client release of DnaK is retarded [20].
IbpA/IbpB disburden DnaK and reduce degradation
Ongoing attempts to fold α-glucosidase will finally result in its degradation. IbpA/IbpB, which are postulated to recognise similar traits of aggregation prone proteins as DnaK [19], will withdraw α-glucosidase from the futile cycle of DnaK binding and release. Thereby, IbpA/IbpB on the one hand disburden the DnaK system to make resources available to repress stress responses, and on the other hand protect α-glucosidase from degradation. These functions might be related, as the heat-shock response includes also proteases that may accumulate to lower levels with ibpAB overexpression. Deletion of ibpAB, conversely, lowers the amount of α-glucosidase and other recombinant proteins [27,49]. Consequently, unlike to incubation at 50°C, ibpAB deletion did not increase aggregation during α-glucosidase production, rather reduced accumulation of insoluble α-glucosidase. Functional replacement of DnaK by IbpA/IbpB is also reflected by the amount of DnaK associated with inclusion bodies and aggregated cellular proteins, that is increased in the ibpAB deletion mutant and decreased by ibpAB overexpression [[19], unpublished results].
IbpA/IbpB prevent degradation in a temperature-dependent manner
Disaggregation and degradation of inclusion bodies took place after arrest of protein synthesis. The disaggregation at and above 30°C was retarded by high levels of IbpA/IbpB. In wildtype cells, IbpA/IbpB accumulation is therefore restricted to requiring conditions, e.g. by degradation of IbpA/IbpB after dissociation from protein aggregates [33] or repression of ibpAB expression despite an otherwise active heat-shock response [14].
Temperatures during both, formation and disaggregation of inclusion bodies, were important for the rate of disaggregation and yield of reactivation. Irreversible structural changes of IbpA/IbpB at low temperatures [25] may impair the protective function, making the disaggregation kinetics at 15°C independent of the IbpA/IbpB level. Hence with other systems, depending on the conditions, no influence of the ibpAB deletion on disaggregation kinetics or even delayed disaggregation in the mutant were observed [19,33,34,50]. The reactivation rate is determined by the difference of disaggregation and degradation rates, which are influenced in parallel by the availability of the sHsps. Hence reactivation was hardly effected by ibpAB deletion or overexpression, in accordance with observation for other proteins [32,34].
With high temperature during production, degradation during the subsequent disaggregation phase was nearly completely avoided in ibpAB proficient strains. In the ibpAB deletion strain, on the contrary, production temperature did not ameliorate the recovery of soluble α-glucosidase. Consequently, the reactivation phase was prolonged with higher IbpA/IbpB concentrations, and the final α-glucosidase activities were increased from 4 in the wildtype cells to 5 Umg-1 with ibpAB overexpression. As the yield of reactivated protein is more important for heat-stress survival than the rate of disaggregation [56], the reduction of degradation may also contribute to the cell protective function of the sHsps.
Conclusions
Deletion of ibpAB renders E. coli more susceptible to growth impairment by production of α-glucosidase. Without recombinant protein production, ibpAB deletion affects growth only at extreme temperatures or in combination with mutations of dnaK. Thus, production of an aggregation prone protein constitutes a good model system to study the functions of IbpA/IbpB under non-lethal conditions. Two major functions were found for IbpA/IbpB: First, the sHsps protect cells during α-glucosidase production and disburden the DnaK system. In the ibpAB deletion strain, insufficient free DnaK was available to control ClpB accumulation, which was strongly intensified. Also enolase and PNP showed a similar pattern.
Second, IbpA/IbpB reduce degradation of α-glucosidase during disaggregation from inclusion bodies. The IbpA/IbpB function depends on elevated temperature. Improved reactivation and protection from degradation were found after production at higher temperature, in accordance with the known heat-activation of sHsps. Also during disaggregation, a higher temperature was beneficial, whereas no protection from degradation was found at 15°C. In the ibpAB deletion strain, however, removal of inclusion bodies was accelerated, possibly due to the higher levels of ClpB and DnaK that were found to be essential for reactivation and degradation of α-glucosidase. Thus, the sHsp level is in a delicate balance: IbpA/IbpB are required to protect the cell, but abundant IbpA/IbpB impede the necessary adaptation and retards removal of aggregates.
Methods
Strains and Plasmids
Escherichia coli MC4100 araD139 Δ (argF-lac)U169 rpsL150 relA1 flbB5301 deoC1 ptsF25 rbsR and JGT17 MC4100 Δ ibpAB [36] were used to produce the yeast α-glucosidase encoded on the plasmid pKK177-3/GLUCP1 [38]. Plasmid pUBS520, supplying the minor argU tRNA and carrying the lacI repressor gene [51] was cotransformed for improving the production of α-glucosidase [52]. The sequence from plasmid pIbp [36] between the two HindIII restriction sides, coding for the ibpA and ibpB genes including their native promoter, was cloned into the HindIII site of the plasmid pKK177-3/GLUCP1 [38], located between the α-glucosidase GLUCPI gene and the 5ST1T2 terminator, to give the new plasmid named pKK177-3/GLUCP1_ibpAB, in which the ibpAB operon is under the control of its native promoter and of the tac-promoter upstream of the α-glucosidase gene. Proper orientation of the insert was tested by EarI digestion.
Culture conditions
The cultures were incubated on Luria-Bertani (LB) medium, supplemented with appropriate antibiotics (ampicilline 100 μg mL-1, chloramphenicol 50 μg mL-1), on a rotary shaker at 37°C to an OD600 of 0.5, induced with 1 mM IPTG and transferred to different temperatures as indicated in the results section. For reactivation experiments, tetracycline was added to a final concentration of 25 μgmL-1 two hours after induction to arrest protein synthesis, and the cultures were transferred to the indicated temperatures.
Product analysis
For SDS-PAGE analysis, cell pellets resuspended in phosphate buffer pH 7 were incubated on ice with 33 mgL-1 lysozyme for 30 min and disrupted by sonication for 20 s. Soluble and insoluble fractions were separated by centrifugation for 20 min at 13,000 rpm. The insoluble fractions were washed with phosphate buffer twice. SDS-PAGE analysis was performed according to the method of Laemmli [53]. Levels of α-glucosidase were estimated by densitometry of Coomassie-stained gels.
α-glucosidase activity was measured as described [54]. For the calculation of specific activities, the protein concentration was determined according to Bradford [55] with bovine serum albumin as standard. Specific activities are expressed as units of α-glucosidase per milligram of total protein (U mg-1).
Two-dimensional gel electrophoresis
Cell pellets from a culture volume of 8/OD600 mL were dissolved in 200 μL lysis buffer containing 8 M urea, 4% CHAPS, 60 mM DDT, 2% Pharmalyte 3–10 and 0.002% bromophenol blue and incubated for one hour at room temperature. The first dimension was carried out on an IPGphor using 24 cm pH gradient 3–10 strips (amersham pharmacia biotech), with the voltage profile recommended by the manufacturer. The second dimension was run in an Ettan Dalt six (amersham pharmacia biotech) on 12.5% polyacrylamide gels. Gels were stained with Coomassie brilliant blue for quantification or by silver staining for visualisation. Gels for cultures incubated at 42°C were performed in quadruplicate, for other temperatures once. Protein spots were identified by comparison with the E. coli 2D database . Spot identities were verified by N-terminal sequencing for ClpB (RLDRLTN) and enolase (SKIVKII). α-glucosidase was not dissolved from the inclusion bodies and is not visible in the gels.
Acknowledgements
We thank F. Baneyx for providing strain MC4100 and JGT17 and plasmid pIbp, B. Bukau for providing a dnaK mutant strain, A. Taylor for providing a clpB mutant strain, Roche for plasmids pUBS520 and pKK177-3/GLUCP1, P. Rücknagel for N-terminal sequencing, U. Rinas for helpful comments and H. Lilie for critically reading an early version of the manuscript. Ha LeThanh is recipient of a grant from Deutscher Akademischer Auslandsdienst (DAAD).
Figures and Tables
Figure 1 Impact of IbpA/IbpB levels on growth during α-glucosidase production at various temperatures. Cultures were grown on complex Luria-Bertani medium at 37°C to an OD600 of 0.5 and after induction of α-glucosidase synthesis by addition of 1 mM IPTG incubated at 24°C, 30°C, 37°C, or 42°C. Growth was linear after induction, and optical density OD600 values after four hours of α-glucosidase production are shown for wildtype strain with (black bars) or without overexpression of ibpAB (grey bars) and the ibpAB deletion strain (white bars). Mean values and 95% confidence intervals from at least three independent experiments are shown.
Figure 2 Effect of ibpAB availability on the proteome composition after two hours of α-glucosidase production at 42°C. After growth at 37°C to OD600 of 0.5, cultures were induced with 1 mM IPTG, and transferred to 42°C. Two hours later, cells were harvested and the proteome was separated by two-dimensional gel-electrophoresis. Silver-stained two-dimensional gels of (A) wildtype strain with or (B) without ibpAB overexpression, and of (C) the ibp AB deletion strain. Reference spots used for normalisation of spot intensities after densitometry are indicated by circles. Positions of selected proteins (cf. text) are indicated.
Figure 3 Level of stress proteins after production of α-glucosidase at different temperatures. Cultures were grown to an OD600 of 0.5 at 37°C, induced with 1 mM IPTG and transferred to the indicated temperatures for two hours. After two-dimensional gel electrophoresis of the proteomes, spot volumes quantified by densitometry of Coomassie-stained gels were normalised with the summed volumes of three reference spots marked in Fig. 2 by circles. Concentrations c in the wildtype strain with ibpAB overexpression (black bar), without overexpression (grey bar), and in the ibpAB deletion mutant (white bar) are given relative to the maximum concentration cmax determined for the respective protein to facilitate comparison. The 95% confidence interval from four experiments at 42°C are shown. Protein designation are given above the panels: chaperones DnaK and ClpB, the thermo-unstable elongation factor (EF-Tu), enolase (Eno), polynucleotide phosphorylase (PNP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Production temperatures are given below the bars.
Figure 4 Kinetics of disaggregation, degradation and reactivation of α-glucosidase from inclusion bodies at various temperature. Cultures producing α-glucosidase in the wildtype strain with overexpression of ibpAB (black triangles, solid lines) or without (grey circles, dashed lines) and in the ibpAB deletion mutant (white squares, dotted lines) were incubated at 37°C to an OD600 of 0.5 and then induced with 1 mM IPTG. Two hours after induction, tetracycline was added to a final concentration of 25 μg mL-1, and the cultures were transferred to 15°C (A, E, I), 24°C (B, F, J), 30°C (C, G, K) or 37°C (D, H, L), respectively. Mean value and confidence interval of at least 2 (15 and 24°C) or 3–5 (30 and 37°C) independent experiments. Lines are from linear regression. (A-D) The amount of insoluble α-glucosidase was estimated by densitometry of Coomassie-stained SDS-PAGE gels and was referred to the value at the time of reactivation start (54 mg g-1 in the wildtype, 58 mg g-1 with overexpression of ibpAB and 49 mg g-1 in the deletion mutant). (E-H) α-Glucosidase activity and protein content were determined in cell extracts after disruption. Specific activity of α-glucosidase is given in units per mg of protein. (I-L) Degradational loss of α-glucosidase calculated as difference of the sum of soluble and insoluble α-glucosidase to the value obtained before addition of tetracycline. Time is given in hours after induction.
Figure 5 Disaggregation and resolubilisation of inclusion bodies produced at 42°C Inclusion bodies of α-glucosidase were produced for two hours at 42°C, followed by addition of tetracycline and subsequent incubation at 30°C. Coomassie-stained SDS-PAGE gel of (A) the insoluble cell fractions and (B) the soluble cell fractions extracted from identical amounts of biomass; times relative to induction and ibpAB genotype are given above the lanes. Positions of α-glucosidase, IbpA/IbpB and membrane proteins are indicated. First lane contains molecular weight markers of 97.4, 66.2, 45, 31, 21.5 and 14.4 kDa, respectively.
Figure 6 Degradation of α-glucosidase after production at 42°C. Cultures of ibpAB deletion mutant (white square), wildtype strain without (grey circle) or with ibpAB overproduction (black triangle) produced α-glucosidase for two hours at 42°C and were transferred to 30°C after addition of tetracycline. Degradational loss of α-glucosidase is shown relative to the amount of α-glucosidase present at the time of tetracycline addition. Time is given in hours after induction.
Figure 7 Impact of production temperature on reactivation of α-glucosidase from inclusion bodies. Inclusion bodies of α-glucosidase were produced for two hours at 37°C (A) or 42°C (B) in ibpAB deletion mutant (white square), wildtype strain without (grey circle) or with ibpAB overproduction (black triangle). Specific activity of α-glucosidase was measured during incubation at 30°C after addition of tetracycline. Mean values and 95% confidence intervals from six (37°C) or four (42°C) independent experiments. Time is given in hours after induction.
Table 1 Relative levels of IbpA and IbpB after two hours of α-glucosidase production at various temperatures with and without ibpAB overexpression from plasmid
Temperature wildtype ibpAB++
IbpAa 42°C 3.3 ± 1.3 84 ± 9.5
37°C 5.0 50
30°C 1.6 19
IbpB 42°C 1 ± 0.3 12 ± 1.3
37°C 1.4 7
30°C 0.7 3
a The level of IbpA and IbpB was estimated by densitometry of Coomassie-stained two-dimensional gels. Spot volumes were normalised using three spots as internal standards (indicated by circles in Fig. 2) and are given relative to the value of chromosomally encoded IbpB at 42°C.
Table 2 Effect of temperature and IbpA/IbpB on accumulation and activity of α-glucosidase four hours after induction
Genotype 24°C 30°C 37°C 42°C
Total α-glucosidase/ (mg g-1) ibpAB++ 38 ± 5 52 ± 1 66 ± 11 47 ± 11
wt 39 ± 2 47 ± 5 56 ± 16 38 ± 7
ibpAB- 36 ± 4 43 ± 2 49 ± 10 28 ± 4
Vol. activity/ (U mL-1) ibpAB++ 0.56 ± 0.17 0.80 ± 0.29 0.13 ± 0.04 0.15 ± 0.01
wt 0.61 ± 0.14 0.76 ± 0.26 0.10 ± 0.02 0.10 ± 0.01
ibpAB- 0.55 ± 0.02 0.74 ± 0.20 0.11 ± 0.04 0.10 ± 0.02
Mean values and 95% confidence intervals from at least three independent experiments. The variance between experiments is mainly caused by different times needed to reach the set temperature after induction depending on the volume of the cultures.
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| 15707488 | PMC552319 | CC BY | 2021-01-04 16:05:47 | no | Microb Cell Fact. 2005 Feb 11; 4:6 | utf-8 | Microb Cell Fact | 2,005 | 10.1186/1475-2859-4-6 | oa_comm |
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Cancer Cell IntCancer Cell International1475-2867BioMed Central London 1475-2867-5-41571590910.1186/1475-2867-5-4ReviewCancer/testis antigens and gametogenesis: a review and "brain-storming" session Kalejs Martins [email protected] Jekaterina [email protected] Biomedical Research and Study Centre of the Latvian University, Riga, Latvia2005 16 2 2005 5 4 4 9 12 2004 16 2 2005 Copyright © 2005 Kalejs and Erenpreisa; licensee BioMed Central Ltd.2005Kalejs and Erenpreisa; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Genes expressed both in normal testis and in malignancies (Cancer/ Testis associated genes – CTA) have become the most extensively studied antigen group in the field of tumour immunology. Despite this, many fundamentally important questions remain unanswered: what is the connection between germ-cell specific genes and tumours? Is the expression of these genes yet another proof for the importance of genome destabilisation in the process of tumorigenesis?, or maybe activation of these genes is not quite random but instead related to some programme giving tumours a survival advantage?
This review collates most of the recent information available about CTAs expression, function, and regulation. The data suggests a programme related to ontogenesis, mostly to gametogenesis. In the "brain-storming" part, facts in conflict with the hypothesis of random CTA gene activation are discussed. We propose a programme borrowed from organisms phylogenetically much older than humans, which existed before the differentiation of sexes. It is a programme that has served as a life cycle with prominent ploidy changes, and from which, as we know, the germ-cell ploidy cycle – meiosis – has evolved. Further work may show whether this hypothesis can lead to a novel anti-tumour strategy.
CTA genesgametogenesispolyploidy
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Introduction
Cancer/Testis (CT) antigens are a group of tumour antigens with gene expression restricted to male germ cells in the testis and to various malignancies. Their function in tumours is enigmatic and a common between testis genes (gametogenesis) and cancer remains elusive. When the causal link is not evident, it is tempting to believe the association is random, and assign it to general aspects of "genome instability in cancer". However, we believe a more direct link may exist. As suggested in this review, possible clues may be found in the common evolutionary pathway between ploidy cycles in meiosis and polyploidy in tumour cells. The latter, along with CT antigen expression, is a characteristic feature of well-progressed tumours. However, before discussing such possibilities, it is necessary to review the established literature.
The search for tumour antigens began in the 1960's with two groups identifying first alpha-fetoprotein (AFP), a serum marker for hepatoma and germ-cell tumours [1], and then carcinoembryonic antigen (CEA), a serum marker for colon and other epithelial cancers [2]. These antigens were discovered using heterogenous sera acquired by immunizing laboratory animals with human tumour material. However, only during the 90's did both cellular [3,4] and humoral [5] immune responses to human tumours get proper molecular definitions.
The first CTA, MAGEA-1, was identified in 1991 by Boon and colleagues using T-cell epitope cloning, a very complicated and time-consuming method [3]. In 1995 the SEREX (serological expression cloning) technique to identify tumour antigens was developed by Pfreundschuh and colleagues [5], which remains the leading approach to identifying new antigens that elicit humoral immune responses. Besides MAGEA1, BAGE, and GAGE1 discovered by T-cell epitope cloning, SEREX very soon displayed more tumour antigens with a cancer/testis restricted expression profile (SSX2, NY-ESO-1, and SYCP-1). The term "cancer-testis (CT) antigen" was introduced by Chen et al. [6], who recognized this group of genes had little in common except their expression profile.
By initial definition, expression of genes coding for CT antigens should be restricted in normal tissues to male germ cells in the testis and to malignancies of various histological types. However, the criteria proposed in the 90's are not true for all antigens of this group as seen today. Furthermore, for many of the recently discovered gene products with the described expression profile, no T-cell recognized epitopes have hitherto been identified. This is why CTA – "Cancer/Testis Associated" is a more appropriate name for this family of genes (and will be used in this context further in this review), because a lot of its members still need to be proven as possessing antigenic properties in cancer patients.
Attributing genes to the CTA gene family is based on several characteristic features [7,8]:
1. Predominant expression in germ cells of the testis and generally not in other normal tissues.
2. Expression in a number of malignant tumours of various histological types.
3. Mapping of the gene to the X-chromosome
4. Membership of a multigene family.
5. Antigenic properties in tumour-bearing patients.
Some exceptions to these criteria for certain CTAs will be described and discussed later.
Expression of CTA genes
To date, 89 individual CTA genes or isoforms have been described, which are organised in to 44 families (see additional file 1). From these, 19 families are testis-restricted, and 11 show additional expression in one or two somatic tissues. Nine are expressed in 3–6 tissue types besides testis, and 5 are ubiquitously somatically expressed. With the exception of the testis-restricted CTAs, the others also show expression in the pancreas but at levels as much as 10 × lower than in testis (based on mRNA expression levels from [9]). Expression of CTA was first shown in melanoma and all the classic CTA are expressed in this type of tumour, but since the 1980's, expression in various other tumours has been recognised (additional file 2).
The expression pattern of CTAs during spermatogenesis is of special interest. Functional analysis of these genes during gametogenesis might well give some clues about their possible role in tumours. Their expression is restricted exclusively to spermatogenic germ cells with other tubular cells (e.g. Leydig and Sertoli) being negative. This fits well with the findings of Yuasa et al. [10] who demonstrated that CTAs have much higher expression frequencies in the germ cell cancers (seminomas) than non-seminomas..
Different CTAs are expressed during different stages of spermatogenesis (Fig. 1.), so one may imagine that their functions are versatile, starting from regulation of mitotic cycling in spermatogonia, association with the meiotic cycle in spermatocytes, and finalizing with acrosome maturation in sperm.
Figure 1 Expression of CTAs during male germ cell development. References for Fig. 1: [25] [27] [28] [37] [40] [42] [53] [92] [93] [94] [95] [96]
In normal tissues, expression of NY-ESO1, MAGE-A3, -A4, and -A8 through -A11 as well as of several members of the XAGE gene family is found in the placenta. NY-ESO1 and several XAGEs are also expressed in the fetal ovary [11-13].
Regulation of CTA expression
The mechanisms involved in regulation of CTA expression have recently been comprehensively reviewed by Albert Zendman et al [14]. Thus, only a short recitation of some of the main points is provided here.
Mostly, methylation processes are responsible for the ectopic derepression of CTA genes. Using the demethylating agent, 5-aza-2-deoxycytidine (5DC), expression of several CTAs in cultured tumour cell lines can be induced/upregulated [15]. 5DC entraps DNA methyltransferases in a complex with DNA, which leads to progressive loss of DNA methylation, thereby releasing transcriptional blockage. Such upregulated expression has been reported for several MAGE members – LAGE-1, SSX-2, CAGE, NY-ESO-1. For MAGE-A1, demethylation is necessary and sufficient for gene expression, suggesting demethylation is the primary mechanism of transcription control [16]. In this context, a recent discovery of a CTA, viz. 'Boris', is interesting. Boris is reported as being expressed in several types of malignancies, and normally plays a major role in regulating methylation processes during spermatogenesis – it removes imprinting from genes during the last mitotic division of spermatocytes (reviewed in [17]). Several lines of evidence indicate that expression of some CTAs is dependent not only on demethylation, but on other transcriptional mechanisms.
Histone deacetylase (HDAC) inhibitors, on their own or in combination with 5DC, can also induce CTA expression, including MAGE, SSX, and NY-ESO-1 family members [15]. The CTA-rich region in Xp11.21-22 (e.g. SSX, MAGE-B) may escape X-chromosomal inactivation, but these genes are not normally expressed in females [18]. While global hypomethylation is common and prominent in colorectal cancer, few CTAs have ever been reported as expressed in this type of cancer [19]. Non-demethylation dependent induction of MAGE expression has been demonstrated by Park et al. [20], demonstrating that 40 mM NaCl induces the transcriptional and translational activation of MAGE-B1 and -B2 in specific tissues at hypertonic conditions.
There exist definite expression patterns (sets) of different CTAs in certain tumours. Marked heterogeneity of CTA expression is found in cells of some tumours, which cannot easily be explained by a global demethylation process [21-24]. The mechanisms of ectopic transcriptional activation of CTA genes clearly needs more investigation.
Function
Information regarding the function and cellular localization of CTAs is far less comprehensive. Often the proposed function is based purely on sequence homology with another protein of a known function. The only CTA proteins functionally established in gametogenesis are SCP-1, involved in chromosome pairing during meiosis [7], OY-TES-1 which functions in acrosin packaging in the acrosome of sperm heads [25], SPO11 acting as a meiosis-specific endonuclease [26], and BORIS, which is involved in cancellation of imprinting by epigenetic reprogramming during the final round of mitosis in spermatogenesis [27]. However, contrary to the situation in meiosis where it is rapidly degraded after the meiotic prophase in spermatocytes, SCP-1 expression in tumours is not cell cycle restricted [7]. BORIS is a paralog of CTCF. CTCF is a highly versatile 11 zinc-finger factor involved in various aspects of gene regulation – X chromosome inactivation, reading of imprinting sites, etc. [27]. During spermatogenesis, Boris is expressed later than many other CTA genes ([27]; Fig. 1.).
Suggestions for the functions of other CTAs mostly arose from studies of their homology with some well-known proteins and their domains, TSP50 being protease-like, CT17 phospholipase-like, and CT15 metalloproteinase-like [28]. The CT15 gene encodes a disintegrin and metalloproteinase (ADAM) domain 2, which is a member of the ADAM protein family [29]. Members of this family are membrane-anchored proteins structurally related to snake venom disintegrins, which have been implicated in a variety of biologic processes involving cell-cell and cell-matrix interactions, including fertilization, muscle development, and neurogenesis. This member is a subunit of an integral sperm membrane glycoprotein called fertilin, which plays an important role in sperm-egg interactions [30]. It is a membrane metalloproteinase with a possible role in tumour evasion and metastasis.
LDHC, the germ cell-specific member of the lactate dehydrogenase family, escapes from transcriptional repression, resulting in significant expression levels in virtually all tumour types tested. It might contribute to the constitutive activation of an anaerobic pathway in tumours, because its expression in tumours is not dependent on hypoxia [31].
Suggestions as to the role of MAGEs with a CTA expression profile mainly depend on studies of their ubiquitously expressed family members (e.g. Necdin, MAGE-D1, NRAGE, Dlxin-1). In general, the data point to a role for MAGEs via transcriptional regulation in cell cycle control and apoptosis. F.ex, Necdin-related MAGE proteins differentially interact with the E2F1 transcription factor and the p75 neurotrophin receptor [32]. The high level of homology among members of the MAGE family in both mouse and human suggests an important function both in testis and cancer.
The products of the SSX genes belong to the family of highly homologous synovial sarcoma X (SSX) breakpoint proteins. These proteins may function as transcriptional repressors; SSX1, SSX2 and SSX4 genes have been involved in the t (X;18) translocation characteristically found in all synovial sarcomas [33-35]. This translocation results in the fusion of the synovial sarcoma translocation gene (SYT) on chromosome 18 to one of the SSX genes on chromosome X. The encoded hybrid proteins are probably responsible for transforming activity. In the nucleus of sarcoma cells, both diffuse and speckled localisations of SSX protein have been reported [36,37].
The HOM-TES-85 protein has structural peculiarities that are shared exclusively with the N-myc oncoprotein. However, functional studies are required for confirmation [38]. Besides, the C-myc proto-oncogene is a normal participant of spermatogenesis (Fig. 2).
Figure 2 Oncogene expression during spermatogenesis (mainly in mice). From [70].
BRDT is similar to the RING3 protein family. It possesses 2 bromodomain motifs and a PEST sequence (a proline, glutamic acid, serine, and threonine cluster) characteristic of proteins that undergo rapid intracellular degradation). The bromodomain is found in proteins that regulate transcription [39].
PLU-1, a large multi-domain nuclear protein also has a strong transcriptional repression activity. It is a member of the ARID family of DNA-binding proteins. Plu-1 mRNA and PLU-1 protein are both highly expressed in the mitotic spermatogonia. The expression is reduced in the early prophase I stages (leptotene, zygotene), but reappears at pachytene, still being detectable in diplotene cells. It is located diffusely over the nucleus. PLU-1 might have a role in regulating meiotic transcription, restricted to certain meiotic stages [40].
The protein encoded by the SPANX gene targets the nucleus and associates with nuclear vacuoles and the redundant nuclear envelope in sperm cells [41]. In situ hybridization of human testis sections showed SPAN-X mRNA expression in round and elongated spermatids [42]. These redundant nuclear envelopes have a unique structure of limited chromatin sheets continued as annulate lamellae. Both these enigmatic structures have also been described in intact lymphomas [43] and irradiated lymphomas [44].
The protein encoded by IL13RA1 gene is a subunit of the interleukin-13 receptor. This subunit forms a receptor complex with IL-4 receptor alpha, a subunit shared by IL-13 and IL-4 receptors. This subunit serves as a primary IL-13-binding subunit of the IL-13 receptor, and may also be a component of IL-4 receptors. This protein binds tyrosine kinase TYK2, and thus may mediate the signalling processes that lead to the activation of JAK1, STAT3 and STAT6 induced by IL-13 and IL-4 [45].
SGY-1 (soggy-1), a secreted protein related to the Dickkopf protein family, is involved in suppressing the Wnt signal-transduction pathway controlling transcription activation of genes such as c-myc, c-jun, Fra, and cyclin D1 by preventing the accumulation of beta-catenin [46,47]. Wnt proteins are implicated in a wide variety of biologic processes including cell fate determination and patterning in early embryos, and in cell growth and/or differentiation in certain adult mammalian tissues [48]. Wnts can induce proliferation in different types of stem cells [49,50]. The importance of Wnt signalling during tumorigenesis has been recently emphasised [51,52].
NY-ESO-1 is one of the most immunogenic and therapeutically promising CTAs but functional studies on this gene are severely lagging behind its practical application. Unlike the majority of CTA genes, NY-ESO-1 stops its expression in well-progressed tumours, so it can be used as a marker to follow the early progression of testicular tumorigenesis [53].
CAGE [54] and HAGE [55] code for proteins with helicase-like features. Probably, it might be involved in recombination exchange in testis and recombination DNA repair in tumours.
TPX-1 is now seen as an integral protein of the outer dense fibres and the acrosome of spermatids in rats [56].
In summary, we see that the functions of individual CTA, when known, are very diverse, including, for example, both activators and repressors of proliferation and transcription.
Immunogenicity
Immunogenicity in cancer patients is elicited only by short peptide sequences of CTA epitopes, which are presented on the tumour cell surface by HLA Class I molecules in the case of cytotoxic T lymphocyte (CTL) mediated immune responses and by HLA Class II molecules on the surface of APC (antigen presenting cells), in the case of T-helper cell (TH) mediated immune responses. Identification of these epitopes is one of the main goals of CTA research. Knowledge of epitopes recognized by the immune system allows the creation of the tumour-specific vaccines. In the vaccination process, T-cell epitopes are often administered together with different adjuvants or cytokines, or delivered using peptide pulsed autologous dendritic cells, all of which are aimed at enhancing the immune reaction [57]. Currently efforts are being made to identify HLA class II restricted epitopes in order to promote TH responses, which are required to support the activity of CTLs, and provide a more "complete" immune response. However, vaccines designated to prime the immune system against tumours expressing various CTA have so far shown only partial clinical success [57-59].
Since the technique used to identify candidate tumour antigens has changed from T-cell epitope cloning [3,60], and SEREX (serological analysis of cDNA expression libraries) [5,61,62] to differential gene expression analysis by various techniques like RDA (representative difference analysis) [63], DD (differential display) [64] and SSH (suppression subtractive hybridization) [65] – and even further to bioinformatics – we cannot really be sure about the adequate use of the definition "antigen" in relation to CTA gene products. Whilst the former two techniques are dependent on the immunogenicity of specific epitopes in cancer patients, the latter ones are simply based on mRNA expression levels or even sequence homologies detected via search engines and provide no answer about immunoreactivity. In additional file 1, one can see that an immune response is documented against only 19 of 44 CTAs. Immune recognition of the majority of these CTAs is cancer-related, occurring spontaneously in cancer patients, not in cancer-free individuals. The exceptions to this are humoral immune responses to MAGEB1/CT3.1 in systemic lupus erythematosus patients [66] and to SPA17/CT22 in vasectomised men [67].
CTA can be considered to be tumour specific. There exists the blood-testis barrier [68], which prevents the immune system from contacting with CTA gene products. Besides this, germinative cells do not express HLA class Ia molecules [69], so they cannot present their expressed proteins to the immune system. For these reasons, the immune system never comes into contact with these proteins and recognizes them as "non-self" structures.
Brain-storming
The function of most CTAs is unknown, although some role in regulation of gene expression (both activating and repressing) seems likely [8]. Are CTAs oncogenes? By definition, oncogenes are normal cellular genes participating in proliferation cascades, which are abnormally activated in tumours [70]. Therefore, with the exception of one (HOM-TES-85, which has structural homology with the myc-oncogene) CTAs can not formally be regarded as oncogenes. Are CTAs simply activated by the imbalanced genome, due to its instability in tumours? Some, such as SSX, whose ectopic expression is caused by the SYT-SSX fusion due to translocation t (X;18) in synovial sarcomas, certainly are. However, a large number of CTA genes are only located on the X-chromosome and this chromosome is not a specific site of chromosome breaks and translocations usually associated with tumours [71]. However, the chromosome region 20q13.2 containing the BORIS gene is commonly amplified or exhibits moderate gains of material in many human cancers. This has strengthened the idea that this region contains a major oncogene [72,73]. A preliminary report using RT-PCR has found that BORIS expression is detectable in over half of ~200 cancer cell lines studied, representing most of the major forms of human tumours, discussed in [17]. However, this observation awaits further confirmation. Theoretically, the aberrant expression of the amplified (by chance) Boris, which by analogy with CTCF may demethylate CTA genes located on X-chromosome, may in turn activate a large body of CTA genes in human malignancies. However, why are other gametogenesis-related CTA genes also activated from other chromosomes? It does not look like a chance event and therefore amplification of "Boris" may still not be random.
Diversity of functions, including genes involved in ontogenesis suggest that CTAs are activated either as a result of the genome instability (however, why testicular genes?) or as part of a complex program. Also from this point, this looks like a program related to gametogenesis. The same idea was proposed by Old [8].
If CTAs generally do not enhance tumour growth except by stimulating proliferation, like oncogenes, another possibility is that they could do it by stimulating DNA repair or inhibiting apoptosis (combination of all three is possible). Indeed, some CTAs have relation to the DNA repair factors by homologous recombination. These are SPO11, SYCP1, helicase-like CAGE and HAGE acting in meiotic prophase of gametogenesis. In turn, homologous recombination in tumours was shown to act anti-apoptotically [74].
Are these CTAs restricted only to prophase of meiosis, where recombination takes place? It appears not. For example, some are associated with the spermatogonial stage (Plu-1 mRNA and PLU-1), and some with maturation of the acrosome (see Fig. 1). It is a program again, and why only male gametogenesis? But it also occurs in oogenesis [8]. Thus, prophase of meiosis (pairing and recombination) may be still the most important.
So, ectopic activation of CTA genes is not entirely random, being induced from the sexual X-chromosome but also from loci on other chromosomes, relating to gametogenesis. However, it is curious then that the very common proto-oncogenes of proliferatives cascades also participate in gametogenesis, e.g. myc, ras, jun, etc. This can be seen in Fig 2, (taken from the Janis Erenpreiss [70]). He and others postulated a link between gametogenesis and cancerogenesis before CTAs were revealed [75-77].
In turn, Old [8] looked at the problem from a new angle and suggested that CTAs provide a causal link between gametogenesis and cancer. This seems plausable, but does this illegitimate program in tumours embrace, even mosaically, only gametogenesis? May be only the DNA recombination repair component is common? But the CTA genes are repressing a wide Wnt family, which also functions in early embryogenesis.
The program sounds more like an ontogenetic (life-cycle) one. Let us remember the experiments by Mintz and Illmeisee [19] who cloned normal genetic mosaic mice by introducting the nuclei of malignant teratocarcinoma into enucleated eggs. Both gametic, parthenogenetic and trophoblastic theories of cancer have also been proposed in the past [8,70] and can be viewed as embryonal or ontogenetic theories of cancer.
Another question is whether CTA genes govern a life-cycle-like program with its key events similar to meiosis? In turn, to which process does meiosis provide a key – recombination and reduction division? Are tumours capable of these? We also have to consider why X-chromosomes are involved, and not Y-chromosomes, which are responsible for sex? Perhaps this is because the X-chromosome is better conserved evolutionary and appeared prior to sex discrimination.
So, this looks like an ontogenetic program, which evolutionary preceded sexual (amphimictic) life-cycle, with the key in recombination and reduction division; but what is it? The answer is evident – ancient ploidy cycles [78,79]. In general, ploidy cycles are displayed as a cyclic increase and reduction of ploidy (chromosome number), and these involve the pairing of homologous chromosomes and their segregation, omitting one round of DNA replication.
There may be reduction of ploidy 2n-n (sexual meiosis) or from nx to 2n DNA numbers, in the asexual ploidy cycles characteristic for a few protists, as in Amoebae and foraminiferans [80,81] as well as part of ontogenetic programs in the more developed taxons [82,83]. Contrary to gametic reduction in sexual meiosis, the reduction of ploidy in polyploid somatic cells is called "somatic reduction".
In this context, expression of some CTAs by the placenta is of interest because trophoblast and decidua are the only mammalian tissue capable of endoreduplication creating enormously large ploidies [84]. an ability shared by many tumour cells. The latest studies on a silver fox revealed somatic reduction in the giant cells of a trophoblast [85].
Now let us return to DNA recombination repair in tumours as a means of survival, as already mentioned. Repair by homologous recombination can protect malignant tumour cells from apoptosis. In particular, as shown in our laboratory, endopolyploid cells employ this mechanism [86]. Likewise, expression of CTAs – endopolyploidy – is a hallmark of malignant tumour progression where there is deficient TP53 function [87].
In turn, some giant tumour cells show the capability to segregate their genomes and return to mitosis ([86,88-91], see Fig. 3).
Figure 3 Reductional mitotic divisions generating low ploidy cells from one large polyploid cell. Non-treated Burkitt's lymphoma cell line, DNA staining with Toluidine-blue. × 2,500.
Conclusion
A hypothesis is put forward that activation of at least some of the CTA genes in p53-deficient human tumours could be due to the genetic program running "relic" ploidy cycles in tumour cells. This hypothesis offers new opportunities for the design of novel tumour treatment strategies. In particular, passive therapy using CTAs to prime the host immune system against the tumour could be replaced with gene therapy aimed to block the function of CTA gene products or even their expression. This approach is promising, because normally only the germ cells in the testis express CTA genes and they are well protected by the blood-testis barrier. Thus, there should not be any problem with tumour-specific priming, and respectively we would predict there to be no side effects.
Supplementary Material
Additional File 1
"Chromosomal localization, type of immune response, identification method and identification references for all known 44 CTA gene families".
Click here for file
Additional File 2
" CTA Frequency (%) of expression in various tumour types".
Click here for file
Acknowledgements
The authors gratefully thank Denys Wheatley and Jamie Honeychurch for his help in editing the article.
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Hidaka S Yasutake T Takeshita H Kondo M Tsuji T Nanashima A Sawai T Yamaguchi H Nakagoe T Ayabe H Tagawa Y Differences in 20q13.2 copy number between colorectal cancers with and without liver metastasis Clin Cancer Res 2000 6 2712 2717 10914715
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Zybina TG Zybina EV Kiknadze II Zhelezova AI Polyploidization in the trophoblast and uterine glandular epithelium of the endotheliochorial placenta of silver fox (Vulpes fulvus Desm.), as revealed by the DNA content Placenta 2001 22 490 498 11373160 10.1053/plac.2001.0675
Ivanov A Cragg MS Erenpreisa J Emzinsh D Lukman H Illidge TM Endopolyploid cells produced after severe genotoxic damage have the potential to repair DNA double strand breaks J Cell Sci 2003 116 4095 4106 12953071 10.1242/jcs.00740
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| 15715909 | PMC552320 | CC BY | 2021-01-04 16:40:09 | no | Cancer Cell Int. 2005 Feb 16; 5:4 | utf-8 | Cancer Cell Int | 2,005 | 10.1186/1475-2867-5-4 | oa_comm |
==== Front
Malar JMalaria Journal1475-2875BioMed Central London 1475-2875-4-121572071310.1186/1475-2875-4-12CommentaryMalaria and urbanization in sub-Saharan Africa Donnelly Martin J [email protected] PJ [email protected] Christian [email protected] Imelda [email protected]'Alessandro Umberto [email protected] Guy [email protected] Flemming [email protected] Eveline [email protected] Harold [email protected] Jean-Francois [email protected] Ian M [email protected] Clifford [email protected] Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA. UK2 Swiss Tropical Institute, P.O. Box, 4002 Basel, Switzerland3 Prince Leopold Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium4 International Water Management Institute Sri Lanka127, Sunil Mawatha, Pelawatte, Battaramulla, Sri Lanka5 Institute of Public Health, Department of International Health, University of Copenhagen, Blegdamsvej 32200 København, Denmark6 International Water Management Institute (West Africa), PMB CT 112, Cantonments, Accra, Ghana7 Institut de Recherche pour le Développement, BP 1386, CP 18524, Dakar, Sénégal8 Systemwide Initiative on Malaria and Agriculture, International Water Management Institute, Private Bag X813, Silverton 0127, South Africa2005 18 2 2005 4 12 12 2 2 2005 18 2 2005 Copyright © 2005 Donnelly et al; licensee BioMed Central Ltd.2005Donnelly et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
There are already 40 cities in Africa with over 1 million inhabitants and the United Nations Environmental Programme estimates that by 2025 over 800 million people will live in urban areas. Recognizing that malaria control can improve the health of the vulnerable and remove a major obstacle to their economic development, the Malaria Knowledge Programme of the Liverpool School of Tropical Medicine and the Systemwide Initiative on Malaria and Agriculture convened a multi-sectoral technical consultation on urban malaria in Pretoria, South Africa from 2nd to 4th December, 2004. The aim of the meeting was to identify strategies for the assessment and control of urban malaria. This commentary reflects the discussions held during the meeting and aims to inform researchers and policy makers of the potential for containing and reversing the emerging problem of urban malaria.
==== Body
Introduction
Africa's population will almost triple by the year 2050. This expansion will occur primarily in urban areas and by 2025, 800 million people will live in urban communities. Especially affected will be West Africa, where the urban population annual growth rate of 6.3% is more than twice the rate of the total population growth. Today in the humid forest zone, more people live in cities than in rural areas and in twenty years time, two out of three West Africans will live in urban centres. While many of Africa's health problems are common to both urban and rural environments, recognizing and meeting the public health challenges in these growing cities is becoming increasingly urgent. Malaria has been considered a predominantly rural disease in Africa, primarily because suitable vector breeding sites are scarce in highly populated areas. Yet, although studies have shown that Anopheles mosquito breeding decreases with increasing proximity to the centre of urban areas [1-3], transmission of malaria still occurs. Clearly, the complex factors contributing to malaria risk in urban areas are not fully understood [3] but evidence is rapidly accumulating that the urban poor are at far higher risk from malaria than previously acknowledged [4,5].
The Malaria Knowledge Programme of the Liverpool School of Tropical Medicine and the International Water Management Institute/ Systemwide Initiative on Malaria and Agriculture convened a meeting in Pretoria 2nd-4th December 2004 to develop an evidence-based approach for evaluating and controlling urban malaria. Participants were drawn from seven sub-Saharan countries, Europe, North America and South Asia (see additional file). Recognizing the need for extensive cross-sectoral involvement and collaboration in dealing with the challenge of urban malaria, representatives from the research/ academic, NGO, development, policy-making and donor communities co-operated in the process to identify key knowledge gaps and opportunities for control. Included in the group were sociologists, clinical epidemiologists, entomologists and control specialists.
Discussion
Identifying the populations at risk in urban areas
Urbanization is a recent phenomenon in Africa: in 1960 there were no African cities with one million inhabitants, today there are forty. Has malaria become a serious problem within these huge cities and their peri-urban environs? Data presented from studies in a number of sub-Saharan African cities (Brazzavile, Congo; Dakar, Senegal; Abidjan, Cote d'Ivoire; Cotonou, Benin; Ouagadougou, Burkina Faso; Dar es Salaam, Tanzania, and Accra and Kumasi, Ghana) showed clearly that malaria is a considerable urban health problem in Africa. The studies demonstrated great heterogeneities in malariometric indices both between and within cities. It was recognized that not only the major cities of Africa, but also many medium sized regional towns, home to a large proportion of the Africa population, have considerable levels of malaria [5]. With malaria risk unevenly distributed across urban environments, interventions must be preceded by the identification and prioritization of the most vulnerable. Vulnerability is not simply the result of low socio-economic status [6], although this is often a major contributory factor, but reflects factors beyond the individual level such as the proximity of the household to sites of urban agriculture or environmental/cultural factors working at the community level. Discussion focussed on research to define this risk, to improve access to correct diagnosis and appropriate treatment and effective preventative measures, and to identify accurate monitoring and evaluation tools tailored to the urban context.
Prioritizing improved diagnosis and treatment for the vulnerable
Misdiagnosis of malaria is a serious problem everywhere, but in areas of low malaria endemicity presumptive treatment of all fevers as malaria can result in over 75% of cases being misdiagnosed as malaria [7]. The effect of malaria misdiagnosis on the vulnerable will result in more ill health due to delayed diagnosis and repeat visits, overburdened health services, more severe malaria, loss of faith in health services, increase in real and perceived malaria resistance, chronic disease secondary to untreated infection, increased cost to patient and to health facilities and consistent misdiagnosis that will encourage detrimental health-seeking behaviour [7].
Effective provision of appropriate treatment also remains a serious challenge in urban settings. The Abuja Declaration stated that by 2005 "At least 60% of those suffering from malaria have prompt access to and are able to use correct, affordable and appropriate treatment within 24 hours of onset of symptoms." Despite the fact that access to quality health care is better on average in urban compared to rural zones, the formal public health facilities are often the last source of treatment used along the pathway to cure. Often malaria care initially involves leftover medicines from the home (from previously incomplete malaria or other treatment regimes), the purchase of cheaper herbal medicines or unprescribed conventional medicines. The problems of obtaining treatment from a health facility may be exacerbated by the need to obtain permission from an authority figure, absence from work and loss of income, the need to raise money to fund both the treatment and associated costs such as travel [6]. As a result, in Africa over 70% of malaria episodes in rural and over 50% in urban areas are self-diagnosed and self-treated [8]. With Home Management of Malaria proposed as an integral part of the Roll Back Malaria strategy, the consequences of presumptive treatment policies for malaria in the context of the introduction of newer and more expensive anti-malarial drug combinations urgently require further investigation [9].
Ensuring malaria prevention measures reach the vulnerable
The highly focal nature of urban malaria requires targeting of interventions to specific urban districts, and therefore, requires detailed information on each area in advance. However, relationships between administrative boundaries, environment and population distribution are complex in urban areas, which makes them difficult to sample and characterize in a representative way. Strategies for population-representative sampling must incorporate a range of environments and populations to identify accurately environmental and other risk factors. This may be further complicated as urban populations can be highly mobile and in peri-urban areas there may be a high rate of turnover in groups of lower socio-economic status. Presentations from South, East and West Africa clearly demonstrated that Geographic Information Systems (GIS)-based approaches are valuable tools for assessing heterogeneities in risk factors for urban malaria, and for subsequent implementation and monitoring of interventions.
Experienced researchers believe that the urban environment has advantages for the effective delivery of appropriate interventions. A number of studies have demonstrated that higher rates of coverage with insecticide-treated bednets can be achieved in urban areas [10,11], although whether or not the most vulnerable groups benefit, remains to be confirmed. Moreover, there is a growing realization within the commercial sector of the need to engage in health and broader social issues. The management of malaria can bring economic benefits to both businesses and the communities in which they operate. This has been powerfully demonstrated in two public-private partnership programmes in southern Africa that utilised indoor residual spraying to control malaria [12,13].
Larval control, achieved either by source reduction or larviciding, can be community directed and may be feasible in certain settings as part of a comprehensive, integrated vector management strategy. There is optimism in some communities about its efficacy and the results of further research into the costs and benefits of such interventions are awaited with interest. Environmental modifications may also be feasible if partners from the community and outside the health sector are engaged. Work from Sri Lanka has demonstrated how a very effective scheme to control malaria by modification of irrigation structures was accepted by the agricultural community because of the financial and water savings that the scheme introduced [14]. However, it was clear that obvious benefits from the intervention must exist to attract the involvement of non-health sectors.
Conclusions
The conclusions of the meeting have been summarised in the Pretoria Statement on Urban Malaria (Figure 1). While it is clear that urban malaria represents a major challenge for public health in Africa, the statement highlights that the unique nature of the urban environment provides an opportunity for malaria control. There are a number of reasons for this: the high population density in urban areas may facilitate increased coverage and impact of both interventions and health education programmes; the activities of departments in urban municipal authorities are typically better resourced and more easily mobilized than in rural areas; the extensive private health sector found in urban settings can be engaged to improve diagnosis, treatment and prevention of malaria. Solutions to the urban malaria problem must include groups from outside the health sector. The disease burden in the most vulnerable communities is a major obstacle to the economic growth of sub-Saharan countries and the challenge is to engage stakeholders at all levels in effective and sustainable intersectoral collaboration [15]. Urban malaria is uniquely amenable to prevention and control as the existing health, urban planning, agricultural and governance structures present opportunities for collaborative approaches that can include both the community and the substantial private sector.
Figure 1 The Pretoria Statement on Urban Malaria.
Authors' contributions
All authors participated as session chairs in the technical workshop and were instrumental in producing the summary conclusions. All authors read and approved the final manuscript.
Supplementary Material
Additional File 1
List of attendees and affiliations
Click here for file
Acknowledgements
We acknowledge the contribution of all workshop participants. The workshop was funded by the DfID-funded Malaria Knowledge Programme of the Liverpool School of Tropical Medicine, the Environmental Health Project (USAID) and the International Development Research Centre (Canada). However, The Department for International Development of the UK government can accept no responsibility for any information or views expressed herein and the content of the commentary is solely the responsibility of the authors.
==== Refs
Coene J Malaria in urban and rural Kinshasa: the entomological input Med Vet Entomol 1993 7 127 37 8481529
Warren M Billig P Bendahmane DB Wijeyaratne P Malaria in urban and peri – urban areas in sub -Sahara Africa EHP activity report 71 1999
Robert V Macintyre K Keating J Trape JF Duchemin JB Warren M Beier JC Malaria transmission in urban sub-Saharan Africa Am J Trop Med Hyg 2003 68 169 76 12641407
Klinkenberg E McCall PJ Hastings IM Wilson MD Amerasinghe FP Donnelly MJ High malaria prevalence and irrigated urban agriculture in Accra, Ghana [abstract] Am J Trop Med Hyg 2004 9
Keiser J Utzinger J de Castro MC Smith TA Tanner M Singer BH Urbanization in sub-saharan Africa and implication for malaria control Am J Trop Med Hyg 2004 118 127 15331827
Bates I Fenton C Gruber J Lalloo D Lara AM Squire SB Theobald S Thomson R Tolhurst R Vulnerability to malaria, tuberculosis, and HIV/AIDS infection and disease. Part 1: determinants operating at individual and household level Lancet Infect Dis 2004 4 267 277 15120343 10.1016/S1473-3099(04)01002-3
Amexo M Tolhurst R Barnish G Bates I Malaria misdiagnosis: effects on the poor and vulnerable Lancet 2004 364 1896 1898 15555670 10.1016/S0140-6736(04)17446-1
McCombie SC Treatment seeking for malaria: a review of recent research Soc Sci Med 1996 43 933 945 8888463 10.1016/0277-9536(95)00446-7
D'Alessandro U Talisuna A Boelaert M Should artemisinin-based combination treatment be used in the home-based management of malaria? Trop Med Int Health 2005 10 1 2 15655007 10.1111/j.1365-3156.2004.01375.x
Feilden RM Lengeler C, Cattani J, de Savigny D Experiences of implementation. in Net Gain: A New Method for Preventing Malaria Deaths 1996 IDRC
Holtz TH Marum LH Mkandala C Chizani N Roberts JM Macheso A Parise ME Kachur SP Insecticide-treated bednet use, anaemia, and malaria parasitaemia in Blantyre District, Malawi Trop Med Int Health 2002 7 220 230 11903984 10.1046/j.1365-3156.2002.00846.x
Conteh L Sharp BL Streat E Barreto A Konar S The cost and cost-effectiveness of malaria vector control by residual insecticide house-spraying in southern Mozambique: a rural and urban analysis Trop Med Int Health 2004 9 125 132 14728616 10.1046/j.1365-3156.2003.01150.x
Sharp B van Wyk P Sikasote JB Banda P Kleinschmidt I Malaria control by residual insecticide spraying in Chingola and Chililabombwe, Copperbelt Province, Zambia Trop Med Int Health 2002 7 732 736 12225502 10.1046/j.1365-3156.2002.00928.x
Konradsen F Matsuno Y Amerasinghe FP Amerasinghe PH van der Hoek W Anopheles culicifacies breeding in Sri Lanka and options for control through water management Acta Trop 1998 71 131 138 9821462 10.1016/S0001-706X(98)00060-6
Macroeconomics and Health: Investing in Health for Economic Development 2001 WHO, Geneva
| 15720713 | PMC552321 | CC BY | 2021-01-04 16:37:30 | no | Malar J. 2005 Feb 18; 4:12 | utf-8 | Malar J | 2,005 | 10.1186/1475-2875-4-12 | oa_comm |
==== Front
Malar JMalaria Journal1475-2875BioMed Central London 1475-2875-4-121572071310.1186/1475-2875-4-12CommentaryMalaria and urbanization in sub-Saharan Africa Donnelly Martin J [email protected] PJ [email protected] Christian [email protected] Imelda [email protected]'Alessandro Umberto [email protected] Guy [email protected] Flemming [email protected] Eveline [email protected] Harold [email protected] Jean-Francois [email protected] Ian M [email protected] Clifford [email protected] Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA. UK2 Swiss Tropical Institute, P.O. Box, 4002 Basel, Switzerland3 Prince Leopold Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium4 International Water Management Institute Sri Lanka127, Sunil Mawatha, Pelawatte, Battaramulla, Sri Lanka5 Institute of Public Health, Department of International Health, University of Copenhagen, Blegdamsvej 32200 København, Denmark6 International Water Management Institute (West Africa), PMB CT 112, Cantonments, Accra, Ghana7 Institut de Recherche pour le Développement, BP 1386, CP 18524, Dakar, Sénégal8 Systemwide Initiative on Malaria and Agriculture, International Water Management Institute, Private Bag X813, Silverton 0127, South Africa2005 18 2 2005 4 12 12 2 2 2005 18 2 2005 Copyright © 2005 Donnelly et al; licensee BioMed Central Ltd.2005Donnelly et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
There are already 40 cities in Africa with over 1 million inhabitants and the United Nations Environmental Programme estimates that by 2025 over 800 million people will live in urban areas. Recognizing that malaria control can improve the health of the vulnerable and remove a major obstacle to their economic development, the Malaria Knowledge Programme of the Liverpool School of Tropical Medicine and the Systemwide Initiative on Malaria and Agriculture convened a multi-sectoral technical consultation on urban malaria in Pretoria, South Africa from 2nd to 4th December, 2004. The aim of the meeting was to identify strategies for the assessment and control of urban malaria. This commentary reflects the discussions held during the meeting and aims to inform researchers and policy makers of the potential for containing and reversing the emerging problem of urban malaria.
==== Body
Introduction
Africa's population will almost triple by the year 2050. This expansion will occur primarily in urban areas and by 2025, 800 million people will live in urban communities. Especially affected will be West Africa, where the urban population annual growth rate of 6.3% is more than twice the rate of the total population growth. Today in the humid forest zone, more people live in cities than in rural areas and in twenty years time, two out of three West Africans will live in urban centres. While many of Africa's health problems are common to both urban and rural environments, recognizing and meeting the public health challenges in these growing cities is becoming increasingly urgent. Malaria has been considered a predominantly rural disease in Africa, primarily because suitable vector breeding sites are scarce in highly populated areas. Yet, although studies have shown that Anopheles mosquito breeding decreases with increasing proximity to the centre of urban areas [1-3], transmission of malaria still occurs. Clearly, the complex factors contributing to malaria risk in urban areas are not fully understood [3] but evidence is rapidly accumulating that the urban poor are at far higher risk from malaria than previously acknowledged [4,5].
The Malaria Knowledge Programme of the Liverpool School of Tropical Medicine and the International Water Management Institute/ Systemwide Initiative on Malaria and Agriculture convened a meeting in Pretoria 2nd-4th December 2004 to develop an evidence-based approach for evaluating and controlling urban malaria. Participants were drawn from seven sub-Saharan countries, Europe, North America and South Asia (see additional file). Recognizing the need for extensive cross-sectoral involvement and collaboration in dealing with the challenge of urban malaria, representatives from the research/ academic, NGO, development, policy-making and donor communities co-operated in the process to identify key knowledge gaps and opportunities for control. Included in the group were sociologists, clinical epidemiologists, entomologists and control specialists.
Discussion
Identifying the populations at risk in urban areas
Urbanization is a recent phenomenon in Africa: in 1960 there were no African cities with one million inhabitants, today there are forty. Has malaria become a serious problem within these huge cities and their peri-urban environs? Data presented from studies in a number of sub-Saharan African cities (Brazzavile, Congo; Dakar, Senegal; Abidjan, Cote d'Ivoire; Cotonou, Benin; Ouagadougou, Burkina Faso; Dar es Salaam, Tanzania, and Accra and Kumasi, Ghana) showed clearly that malaria is a considerable urban health problem in Africa. The studies demonstrated great heterogeneities in malariometric indices both between and within cities. It was recognized that not only the major cities of Africa, but also many medium sized regional towns, home to a large proportion of the Africa population, have considerable levels of malaria [5]. With malaria risk unevenly distributed across urban environments, interventions must be preceded by the identification and prioritization of the most vulnerable. Vulnerability is not simply the result of low socio-economic status [6], although this is often a major contributory factor, but reflects factors beyond the individual level such as the proximity of the household to sites of urban agriculture or environmental/cultural factors working at the community level. Discussion focussed on research to define this risk, to improve access to correct diagnosis and appropriate treatment and effective preventative measures, and to identify accurate monitoring and evaluation tools tailored to the urban context.
Prioritizing improved diagnosis and treatment for the vulnerable
Misdiagnosis of malaria is a serious problem everywhere, but in areas of low malaria endemicity presumptive treatment of all fevers as malaria can result in over 75% of cases being misdiagnosed as malaria [7]. The effect of malaria misdiagnosis on the vulnerable will result in more ill health due to delayed diagnosis and repeat visits, overburdened health services, more severe malaria, loss of faith in health services, increase in real and perceived malaria resistance, chronic disease secondary to untreated infection, increased cost to patient and to health facilities and consistent misdiagnosis that will encourage detrimental health-seeking behaviour [7].
Effective provision of appropriate treatment also remains a serious challenge in urban settings. The Abuja Declaration stated that by 2005 "At least 60% of those suffering from malaria have prompt access to and are able to use correct, affordable and appropriate treatment within 24 hours of onset of symptoms." Despite the fact that access to quality health care is better on average in urban compared to rural zones, the formal public health facilities are often the last source of treatment used along the pathway to cure. Often malaria care initially involves leftover medicines from the home (from previously incomplete malaria or other treatment regimes), the purchase of cheaper herbal medicines or unprescribed conventional medicines. The problems of obtaining treatment from a health facility may be exacerbated by the need to obtain permission from an authority figure, absence from work and loss of income, the need to raise money to fund both the treatment and associated costs such as travel [6]. As a result, in Africa over 70% of malaria episodes in rural and over 50% in urban areas are self-diagnosed and self-treated [8]. With Home Management of Malaria proposed as an integral part of the Roll Back Malaria strategy, the consequences of presumptive treatment policies for malaria in the context of the introduction of newer and more expensive anti-malarial drug combinations urgently require further investigation [9].
Ensuring malaria prevention measures reach the vulnerable
The highly focal nature of urban malaria requires targeting of interventions to specific urban districts, and therefore, requires detailed information on each area in advance. However, relationships between administrative boundaries, environment and population distribution are complex in urban areas, which makes them difficult to sample and characterize in a representative way. Strategies for population-representative sampling must incorporate a range of environments and populations to identify accurately environmental and other risk factors. This may be further complicated as urban populations can be highly mobile and in peri-urban areas there may be a high rate of turnover in groups of lower socio-economic status. Presentations from South, East and West Africa clearly demonstrated that Geographic Information Systems (GIS)-based approaches are valuable tools for assessing heterogeneities in risk factors for urban malaria, and for subsequent implementation and monitoring of interventions.
Experienced researchers believe that the urban environment has advantages for the effective delivery of appropriate interventions. A number of studies have demonstrated that higher rates of coverage with insecticide-treated bednets can be achieved in urban areas [10,11], although whether or not the most vulnerable groups benefit, remains to be confirmed. Moreover, there is a growing realization within the commercial sector of the need to engage in health and broader social issues. The management of malaria can bring economic benefits to both businesses and the communities in which they operate. This has been powerfully demonstrated in two public-private partnership programmes in southern Africa that utilised indoor residual spraying to control malaria [12,13].
Larval control, achieved either by source reduction or larviciding, can be community directed and may be feasible in certain settings as part of a comprehensive, integrated vector management strategy. There is optimism in some communities about its efficacy and the results of further research into the costs and benefits of such interventions are awaited with interest. Environmental modifications may also be feasible if partners from the community and outside the health sector are engaged. Work from Sri Lanka has demonstrated how a very effective scheme to control malaria by modification of irrigation structures was accepted by the agricultural community because of the financial and water savings that the scheme introduced [14]. However, it was clear that obvious benefits from the intervention must exist to attract the involvement of non-health sectors.
Conclusions
The conclusions of the meeting have been summarised in the Pretoria Statement on Urban Malaria (Figure 1). While it is clear that urban malaria represents a major challenge for public health in Africa, the statement highlights that the unique nature of the urban environment provides an opportunity for malaria control. There are a number of reasons for this: the high population density in urban areas may facilitate increased coverage and impact of both interventions and health education programmes; the activities of departments in urban municipal authorities are typically better resourced and more easily mobilized than in rural areas; the extensive private health sector found in urban settings can be engaged to improve diagnosis, treatment and prevention of malaria. Solutions to the urban malaria problem must include groups from outside the health sector. The disease burden in the most vulnerable communities is a major obstacle to the economic growth of sub-Saharan countries and the challenge is to engage stakeholders at all levels in effective and sustainable intersectoral collaboration [15]. Urban malaria is uniquely amenable to prevention and control as the existing health, urban planning, agricultural and governance structures present opportunities for collaborative approaches that can include both the community and the substantial private sector.
Figure 1 The Pretoria Statement on Urban Malaria.
Authors' contributions
All authors participated as session chairs in the technical workshop and were instrumental in producing the summary conclusions. All authors read and approved the final manuscript.
Supplementary Material
Additional File 1
List of attendees and affiliations
Click here for file
Acknowledgements
We acknowledge the contribution of all workshop participants. The workshop was funded by the DfID-funded Malaria Knowledge Programme of the Liverpool School of Tropical Medicine, the Environmental Health Project (USAID) and the International Development Research Centre (Canada). However, The Department for International Development of the UK government can accept no responsibility for any information or views expressed herein and the content of the commentary is solely the responsibility of the authors.
==== Refs
Coene J Malaria in urban and rural Kinshasa: the entomological input Med Vet Entomol 1993 7 127 37 8481529
Warren M Billig P Bendahmane DB Wijeyaratne P Malaria in urban and peri – urban areas in sub -Sahara Africa EHP activity report 71 1999
Robert V Macintyre K Keating J Trape JF Duchemin JB Warren M Beier JC Malaria transmission in urban sub-Saharan Africa Am J Trop Med Hyg 2003 68 169 76 12641407
Klinkenberg E McCall PJ Hastings IM Wilson MD Amerasinghe FP Donnelly MJ High malaria prevalence and irrigated urban agriculture in Accra, Ghana [abstract] Am J Trop Med Hyg 2004 9
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| 0 | PMC552322 | CC BY | 2021-01-04 16:37:34 | no | Biomed Eng Online. 2005 Feb 10; 4:8 | latin-1 | Biomed Eng Online | 2,005 | 10.1186/1475-925X-4-8 | oa_comm |
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J NanobiotechnologyJournal of Nanobiotechnology1477-3155BioMed Central London 1477-3155-3-21571793510.1186/1477-3155-3-2ResearchRapid self-assembly of DNA on a microfluidic chip Zheng Yao [email protected] Tim [email protected] Dammika P [email protected] Christopher James [email protected] Department of Electrical and Computer Engineering, 2nd Floor, ECERF Building (9107 – 116St.) University of Alberta, Edmonton, Alberta, T6G 2V4 Canada2 Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada2005 18 2 2005 3 2 2 21 7 2004 18 2 2005 Copyright © 2005 Zheng et al; licensee BioMed Central Ltd.2005Zheng et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
DNA self-assembly methods have played a major role in enabling methods for acquiring genetic information without having to resort to sequencing, a relatively slow and costly procedure. However, even self-assembly processes tend to be very slow when they rely upon diffusion on a large scale. Miniaturisation and integration therefore hold the promise of greatly increasing this speed of operation.
Results
We have developed a rapid method for implementing the self-assembly of DNA within a microfluidic system by electrically extracting the DNA from an environment containing an uncharged denaturant. By controlling the parameters of the electrophoretic extraction and subsequent analysis of the DNA we are able to control when the hybridisation occurs as well as the degree of hybridisation. By avoiding off-chip processing or long thermal treatments we are able to perform this hybridisation rapidly and can perform hybridisation, sizing, heteroduplex analysis and single-stranded conformation analysis within a matter of minutes. The rapidity of this analysis allows the sampling of transient effects that may improve the sensitivity of mutation detection.
Conclusions
We believe that this method will aid the integration of self-assembly methods upon microfluidic chips. The speed of this analysis also appears to provide information upon the dynamics of the self-assembly process.
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Background
There has been a rapid growth in the number of applications that are based upon DNA self-assembly, ranging from DNA microarrays (e.g. Affymetrix [1]) in the life sciences, through conformation-based mutation detection methods [2,3], to the ongoing development of DNA scaffolding methods of nanoassembly [4]. The control of the degree of DNA hybridisation requires elaborate and time consuming sample preparation (eg [5]) with methods that may require hours to achieve hybridisation [6], and on the order of an hour even within miniaturised systems [1,7]. However, a rapid method of controlling denaturation and renaturation within a microfluidic device would enable an inexpensive mutation detection method that could be performed within minutes.
Microfluidic devices or 'microchips' are photolithographically-defined networks of microchannels in glass where the microchannels are similar in size to conventional capillaries. These microchips provide compelling advantages in terms of speed, reagent usage and integration over conventional capillary or gel-based methods. The potential of the microfluidic chip has led to the use of terms such as "micro-total analysis systems" and "lab-on-a-chip". These microchips have been demonstrated in conjunction with a range of applications that integrate the polymerase chain reaction (PCR) and capillary electrophoresis (CE) methods with some reaching nanolitre or smaller scale volumes. A powerful advantage of the microchip approach is that it can implement much the same molecular biology protocols and reagents as used with conventional equipment, thereby allowing a wealth of established expertise to be transferred to the microscale.
Although the most effective method of mutation detection is sequencing, it is also by far the most expensive [8]. The microarray [8] technique, although powerful, is still handicapped by significant false positive rates and high cost [9]. Alternative methods based on DNA self-assembly are much faster than sequencing and these include single-strand conformation polymorphism (SSCP), denaturing high performance liquid chromatography (DHPLC) and heteroduplex analysis (HA). Although their cost has been shown to be far lower than sequencing, the achievable sensitivities (the percentage of mutations that are successfully detected) are only about 90 % [10,9].
Microfluidic chips may enable extremely high throughputs and high levels of integration. The achievement of this goal has been hindered by the lack of successful integrations of methods of mutation analysis based on single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) – likely due to the difficulties in controlling the degree of hybridisation on chip without time consuming thermal processing. A great advantage would be provided by a method of enabling microchip-based control of a rapid DNA self-assembly process.
The term wildtype is used to describe any given genetic sequence that does not contain mutations. Since individuals usually carry two copies of each gene, the genetic sequence of the two copies may be identical (homozygous) or may differ (heterozygous). DNA is normally double stranded, but under some conditions (e.g. high temperature), melts into single strands. Under other conditions, such as a lower temperature, these single strands will self-assemble into the double-stranded form again. The resulting double-stranded DNA is referred to as a homoduplex if the sequences are perfectly complementary, or a heteroduplex if the sequences are nearly complementary (i.e. a mutant sequence paired with a wildtype sequence). The misfit in a heteroduplex creates a "bulge" or "bubble" where the bases do not match and this affects the shape of the assembled molecule, typically lowering its velocity during electrophoretic movement, i.e. the heteroduplexes typically migrate more slowly than the homoduplexes. Any heterozygous sample will generate four different duplexes, two homoduplexes and two heteroduplexes, although the molecules often co-migrate so that fewer than 4 separate electropherogram peaks are resolved.
In the HA method, electrophoretic conditions are chosen in order to enhance the velocity differences between the duplexes so that the process of duplex self-assembly can be used to determine the presence of a heterozygous state (hence indicating the presence of a mutation). In SSCP, isolated strands of ssDNA find near-complementary sequences on the same strand, with the result that the strand folds upon itself in a sequence dependent manner forming new conformations. This is a simplistic description since ssDNA without self-similar sequences, and homoduplex dsDNA, may also take complex forms. Techniques such as HA that aim to separate homoduplex fragments from heteroduplex fragments often use some combination of thermally and chemically denaturing conditions to cause the partial melting of the duplex, resulting in a shift in mobility or chromatography column retention time that increases with the degree of mismatch.
Many medical diagnostics could be implemented on microchips if an effective implementation of a highly sensitive mutation analysis method could be integrated with PCR/CE. Considerable work has been done in developing SSCP [11] and HA [2,3,12]. An excellent review of such methods has been produced by Jin et al. [13]. The main drawback is the lower sensitivity of these methods. In macroscopic work Kozlowski and Krzyzosiak [5] and Kourkine et al. [14] greatly improved their sensitivities by combining SSCP and HA methods to develop capillary-based electrophoretic techniques with sensitivities of 90–94 % for SSCP and 75–81 % for HA. In a landmark analysis, Kourkine et al. achieved 100 % sensitivity by analysing denatured and non-denatured fragments in tandem. Despite being highly effective, the additional sample preparation required by these methods (i.e. separately preparing both single and double stranded DNA and maintaining this strandedness) complicates their implementation on microchips.
In this work, we present an electrophoretic method in which DNA is denatured in a microchip (with formamide) and, depending upon the sequence of applied voltages, can be prepared with a widely varying degree of hybridisation (i.e. from almost entirely ssDNA to almost entirely dsDNA). Given the small volumes involved within the microchip, diffusion time plays a small role and the reassembly process can be fast, with dsDNA obtained within minutes. The rapidity of the manipulation possible on this system allows some investigation of the dynamics of the reassembly, indicating that there are well-defined intermediate states where both ssDNA and dsDNA exist in the reassembly process.
We have applied our methods to the H63D and S65C mutations from the HFE gene associated with hereditary hemochromatosis (HH). The denaturation technique used enables a combined microchip-based method of HA and SSCP analysis.
Results
Heteroduplex Analysis
In our electrophoretic analyses with a double-T chip (described below), the dsDNA arrives at the detection point before the ssDNA (after about 105 s of separation, versus 190 s of separation for the ssDNA). As shown in Fig. 1, to demonstrate that our analysis conditions allow for the detection of mutations by means of HA, we analysed (undenatured) PCR products of a homozygous wildtype, a heterozygous and a homozygous H63D mutant. We found, as expected, that the heterozygous sample had two distinct peaks due to the transport of heteroduplexes as well as homoduplexes. However, the wildtype and homozygous mutant samples looked very similar, with the exception of a small peak following the main peak of the mutant sample. This small peak was only apparent with this sample and seems to indicate a PCR artefact. The size and shape of the bump remained consistent throughout the experiments and did not affect the peak intensities of either dsDNA or ssDNA. The bump is too small to add ambiguity when resolving the H63D mutation by HA and it should be noted that the emphasis of this work is on inducing the formation of dsDNA and ssDNA on-chip rather than upon improving mutation detection.
Figure 1 Double-stranded DNA peak profiles prior to the addition of formamide (fluorescence in relative fluorescence units (RFU) vs. time). a) wildtype, b) homozygous H63D mutant, c) heterozygous H63D mutant.
Simultaneous Analysis of ssDNA and dsDNA
As expected, after the addition of formamide the electropherograms showed the presence of ssDNA peaks in addition to the dsDNA peaks (Fig. 2). The dsDNA profiles seen here are identical to those seen prior to the addition of formamide (Fig 1). The ssDNA peaks show differences in relative peak heights and in peak profile – and most notably the heterozygous sample shows a clefted peak. A comparison of the ssDNA profiles for the wildtype, homozygous mutant and heterozygous mutant would constitute a demonstration of SSCP analysis. Although the relative spacing of the ssDNA peaks differs between the wildtype and homozygous mutant, the most obvious difference is the clefted peak seen in the electropherogram of the heterozygous sample. This clefted peak was not present in the corresponding profiles of the homozygous samples (neither wildtype nor mutant). Under these conditions of electrophoresis, the mutational status of H63D is readily apparent. We have developed a combined HA and SSCP method and will report on it elsewhere (that report includes the detection of the common C282Y mutation). To our knowledge this is the first report of a method for performing combined on-chip HA and SSCP. Our emphasis here is on the ability to achieve rapid denaturation and renaturation processes on-chip.
Figure 2 Electropherograms of H63D (ss and ds DNA) following addition of formamide (fluorescence in relative fluorescence units (RFU) vs. time). a) wildtype, b) homozygous H63D mutant, and c) heterozygous H63D mutant.
Reassembly of dsDNA
In order to confirm that we are reassembling DNA on chip rather than denaturing to varying degrees we investigated the on-chip production of heteroduplexes from two samples of homoduplexes (i.e. homozygous) samples. Fig. 3a) shows the results of the analysis of a mix of the dsDNA from a homozygous H63D and its corresponding wildtype. This first analysis (done without the addition of formamide) showed a peak profile similar to that seen for the pure wildtype or homozygous mutant in Fig. 1 – i.e. no heteroduplexes are evident. We then added formamide to form a mixture in the sample well of homozygous mutant ssDNA with wildtype ssDNA. Once electrophoretically extracted from the sample well, the ssDNA reanneals to form heteroduplex mutants for analysis. As expected, the dsDNA profile of Fig. 3b) is that of the heteroduplex profile seen in Fig. 1 (the signal to noise ratio of this electropherogram is low because the sample is still primarily ssDNA). This indicates that the DNA is extracted from the formamide-rich sample well as ssDNA and reassembles to dsDNA in the microchip channels.
Figure 3 Double-stranded DNA peak profile of the mixture of H63D homozygous mutant with wildtype prior to and after the addition of formamide (fluorescence in relative fluorescence units (RFU) vs. time). a) prior to and b) after
The reassembly of ssDNA can also be shown by denaturating the wildtype and homozygous mutant in separate wells. The two denatured samples were injected simultaneously and their ssDNA mixed in the injection channel of a Y-chip (described below). Subsequent separation and detection showed peak profiles (Fig. 4) similar to those obtained with the heterozygous mutant for both HA and SSCP. This suggests that the method of denaturation used here is a powerful tool for comparing test samples, either in the same or in separate sample wells. The testing of the wildtype, homozgyous and heterozygous mutants could be conducted by injecting samples from the desired wells without reloading the chip. This would greatly improve throughput.
Figure 4 Separate injections of wildtype and homozygous H63D samples recombining on-chip (fluorescence in relative fluorescence units (RFU) vs. time)
As will be described in the following section, by varying the electrophoretic parameters we can control the relative amount of dsDNA formed – a significantly larger amount could be obtained.
Dynamics of DNA Reassembly
After demonstrating that reassembly occurred within the microchannels after extraction from the formamide-rich sample well, it was of interest to investigate how the sequence and timing of the sample extraction and analysis might affect the degree of rehybridisation. In the work presented thus far we used a 60 s injection (although 20 s would probably have sufficed) as a means of drawing sample directly from the sample well to the intersection, from whence it could be analysed.
After the addition of formamide and an analysis of the resulting sample (60 s injection and 180 s separation), a series of analyses were performed wherein each short injection (10 s) with a lower electric field was followed by a 180 s separation. These short injections sampled DNA that had remained in the microchannel since its extraction during the 60 s injection from the first analysis. The time required for the DNA to travel from the sample well to the intersection with the applied electric field during the 10 s short injections was calculated to be approximately 53 s. (The short injections are carried out at a lower field than the initial injection.) Thus, the two short injections of 10 s each were not enough to bring in fresh samples from the sample well. Fig. 5 indicates that after the initial 60 s injection the dsDNA concentration steadily increases as rehybridization occurs in the microchannel. Depending on extraction timing (e.g. short injections vs. longer), the relative intensities of the ssDNA and the dsDNA can be varied by a factor of approximately 10, ranging from primarily ssDNA to primarily dsDNA. Further optimisation is possible with changes in microchip geometry. (Shorter injection channels would allow for more ssDNA to be introduced).
Figure 5 Electropherograms after successive short injections of H63D heterozygous mutant DNA that show the change of ssDNA to dsDNA in the channels after leaving the formamide-rich environment of the sample well. (fluorescence in relative fluorescence units (RFU) vs. time). a) H63D immediately after a 60 s injection. b) H63D after a subsequent 10 s injection c) H63D after a second subsequent 10 s injection
Another interesting feature of Fig. 5 is that following the addition of formamide, the first peak of the ssDNA (marked *) seen in the first analysis after a 60 s (Fig. 5a) injection is never present after a subsequent 10 s injection (Fig. 5b) although it can be recovered by another 60 s injection (not shown). The strength of this peak is strongly dependent upon the sample tested (as discussed below). This interesting phenomenon was observed with wildtype, homozygous and heteroduplex samples corresponding to H63D and S65C (data not shown) and the transient peak was clefted for heterozygous S65C (Fig. 6) and not clefted for H63D (Fig. 5a). It appears that this intermediate state may be used to investigate the dynamics of reassembly by a rapid microchip-based method.
Figure 6 Electropherogram (ss and ds DNA) after initial 60 s injection of S65C heterozygous mutant DNA (fluorescence in relative fluorescence units (RFU) vs. time).
Discussion
The integration onto a microchip of an effective means of mutation detection is perhaps one of the most important technological barriers to the implementation of microchip-based medical diagnostics. The best means of attaining sufficiently high sensitivity is by integrating several existing methods of microchip-based mutation detection. The capillary-based analysis procedure developed by Kourkine et al. [14] is likely to be highly effective in conjunction with the microchip analysis of prepared samples, but since the procedure is based upon the thermal processing (95°C and snap cooling) of diluted PCR products, the integration of this processing onto the microchip may be problematic. The present method allows for such integrations, thereby enabling the mutation analysis throughputs predicted by Medintz et al. [15] – throughputs as much as 100 times higher that those presently attainable. Another issue is that of signal to noise ratios – rather than dilute our sample (possibly weakening its signal strength) we can analyse the sample essentially undiluted. Moreover, we can enhance the signal strength, as we choose, for either the ssDNA or the dsDNA.
As demonstrated here, this method also allows on-chip comparisons of one type of DNA with another. A common problem encountered with HA methods is that they cannot distinguish homozygous mutant from homozygous wildtype – the present technique would allow an on-chip comparison of these samples to produce heteroduplexes that will then indicate the mutational status.
The on chip denaturation is produced through the addition of formamide. The melting temperature for this sequence of DNA following the addition of formamide was found to be approximately room temperature, as determined by
Tm = 81.5 + 16.6(log M) + 0.41 (% G + C) - 0.72 (% formamide) (1)
where Tm is the melting temperature in degrees Celsius, M is the monovalent salt molarity, (% G + C) is the percent of the guanine and cytosine in the DNA strand of interest, and (% formamide) is the percentage of formamide added [16]. The melting of DNA was confirmed by forming heteroduplexes on-chip.
The ability to quickly re-hybridise on chip allows for rapid investigation of self-assembly mechanisms. In addition, this re-hybridisation enables the formation of duplexes made from a sample and a set of DNA references – i.e. DNA self-assembly within a microchip could be used to form duplexes that, under electrophoretic analysis, would show the results of comparing the sample DNA with each type of DNA in the reference set. This could avoid the need for DNA sequencing.
The rapidity of our method appears to provide additional information upon short-lived conformations. Although we have added a thermal re-annealing step as part of our PCR protocol, that step does not affect the results of analysis after adding formamide – i.e. by re-annealing on-chip the thermal reannealing is not needed. The thermal re-annealing stage was added to allow the direct comparison of heterozygous samples from the PCR with heterozygous samples after on-chip reassembly. After adding formamide, the electropherogram of the first separation analysis following any long injection shows a clearly defined transient peak. For H63D samples the transient peak is a single peak, whereas for S65C the transient peak is clefted. We have found that the transient peaks vary in size significantly depending upon the electrophoretic and PCR protocols used. Initially we had assumed that this transient peak indicated that the reassembly of the DNA was not 'random' but instead hybridised first in a high-melting point region, and only slowly thereafter. In this model, the presence of the split-peak would provide information upon the location of the mutation. This suggests that mutation S65C is within the higher melting point domain, while the H63D is not. However, as determined by the Meltmap program (generously provided by L. Lerman (MIT)), neither the H63D nor S65C mutations were within the high melting point region of the exon (data not shown).
Several research groups have reported artefacts that arise from ssDNA-primer interactions [14,17-19]. Kourkine et al. [14,18] reported that primer-ssDNA complexes can give rise to extra peaks during SSCP. They performed tests with samples of PCR-amplified DNA with and without the removal of the PCR-primers after the amplification step and found that the presence of primers led to the appearance of extra peaks [18]. A reduction in primer concentration during PCR also proved to be effective in minimizing the appearance of these peaks. Kozlowski and Krzyzosiak [19] have reported similar effects and suggested that the primer-ssDNA complex may have a different mobility simply because of its changed mass, or perhaps due to a change in conformation induced by the binding. In the context of SSCP, they discussed two approaches for dealing with this effect 1) remove it through purification so as to obtain simpler profiles or 2) use the effect to advantage by achieving higher sensitivity in the detection of mutations. Hennessy et al. [17] performed similar tests and reported that variations in primer concentration are the likely source of irreproducible SSCP profiles. They too suggested that this effect could be used to increase the sensitivity of SSCP.
We therefore suggest that the transient peak is due to the pairing of one product strand with one primer as a result of the renaturation process. The primer-ssDNA complex is primarily ssDNA with a small region of dsDNA at the end(s) of the strand. It is therefore expected to migrate with similar mobility as the ssDNA peaks. The disappearance of the transient peaks with the subsequent short injections may be a result of the complementary single strand binding and displacing the primer. However, the presence of the transient peaks may still provide useful information. The differences in the transient peaks (cleft versus no cleft) between S65C and H63D suggest that their shape may be dependent on the position of the mutation and that the position greatly affects the transport of the transient form of DNA. Thus, the phenomenon of the transient may be a general behaviour that could provide additional mutational information. In corroboration of past work by others [17,19], it therefore appears that the primer effects do provide mutational information. Moreover, this effect can be produced or avoided depending on whether the desire is to avoid the more complex profiles or to use them to achieve higher sensitivity.
Conclusion
We have developed a method of rapidly disassembling and re-assembling DNA within a microfluidic chip, allowing us control over the relative amount of ss and dsDNA and enabling the performance of rapid hybridisations under electrophoretic control. It has been reported that, when combined, HA and SSCP can provide sensitivities of 100% (e.g. [14]). In our work to date we have tested a large number of samples, predominantly of HFE, BRCA1 and BRCA2 sequences, and representing approximately several dozen different sequences. All samples containing a mutation have had their mutational status detected by at least one method. We expect then that the sensitivity of the combined methods will be close to 100%. We are now applying this method as part of a study of the application of DNA self-assembly based mutation detection methods (HA and SSCP) to the implementation of highly integrated microchips for performing medical diagnostics.
The present work is also an early step towards directing and studying DNA self-assembly within microfluidic systems. The method applied here could be improved significantly by shortening the injection and separation channels and ultimately may even assist in providing the control needed to direct the assembly of DNA-based nanosystems within microfluidic channels.
Methods
Samples
Volunteers who had given informed consent donated lymphocytes from which DNA was extracted and purified by using phenol-chloroform-isoamyl alcohol extractions [20] or the QIAmp DNA Blood kit (QIAGEN, Mississauga, ON). The purified DNA was solubilized in a Tris-EDTA buffer (TE, pH 8.0) and stored at 4°C. All genotypes were confirmed on an ABI Prism 377 Slab Gel Sequencer (Applied Biosystems, Streetsville, ON), using an ABI Prism BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit with AmpliTaq DNA Polymerase (Applied Biosystems).
The two mutations tested were H63D and S65C, from HFE Exon 2. PCR was performed on 25 μL reactions of both mutations. Thermal cycling was performed on all the samples as follows: 94 C for 2 min, 35 cycles of (94°C for 30 s, 55°C for 30 s, 72°C for 30 s), and finally 72°C for 10 min, 4°C thereafter. For H63D and S65C, the PCRs are performed with 5 μL of 30 ng/μL of genomic template DNA, 2 μL of 5 μmol/L each of HEX-HFE-2F primer and H63DR primer (Table 1), 2 μL each of 10 mmol/L dNTPs, 0.75 μL of 50 mmol/L of MgCl2, 2.5 μL of 10× PCR reaction buffer and 0.5 μL of Platinum Taq DNA Polymerase. All samples were re-annealed following PCR by first heating at 95°C for 3 min, followed by a subsequent ramping down of temperature by 1°C per minute until 65°C. The samples were then stored at -20°C.
Table 1 Primers Used for PCR
Amplicon – Primer Sequence 5' Label Final Concentration
HFE Exon 2 – forward 5'-TCA GAG CAG GAC CTT GGT CTT TCC-3' HEX 0.4 μM
HFE Exon 2 – reverse 5'-CAT ACC CTT GCT GTG GTT GTG ATT-3' N/a 0.4 μM
Reagents
PCR reagents (polymerases, buffers and primers) were obtained from Invitrogen (Burlington, ON). GeneScan™ polymer was used for microchip electrophoresis and obtained from PE Applied Biosystems (Foster City, CA). A polymer consisting of 5% GeneScan polymer and 10% glycerol (5GS10G), commonly used for SSCP, was made. Tris borate (Fisher Scientific, Fairland, NJ) with EDTA (Merck KGaA, Darmstadt, Germany) was used as the running buffer in concentrations of 1× and 0.1×. Glycerol (Sigma, Saint Louis, MO) is also added to each in 10% and 1% concentrations respectively (1 × TBE10G and 0.1 × TBE1G). De-ionised formamide (minimum 99.5%) was obtained from Sigma (F9037, Saint Louis, MO). The formamide was aliquotted and kept frozen until required.
Microchip Electrophoresis
The microchips were purchased from Micralyne (Edmonton, AB) and unless otherwise mentioned were a 4 port double T design (Fig. 7) consisting of 4 reservoirs (or wells) linked by two microchannels. One microchannel served as a separation channel approximately 80 mm in length and was nominally 50 μm wide and 20 μm deep. In order to demonstrate control of on-chip mixing we also used an 8-port Y-chip with 8 reservoirs, 2 of which are not connected by any channel and with a third reservoir connected by a 58 mm channel that was unused in this work (Fig. 8). Electrophoresis upon microchips was performed using the Microfluidic Tool Kit (μTK, Micralyne) as described previously [2], with a laser induced fluorescence (LIF) system that provides excitation at a wavelength of 532 nm and detection at 578 nm. The LIF signal was recorded by the μTK with sampling at 200 Hz and these data were recorded to a PC running a compiled LabVIEW interface (supplied by Micralyne).
Figure 7 Glass microchip (Micralyne Inc.) with double-T intersection.
Figure 8 Glass microchip (Micralyne Inc.) with Y-shaped intersecting channels.
Microchip Loading and Electrophoresis
The microchip was loaded with 5GS10G polymer without any pre-treatment. The sample well was loaded with 2.6 μL of 0.1 × TBE1G followed by 0.4 μL of DNA sample and mixed. The remaining wells were loaded with 3 μL of 1 × TBE10G. In the case of the Y-chip, 0.4 μL of wildtype DNA was added to 2.6 μL of 0.1 × TBE1G in the first sample well and mixed. The second sample well was filled with 2.6 μL of 0.1 × TBE1G and 0.4 μL of homozygous mutant. The operation of the μTK (injection and separation) was automated through the use of the LabVIEW interface. LIF detection took place 76 mm downstream from the intersection. We have found that the reproducibility of the peak arrival times is within 2 per cent from one run to the next. As such we have not needed to introduce size standards.
Injection
The sample DNA was brought from the sample well to the intersection and onto the sample waste well by applying 500 V/cm for 60 s. No initial injection was done with the Y-chip prior to denaturation. During this process the buffer and buffer waste well are left electrically disconnected. In doing so the intersection of the two (three) channels is filled with the sample DNA. This stage is referred to as an injection due to the injection of DNA into the separation channel in the sharply defined volume of the intersection of the channels.
Separation
Immediately following injection, the DNA caught within the intersection is separated by applying 714 V/cm for 180 s between the buffer and buffer waste wells. During this step, the sample and sample waste wells are left electrically disconnected. The effective separation distance was 76 mm from the intersection.
Denaturation
After the initial run on the 4-port chip, 1.5 μL of the sample mixture was removed and 1.5 μL of formamide was added and mixed. Following Howley et al. [16], this is sufficient to denature the DNA with a melting temperature of approximately 25.7°C. Since Fig. 4 clearly shows formation of heteroduplexes, we take this to indicate that the temperature was high enough to allow strands to interchange. Another run was then done with the same parameters as above. In the case of the Y-chip, denaturation of each sample was done immediately following the addition of the samples to the wells. A voltage of 400 V was applied between the sample and sample waste wells during a 60 s injection followed by a separation of 180 s. Subsequent electrophoretic runs followed with 10 s injections at 125 V/cm and 180 s of separation at 714 V/cm for both the 4-port and Y-chip. No additional mixing of the two samples for the Y-chip were required
Authors' Contributions
YZ performed the experimental work with some assistance from TF. DM performed additional protocol development. CB provided overall direction. All authors contributed to the writing of the manuscript and all made substantial contributions to the work.
Acknowledgements
We gratefully acknowledge the support of the Natural Sciences and Engineering Research Council of Canada.
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| 15717935 | PMC552323 | CC BY | 2021-01-04 16:38:05 | no | J Nanobiotechnology. 2005 Feb 18; 3:2 | utf-8 | J Nanobiotechnology | 2,005 | 10.1186/1477-3155-3-2 | oa_comm |
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World J Surg OncolWorld Journal of Surgical Oncology1477-7819BioMed Central London 1477-7819-3-101570750010.1186/1477-7819-3-10ResearchDiagnosis and treatment of carotid body paraganglioma: 21 years of experience at a clinical center of Serbia Davidovic Lazar B [email protected] Vojko B [email protected] Dragan M [email protected] Radomir P [email protected] Stevo N [email protected] Institute for Cardiovascular Diseases, Clinical Center of Serbia, Belgrade, Serbia and Montenegro2 Institute for Otorhinolaringology and Maxillofacial Surgery, Clinical Center of Serbia, Belgrade, Serbia and Montenegro2005 12 2 2005 3 10 10 28 6 2004 12 2 2005 Copyright © 2005 Davidovic et al; licensee BioMed Central Ltd.2005Davidovic et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The carotid body paraganglioma (chemodectoma) is a relatively rare neoplasm of obscure origin. These are usually benign and commonly present as asymptomatic cervical mass.
Patients and methods
Records of 12 patients (9 female and 3 male) with carotid body tumors treated between 1982 and 2003, treated at our center were retrospectively reviewed. Data on classification, clinical presentation, and surgical treatment were extracted from the case records. Surgical complications and treatment outcome were noted and survival was calculated by actuarial method. The literature on carotid body paraganglioma was reviewed.
Results
The average age of the patients was 52 years (range 30–78 years). Eight of these cases presented as a large asymptomatic non-tender neck mass, and two each presented with dysphagia, and hoarseness of voice. As per Shamblin classification seven of tumors were type II and 5 were types III. In 7 cases subadventitial tumor excision was performed, while in 5 associated resection of both external and internal carotid arteries was carried out. The artery was repaired by end-to-end anastomosis in one case, with Dacron graft in one case, and with saphenous vein graft in 3 cases. There was no operative mortality. After a mean follow-up of 6.2 years (range 6 months to 20 years), there were no signs of tumor recurrence in any of the cases.
Conclusions
Surgical excision is the treatment of choice for carotid body paragangliomas although radiation therapy is an option for patients who are not ideal candidates for surgery. For the tumors that are in intimate contact with carotid arteries, the treatment by vascular surgeon is recommended.
==== Body
Background
Paraganglioma arising from the carotid body are relatively rare tumors but constitute majority of head and neck paragangliomas (60–70%) [1-6]. The term paraganglia was first used by Kohn in the early twentieth century and is the most appropriate nomenclature from an embryologic standpoint [3-5]. Other terms such as carotid body tumor, glomus tumor, chemodectomas, and nonchromaffin tumor are less accurate terms and therefore should be best avoided [7-13]. The neoplasm present as asymptomatic neck mass. We report our experience with surgically treated carotid body tumors.
Patients and methods
Between 1982 and 2003, 12 patients (9 female and 3 male) with carotid body paraganglioma were surgically treated at the Institute for Cardiovascular Diseases, Serbian Clinical Centre. Mean age of the patients was 52 years (range 30–78 years). The records of these patients were retrospectively reviewed for clinical presentation, diagnostic work-up, surgical treatment, and outcome. Descriptive data was presented as frequency and percentage. Survival was calculated by actuarial method. World literature on carotid body paraganglioma was reviewed. The articles were extracted using the key words carotid body and paraganglioma.
All the patients were followed-up periodically every 6 months for the first year, yearly for next 5 years, thereafter only select patients were followed. The patients who had undergone carotid artery repair were followed-up with yearly duplex scanning; two patients were followed by a computerized tomography (CT) scan, and one by regular magnetic resonance (MR) imaging.
Results
Eight cases presented as large non-tender neck masses located just anterior to the sternocleidomastoid muscle, two patients presented with dysphagia due to hypoglossal nerve compression, while two other had hoarseness of voice. The duplex ultrasonography and selective carotid angiography were used for diagnosis in the eleven cases, CT in five, and MR imaging in three cases (Figure 1 and 2). In one case the diagnosis was established on intraoperative exploration.
Figure 1 Selective carotid angiography showed carotid body paraganglioma. The typical separation (''lyre sign") of external and internal carotid arteries, are presented.
Figure 2 Selective carotid angiography showed hypervascularization of the carotid body paraganglioma mostly from the external carotid artery.
Intraoperatively on exploration of the neck seven of the cases showed a medium size tumor intimately associated and compressing carotid vessels (Shamblin II), and a large tumor involving carotid vessels in five cases (Shamblin III). In 7 cases of Shamblin II carotid body paraganglioma a subadventitial tumor excision was performed while in other 5 cases both external and internal carotid arteries were resected. One of these was repaired by end-to-end anastomosis, one with interposition of Dacron® graft, and other 3 were repaired with reversed saphenous vein graft.
The histological examination showed no signs of malignancy in any of the tumors. In two cases transient hypoglossal nerve palsy was noticed. Another patient had unexpected postoperative hoarseness of voice due to the transient vagus nerve palsy. All these three cases subsequently recovered. There was no operative mortality. The patients were followed-up from the 6 months to 20 years (mean 6.2 year) no local, regional or distant metastasis was noticed. The actuarial survival was 100%.
Discussion
The carotid body was first described by von Haller in 1743 [14]. It is highly specialized organ located at the common carotid artery bifurcation. Its feeding vessels run primarily from the external carotid artery. The function of the carotid body is related to autonomic control of the respiratory and cardiovascular systems, as well as blood temperature [3,10,12,15-23]. Paraganglioma is a relatively rare neoplasm occurring in carotid body [1-6].
The carotid body paraganglioma is more common in women [2-5,20,25-28]. The incidence of bilateral carotid body lesions is approximately 10%. Most of these lesions are benign however malignant behavior is often encountered. For diagnosis of malignant carotid body paraganlioma there are no clear histological characteristics that differentiate it from benign lesions. This diagnosis is reserved for the tumors with local, regional and distant metastasis. The rate of malignancy is reported to be 6–12.5% of all cases [3-5,9,11,29-35]. The 7–9% of the cases are hereditary [2,4,20,25-28,36]. None of our cases were bilateral or hereditary.
Carotid body paraganglioma often present as slow growing, non-tender neck masses located just anterior to the sternocleidomastoid muscle at the level of the hyoid. The tumor is mobile in the lateral plane but its mobility is limited in the cephalocaudal direction [3-5,13-15,21,26,36-38]. Occasionally the tumor mass may transmit the carotid pulse or demonstrate a bruit or thrill [39]. Because of its location in close approximation to carotid vessels and X-XII cranial nerves, tumors enlargement causes progressive symptoms such as dysphagia (two of our cases), odynophagia, hoarseness of voice (two of our cases) or other cranial nerve deficits [2-5,14,26,27,32,37,40]. The patients may give a history suggestive of symptoms associated with catecholamine production such as fluctuating hypertension, blushing, obstructive sleep apnea and palpitations [3-5,10,14,15,21-23,37].
Size of the tumor has a great importance not only for its clinical manifestations but also for treatment. In 1971, Shamblin introduced a classification system based on tumors size [41]. They classified small tumors that could be easily dissected away from the vessels as group I. Group II (7 of our cases) included paragangliomas of medium size that were intimately associated and compressed carotid vessels, but could be separated with careful subadventitial dissection. Group III consisted of (5 of our cases) tumors that were large and typically encased the carotid artery requiring partial or complete vessel resection and replacement. Histologically, carotid body paraganglioma resemble the normal architecture of the carotid body. The tumors are highly vascular, and between the many capillaries are clusters of cells called Zellballen [41].
The carotid angiography is the most useful diagnostic test for paragangliomas. The angiography demonstrates tumor blood supply and widening of the carotid bifurcation by a well-defined tumor blush ("lyre sign"), which is classic pathognomonic angiographic finding [5,8,37-39,42,43]. MR and contrast CT are more effective non-invasive imagining modalities comparing with duplex ultrasonography, especially for small tumors [3,37-39,42-45]. Radioimmunodetection of carotid body paraganglioma by 111In labeled anti-CEA antibody is also described in literature [9,46]. The differential diagnosis includes other tumors in this area, carotid artery aneurysms and elongation. For this reason using of precutaneous fine-needle aspiration for preoperative diagnosis of carotid body paraganglioma, can be very dangerous [47].
Resection of carotid body paraganglioma carries inherent risks of injury to the cranial nerves, carotid arteries as well excessive blood loss. Reigner first attempted resection of a carotid body paraganglioma in 1880, but the patients did not survive [48]. Maydel was the first to remove a carotid body paraganglioma successfully in 1886, but the patient became aphasic and hemiplegics due to internal carotid artery ligature [49]. In 1903, Scudder performed the first successful removal of carotid body paraganglioma [50]. The surgical excision with careful subadventitial dissection is treatment of choice for most carotid body paragangliomas (Shamblin I and II) [2-6,14-18,34,37-40,43]. The Shamblin III of carotid body paraganglioma requires resection of the external and/or internal carotid artery. If the internal carotid is encased in tumor or damaged during resection, immediate repair/replacement should be performed [15,37,39,40,42,43,51,52]. The second problem during tumor excision is bleeding, which sometimes can be massive. In such cases clamping of all carotid arteries is useful, with placement of internal carotid shunt [18,35,37]. Having in mind our experience with surgical treatment of both carotid body gangliomas as well as carotid stenosis, we recommend Pruitt-Inahara double balloon occlusive internal carotid shunt [37]. The placement of this shunt through incision on the common carotid artery contributes to the adequate bleeding control from the common and internal carotid arteries, as well as brain protection. This procedure gave a clean and dry operative field during tumor removal [3,37]. Some other articles recommend angiographic embolization preoperatively [3,23,37,42,53-55]. The Preoperative embolization of a carotid body paraganglioma can be performed by ethanol or polyvinyl alcohol. The finally result is a complete devascularization [55]. Earlier the carotid body paragangliomas were considered radioresistant [34]. However, more recent studies indicate good responses to radiation therapy [11,30]. Most authors recommend radiotherapy for giant and recurrent carotid body paragangliomas, and with malignant carotid body paragangliomas metastatic to the regional lymph nodes [8,33-36].
The modern surgical techniques have reduced the risk of postoperative stroke in carotid body paraganglioma resection to less than 5% [37,40,56]. However, the incidence of cranial nerve injury remains strikingly high, ranging from 20% to 40% [37,38,48,56,57]. In 20% of patients the neurological deficits is permanent. We found two (18%) transient hypoglossal, and one transient vagus nerve damage. recurrence after complete resection occurs in approximately 6% of patients [15,37,39,40,42,43,51,52]. In our study however, there were no recurrences. The patients with internal carotid artery reconstruction should undergo duplex scanning periodically to identify graft stenosis.
Conclusion
Early operative management is warranted to avoid the possibility of eventual metastasis and progressive local invasion to the point of inoperability. In case of tumors intimately contact with carotid arteries, the treatment by vascular surgeon is recommended.
Competing interests
The author(s) declare that they have no competing interests.
Funding source
Nil
Authors' contributions
LBD: Preperation of draft manuscript
VBD: Literature search, data collection
DMV: Study design, data analysis, interpretation, preparation of draft
RPS: Study coordination, data interpretation, manuscript preperation
SND: Manuscript editing, preparation of final manuscript for publication
All authors read and approved the manuscript.
==== Refs
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| 15707500 | PMC552324 | CC BY | 2021-01-04 16:39:05 | no | World J Surg Oncol. 2005 Feb 12; 3:10 | utf-8 | World J Surg Oncol | 2,005 | 10.1186/1477-7819-3-10 | oa_comm |
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World J Surg OncolWorld Journal of Surgical Oncology1477-7819BioMed Central London 1477-7819-3-131572369510.1186/1477-7819-3-13ReviewUmbilical metastases: current viewpoint Gabriele Raimondo [email protected] Marco [email protected] Federico [email protected] Mario [email protected] Department of Surgery, "P.Valdoni" – University of Rome "La Sapienza", Rome, Italy2005 21 2 2005 3 13 13 2 8 2004 21 2 2005 Copyright © 2005 Gabriele et al; licensee BioMed Central Ltd.2005Gabriele et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Umbilical metastases from a malignant neoplasm, also termed Sister Mary Joseph's nodule, are not commonly reported in the English literature, and they have usually been considered as a sign of a poor prognosis for the patient. The present article reports on the current view point on umbilical metastasis besides discussing the epidemiology, clinical presentation, pathophysiology and treatment.
Method
A search of Pubmed was carried out using the term 'umblic*' and 'metastases' or metastasis' revealed no references. Another search was made using the term "Sister Joseph's nodule" or sister Joseph nodule" that revealed 99 references. Of these there were 14 review articles, however when the search was limited to English language it yielded only 20 articles. Articles selected from these form the basis of this report along with cross references.
Results
The primary lesions usually arise from gastrointestinal or genitourinary tract malignancies and may be the presenting symptom or sign of a primary tumour in an unknown site.
Conclusion
A careful evaluation of all umbilical lesions, including an early biopsy if appropriate, is recommended. Recent studies suggest an aggressive surgical approach combined with chemotherapy for such patients may improve survival.
==== Body
Background
Cutaneous metastases localised to the umbilicus are named "Sister Mary Joseph's nodules". In 1949 Sir Hamilton Bailey initially used this eponym in his book "Physical Signs in Clinical Surgery" to describe umbilical metastases, in honour of Sister Mary Joseph, the superintendent nurse and surgical assistant of Dr. William Mayo at St. Mary's Hospital in Rochester (presently the Mayo Clinic in Minnesota, USA). Sister Mary Joseph was the first to note the link between umbilical nodules and intra abdominal malignancy [1-4].
Epidemiology
The occurrence of cutaneous metastases from malignant neoplasms occurs in from 1% to up to 9% of individuals, as determined at autopsy. Those metastases to the umbilicus are uncommon and represent only 10% of all secondary tumours which have spread to the skin [5,6]. Epidemiological studies showed that this condition predominates in females [7].
From a review of the literature, umbilical neoplastic nodules can be due to a primary tumour in 38% of cases, due to endometriosis in 32% of individuals, and in 30% are actually secondary tumour deposits from a primary tumour elsewhere [8]. If these nodules are secondary tumour deposits then the source of the primary tumour may be from the gastrointestinal (35–65%) and genitourinary (12–35%) tract. In addition, in 3–6% of cases it originates from haematological malignancies, or lung and breast cancers. In 15% to 30% of patients the source of the primary site of the tumour remains unknown [9-11].
Clinical findings
Sister Mary Joseph's nodules usually present as a painful lump on the anterior abdominal wall. It has irregular margins and a hard fibrous consistency. The surface may be ulcerated and necrotic, with either blood, serous, purulent, or mucous discharge from it. The size of the nodule usually ranges from 0.5 to 2 cm, although some nodules may reach up to 10 cm in size [8]. High-resolution ultrasound (US) helps to clarify the clinical findings by detecting solid umbilical nodules, even if the diagnosis is difficult to make clinically. Moreover, careful examination and imaging of the abdominal contents may also point to the diagnosis [12].
Pathophysiology
A full understanding of the mechanisms whereby the tumour spreads to the umbilicus remains unclear. However, following anatomical criteria, and several hypothesis have been proposed.
The umbilical ring is a scar invaginated on the abdominal wall between the transversalis fascia and peritoneum. After birth, the foetal cord structures develop into ligaments or peritoneal folds: 1) median umbilical ligament secondary to the obliterating urachus, 2) medial umbilical ligaments (which are obliterated umbilical arteries); 3) ligamentum teres (obliterated left umbilical vein) that continues into 4) the falciform ligament. On the lateral umbilical folds the inferior epigastric vessels and, sometimes, a vestigial vitelline duct connecting the umbilicus to the ileum can be recognised. The umbilical region shows a rich arterial supply that includes the inferior epigastric and deep circumflex iliac branches of the external iliac artery, and the superior epigastric branch of the internal mammary artery.
The venous drainage includes several anastomotic branches, coming from cranially the axillary vein, through the internal mammary vein, and caudally, the femoral vein through the superficial epigastric vein. In addition, the umbilicus may be connected with the portal system, through small umbilical veins.
The lymphatic system connects the umbilical region to the axillary, inguinal, and para-aortic lymph nodes. The deep lymphatic system passes along the falciform ligament, pierces the diaphragm and enters the anterior mediastinum or courses to the nodes around the iliac arteries [12-15].
All these systems (arterial, venous and lymphatic) as described represent possible routes by which metastatic tumour cells could implant into the umbilical region.
It is reasonable to suggest that direct extension of tumour through the peritoneum is the preferred route for gastrointestinal tumours. Furthermore, the common association between hepatic and umbilical metastases might suggests that the hypothesis that the tumour spreads from the primary tumour to the liver through the portal system and then through the lymphatic and/or venous channels, they spread to the umbilicus. It is still unclear if the umbilical tumour spread precedes the hepatic spread or vice versa.
Renal cell carcinoma typically spreads via extra-renal extension, lymphatic dissemination or venous invasion by the tumour. Intraperitoneal spread may occur as a result of disruption of the renal capsule [16]. The dissemination of neoplastic cell through the urachus is assumed to be the mechanism for the bladder cancers.
Haematogenous, lymphatic and venous spread all represent valid mechanisms of tumour spread from gynaecological cancers [7,14].
Prognosis and therapy
Usually the presence of an umbilical metastasis indicates a poor prognosis, is a sign of advanced neoplastic disease, and may not be amenable to surgery. The survival of these patients without treatment has been reported to range from 2 to 11 months from the time of initial diagnosis [17-19].
However, recent studies have suggested that there are several factors which are able to influence the prognosis of such patients. Certain data has shown a better survival (mean 9.7 months) in patients who detect an umbilical metastasis before definitive treatment of the primary tumour. In contrast, when the lesion appears after the primary tumour has been treated then the survival for these patients does not exceed the 7.6 months [16,17].
Moreover, the aetiology of the primary malignancy determines the prognosis. For example, a better survival rate for patients with primary ovarian carcinoma has been reported previously [14].
Finally, the type of treatment seems able to influence the patient's prognosis. Despite some authors proposing only palliative treatment because of these patients poor prognosis [10,17,20], recent studies have demonstrated that there is a better survival (21 months) for patients if they are treated with a combination of surgery and adjuvant therapy instead of surgery alone (7.4 months) or chemotherapy alone (10.3 months) [9,11,14,18].
Obviously, the appropriateness of such an aggressive treatment approach is determined by the clinical state of the patient.
Conclusion
A careful examination of all umbilical lesions is recommended, especially in those patients with gastrointestinal and genitourinary tract malignancies. All umbilical mass lesions should be biopsied to determinate the pathological nature of the lesion.
Clinical experience suggests that, whenever it is possible, an aggressive surgical approach combined with chemotherapy treatment may be considered to offer the patient the best survival probability.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
RG contributed to conception, design, literature search and preparation of manuscript.
MC contributed to conception, design, literature search and preparation of manuscript.
FE contributed to conception, design, literature search and preparation of manuscript.
MB contributed to conception, design, literature search and preparation of manuscript.
All authors read and approved the manuscript
==== Refs
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Lookingbill D Spangler N Sexton FM Skin involvement as the presenting sign of internal carcinoma. A retrospective study of 7316 cancer patients J Am Acad Dermatol 1990 22 19 26 2298962
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Barrow MV Metastatic tumors of the umbilicus J Chron Dis 1966 19 1113 1117 5966767 10.1016/0021-9681(66)90144-5
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Gabriele R Borghese M Conte M Basso L Sister Mary Joseph's nodule as a first sign of cancer of the cecum: report of a case Dis Colon Rectum 2004 47 115 117 14719158 10.1007/s10350-003-0018-5
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| 15723695 | PMC552325 | CC BY | 2021-01-04 16:39:05 | no | World J Surg Oncol. 2005 Feb 21; 3:13 | utf-8 | World J Surg Oncol | 2,005 | 10.1186/1477-7819-3-13 | oa_comm |
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World J Surg OncolWorld Journal of Surgical Oncology1477-7819BioMed Central London 1477-7819-3-141573056310.1186/1477-7819-3-14Case ReportA giant subcutaneous leiomyosarcoma arising in the inguinal region Yajima Kazuhito [email protected] Yoshio [email protected] Nobuhiro [email protected] Daisuke [email protected] Hajime [email protected] Katsuyoshi [email protected] Division of Digestive and General Surgery, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Niigata City, 951-8510 Japan2 Division of Pathology, Niigata University Medical and Dental Hospital, 1-754 Asahimachi-dori, Niigata City, 951-8520 Japan2005 24 2 2005 3 14 14 29 11 2004 24 2 2005 Copyright © 2005 Yajima et al; licensee BioMed Central Ltd.2005Yajima et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Subcutaneous leiomyosarcoma is a rare condition that accounts for 1% to 2% of all superficial soft tissue malignancies. Approximately 10% of cases arise in the trunk, although the extremities are the most commonly affected.
Case presentation
We report herein the case of a 31-year-old man with a subcutaneous leiomyosarcoma, measuring 124 × 105 mm, arising in the left inguinal region. A wide local excision (with a resection margin ≥ 20 mm) was performed. Histological examination of the resected specimen revealed a leiomyosarcoma with high cellularity and two mitoses per 10 high-power fields. The patient remains well with no evidence of disease 5 years and 8 months after the operation.
Conclusion
This is the first reported case of subcutaneous leiomyosarcoma arising in the inguinal region and also one of the largest tumors reported. The experience of this case and a review of the English-language literature (PubMed, National Library of Medicine, Bethesda, MD, USA) suggest that a resection margin of ≥ 10 mm is recommended when excising this rare tumor.
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Background
Subcutaneous leiomyosarcoma arises from smooth muscle in the walls of arterioles and veins. It is a rare tumor accounting for 1% to 2% of all superficial soft tissue malignancies [1-4]. It usually occurs in patients between 50 and 70 years of age [1,2,5], with a male predominance ranging from 2:1 to 3:1 [2,4]. Although most tumors present as a subcutaneous nodule in the extremities, usually measuring 30 mm or less in diameter, about 10% of cases arise in the trunk [1,2,4]. We herein report the case of a patient with a giant subcutaneous leiomyosarcoma arising in the inguinal region.
Case presentation
A 31-year-old man presented with a painless left inguinal tumor, which had gradually grown during the past six months. Physical examination on admission revealed a fist-sized subcutaneous tumor in the inguinal region. The overlying skin appeared normal without ulceration (Figure 1), and there was no inguinal lymphadenopathy. Computed tomography depicted a solid tumor with heterogeneous contrast enhancement in the adipose tissue, and no metastases to the liver and lung (Figure 2). With a tentative diagnosis of soft tissue sarcoma of unknown origin, a wide local excision (with a resection margin ≥ 20 mm) was performed.
Figure 1 A fist-sized tumor arising in the subcutaneous adipose tissue in the inguinal region. An arrow indicates the navel.
Figure 2 Computed tomography depicted a solid tumor with heterogeneous contrast enhancement (arrowheads) in the adipose tissue.
The resected tumor, measuring 124 × 105 mm, was solid, encapsulated, and a homogeneous yellowish white in color, without central necrosis and hemorrhage on its cut surface. Routine histological examination with hematoxylin-and-eosin revealed that the tumor comprised spindle-shaped cells with high cellularity in parts (Figure 3) and two mitoses per 10 high-power fields. Immunohistochemistry with mouse monoclonal antibodies against desmin (D33, Dako Cytomation Japan Co. Ltd., Kyoto, Japan), alpha-smooth muscle actin (1A4, Dako Cytomation Japan Co. Ltd., Kyoto, Japan), vimentin (V9, Dako Cytomation Japan Co. Ltd., Kyoto, Japan) and S-100 protein (2A10, IBL-Japan Co. Ltd., Takasaki, Japan) was performed. As the tumor cells showed only immunoreactivity for desmin (Figure 4) and alpha-smooth muscle actin, a diagnosis of leiomyosarcoma of subcutaneous adipose tissue origin was confirmed.
Figure 3 The tumor comprised spindle-shaped cells with high cellularity in parts (hematoxylin-and-eosin; original magnification, × 100).
Figure 4 Spindle-shaped cells showing strong immunoreactivity for desmin in the cytoplasm (Desmin immunohistochemistry; original magnification, × 400).
The patient had an uneventful recovery and was discharged on the 9th postoperative day. As the resection margin was negative, no adjuvant treatment was given. He remains well with no evidence of disease 5 years and 8 months after excision.
Discussion
Although subcutaneous leiomyosarcoma commonly arises in the lower extremities, it occasionally affects the trunk [1,2,4]. A review of the English-language literature (PubMed, National Library of Medicine, Bethesda, MD, USA) suggests that the case reported here is the first one arising in the inguinal region and involves one of the largest tumors reported thus far [2,7]. Earlier authors proposed several prognostic factors for soft tissue sarcomas (including subcutaneous leiomyosarcoma) [2,6-8]. Factors adversely affecting the prognosis include high mitotic index (≥ 5 mitoses per 10 high-power fields) [2], high histologic grade [6], extensive necrosis [7], nodular growth pattern [7], deep tumor [8], and large tumor size [8]. Among them, tumor size (≥ 5 cm) is the strongest independent prognostic factor [8]. In the current case, low mitotic index, low histologic grade and the absence of necrosis favored a good prognosis, while large tumor size, deep tumor and nodular growth pattern were adverse prognostic factors.
As subcutaneous leiomyosarcoma is resistant to radiotherapy and chemotherapy [2,3,9], surgical excision provides the only chance of cure. Prognosis after excision is generally considered poor, and even after a wide excision, local recurrence may occur in 40% to 60% of patients, followed by distant metastases in 20% to 40% of cases [1,2]. Contaminated margins contribute to the frequency of local recurrences [5,10-12]. In the current case, the resection margin of ≥ 20 mm successfully controlled the tumor. McKee et al., [12] demonstrated that resection margins ≥ 10 mm decreased the risk of both local and distant recurrences in patients with soft tissue sarcomas (including subcutaneous leiomyosarcoma). A resection margin of ≥ 10 mm is therefore recommended when excising this rare tumor.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
KY, YS, NF, and DS took part in the operation, performed the literature search and drafted the manuscript for submission. HU performed histological examination. KH supervised the preparation of the manuscript and edited the final version for publication. All authors read and approved the final manuscript.
Acknowledgements
Written consent was obtained from the patient for publication.
==== Refs
Stout AP Hill WT Leiomyosarcoma of the superficial soft tissue Cancer 1958 11 844 854 13561254
Fields JP Helwig EB Leiomyosarcoma of the skin and subcutaneous tissue Cancer 1981 47 156 169 7459804
Wile AG Evans HL Romsdahl MM Leiomyosarcoma of soft tissue: a clinicopathologic study Cancer 1981 48 1022 1032 7272926
Guillen DR Cockerell CJ Cutaneous and subcutaneous sarcomas Clin Dermatol 2001 19 262 268 11479038 10.1016/S0738-081X(01)00177-8
Bernstein SC Roenigk RK Leiomyosarcoma of the skin: treatment of 34 cases Dermatol Surg 1996 22 631 635 8680785 10.1016/1076-0512(95)00186-7
Coindre JM Bui NB Bonichon F Mascarel I Trojani M Histopathologic grading in spindle cell soft tissue sarcomas Cancer 1988 61 2305 2309 3365658
Kaddu S Beham A Cerroni L Humer-Fuchs U Salmhofer W Kerl H Soyer HP Cutaneous leiomyosarcoma Am J Surg Pathol 1997 21 979 987 9298873 10.1097/00000478-199709000-00001
Jensen ML Jensen OM Michalski W Nielsen OS Keller J Intradermal and subcutaneous leiomyosarcoma: a clinicopathological and immunohistochemical study of 41 cases J Cutan Pathol 1996 23 458 463 8915854
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Lewis JJ Leung D Espat J Woodruff JM Brennan MF Effect of reresection in extremity soft tissue sarcoma Ann Surg 2000 231 655 663 10767786 10.1097/00000658-200005000-00005
McKee MD Liu DF Brooks JJ Gibbs JF Driscoll DL Kraybill WG The prognostic significance of margin width for extremity and trunk sarcoma J Surg Oncol 2004 85 68 76 14755506 10.1002/jso.20009
| 15730563 | PMC552326 | CC BY | 2021-01-04 16:39:05 | no | World J Surg Oncol. 2005 Feb 24; 3:14 | utf-8 | World J Surg Oncol | 2,005 | 10.1186/1477-7819-3-14 | oa_comm |
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Virol JVirology Journal1743-422XBioMed Central London 1743-422X-2-101570748910.1186/1743-422X-2-10HypothesisReplicative Homeostasis: A fundamental mechanism mediating selective viral replication and escape mutation Sallie Richard [email protected] Suite 35, 95 Monash Avenue, Nedlands, Western Australia, Australia2005 11 2 2005 2 10 10 23 1 2005 11 2 2005 Copyright © 2005 Sallie; licensee BioMed Central Ltd.2005Sallie; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Hepatitis C (HCV), hepatitis B (HBV), the human immunodeficiency viruses (HIV), and other viruses that replicate via RNA intermediaries, cause an enormous burden of disease and premature death worldwide. These viruses circulate within infected hosts as vast populations of closely related, but genetically diverse, molecules known as "quasispecies". The mechanism(s) by which this extreme genetic and antigenic diversity is stably maintained are unclear, but are fundamental to understanding viral persistence and pathobiology. The persistence of HCV, an RNA virus, is especially problematic and HCV stability, maintained despite rapid genomic mutation, is highly paradoxical. This paper presents the hypothesis, and evidence, that viruses capable of persistent infection autoregulate replication and the likely mechanism mediating autoregulation – Replicative Homeostasis – is described. Replicative homeostasis causes formation of stable, but highly reactive, equilibria that drive quasispecies expansion and generates escape mutation. Replicative homeostasis explains both viral kinetics and the enigma of RNA quasispecies stability and provides a rational, mechanistic basis for all observed viral behaviours and host responses. More importantly, this paradigm has specific therapeutic implication and defines, precisely, new approaches to antiviral therapy. Replicative homeostasis may also modulate cellular gene expression.
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Background
1. Disease burden
Hepatitis C (HCV), HBV and HIV are major causes of premature death and morbidity globally. These infections are frequently life-long; Hepatitis viruses may result in progressive injury to the liver and cirrhosis, and death from liver failure, or hepatocellular carcinoma, while HIV causes progressive immune depletion and death from the acquired immunodeficiency syndrome (AIDS). Together, these infections cause millions of premature deaths annually, predominantly in "developing" countries. Other viruses replicating via RNA intermediaries cause similar morbidity among domestic and wild animal populations. While education, public health measures and vaccination (for HBV) have resulted in significant progress in disease control, therapy of established viral infection remains unsatisfactory.
2. Viral replication
RNA viruses and retroviruses replicate, at least in part, by RNA polymerases (RNApol), enzymes that lack either fidelity or proofreading function [76]. During replication of hepatitis C HCV or HIV each new genome differs from the parental template by up to ten nucleotides [61] due to RNApol infidelity that introduces errors at ~1 × 10-5 mutations / base RNA synthesised.
Viruses replicate by copying antigenomic intermediate templates and hence obey exponential growth kinetics, such that [RNA]t = [RNA](t-1)ek, where [RNA]t is virus concentration at time (t) and k a growth constant. However, because of RNApol infidelity, wild-type (wt) virus will accumulate at [RNAwt]t = [RNAwt](t-1)•(1-ρ)•K1 and variant forms (mt) at [RNAmt]t ≈ ([RNAwt](t-1)•ρ + [RNAmt](t-1))•K1, where ρ is the probability of mutation during replication and K1 = ek. Therefore, while wild-type virus predominates early, replication (and intracellular accumulation) of variant virus and viral proteins will accelerate (in a ratio of ([RNAwt](t-1)•ρ + [RNAmt](t-1))/ [RNAwt](t-1)•(1-ρ) compared to wild type) and variant viral RNAs will rapidly predominate (Figure 1). Mutations progressively accumulate in RNA viruses [17] and ultimately variant RNAs and proteins, if variant RNAs are translated, will become dominant. It is also likely some variant viral proteins will resist cellular trafficking, further accelerating the intracellular accumulation of variant forms relative to wild type.
Figure 1 Effect of RNApol fidelity on replication. Each replication cycle may produce either wild-type (Wt) or variant (Mt) copies of parental template in a ratio determined by polymerase fidelity. If HCV RNApol Mu is 10-5 mutations per base RNA synthesized, Mt:Wt ratio at G1 is ~9:1, by G3 unmutated parental genome is 6.8 × 10-4of total virus population, and by G20 7.5 × 10-22
The paradox of quasispecies stability
Two fundamental problems critical to understanding RNA virus quasispecies biology arise because of RNA polymerase infidelity and the mode of viral replication:
1: Replication kinetics
Hepatitis C, HIV, and HBV and other viruses, have broadly similar kinetics (Figure 2); initial high level viral replication that rapidly declines to relatively constant low-level viraemia [11,12], typically 2–3 logs lower than at peak, for prolonged periods, a kinetic profile attributed to "immune control" [12]. However, immune control is a conceptually problematic explanation for the initial decline in viral load; For example; why would potent host responses (of whatever type; humoral, cell mediated or intracellular immunity, or any combination thereof), having reduced viral load and antigenic diversity by a factor of 102–3 within days, falter once less than 1% of virus remains?
Figure 2 Viral kinetic paradox. Viral replication kinetics (—). If host factors (Ic, black arrows) reduce viral replication acutely (point A), then they must exceed viral forces (Ve, grey arrows). At equilibrium (e.g. points B or C) host forces must balance viral forces; Ic must therefore fall by a factor of 102–3 from A.
Formally
1. Assume immune mechanisms reduce initial viral replication.
2. Let Ic(t) represent the immune forces favouring viral clearance and Ve(t) viral forces promoting quasispecies expansion pressures at time (t).
3. Assume immune pressures Ic required to clear virus are proportional to viral concentration [V], that is; Ve ∝ [V] (or Ve = ke [V] where ke is some constant), so that Ic required to clear one viral particle Ic(1) is less than that Ic required to clear 10 viral particles Ic(10).
4. At equilibrium (e.g. time points B or C, Figure. 2) immune clearance pressures approximate viral antigenic expansion pressures: Ic(b or c) ≈ Ve(b or c). Eq.1
5. If Ic causes the reduced viral load seen between time A and time B or C, [Ve(a)] ⇒ [Ve(b or c)], then immune clearance pressures must exceed viral expansion pressures at that time i.e. Ic(a) > Ve(a). Eq.2
6. As viral antigenic expansion pressures at time A exceed those at time (B or C) by 102–3 [V(a)] ≈ [V(b or c)]• 102–3, and Ic(b or c)= Ve(b or c) then immune clearance pressures at time A exceed those at time (B or C) by102–3 Ic(a) >Ic(b or c)• 102–3. That is, immune pressures fall by 102–3 between time A and B or C, (Figure. 2).
Prompting
i) Why, and by what mechanism, would immune forces, or any other host defense mechanisms, fall by 102–3 over days between time A and B or C?
There is, of course, no evidence immune pressures fall, and very considerable evidence both antibody and adaptive T cell responses are increasing when viral replication is falling [5,12]. These facts are irreconcilable with the notion that immune or other any host mechanisms control initial viral replication and strongly suggest immune or any other host mechanism(s) are not the primary reason viral load falls initially. Further, as down-regulation of viral replication frequently occurs prior to development of neutralising antibody, in the absence of any demonstrable antiviral antibody, or T-cell responses [25,41], and without lysis of infected cells [25], it is difficult to argue, with any conviction, that either humoral or cellular immune responses primarily cause reduced viral replication. Evidence that prior HCV infection does not confer protective immunity against either heterologous HCV infection in chimpanzee [22]or either homotypic [33] or heterotypic [32] human reinfection further undermines the paradigm of "immune control". Inhibition of immune or other host mechanisms is an untenable explanation of this massive apparent fall in immune clearance pressures; if occurred to any degree, an increase, rather than the observed decrease, in viremia would result. In the absence of a rational host mechanism consistent with observed viral kinetic data, the ineluctable conclusion is that non-host (i.e. viral) mechanisms (i.e. viral auto regulation) must be operative.
Chronic viral persistence raises other issues; At steady state (e.g. points B or C, Figure. 2), the rate of HIV and HCV production is estimated at 1010 molecules / day [11,29,52,57] while HBV production may be 1011 molecules/day resulting in an average viral load of 1010 molecules/person [52,57]. However, during peak replication virus production may 102–3 times the basal rate [11,12], indicating enormous reserve replicative capacity. As basal viral replication is clearly sufficient for long-term stability, and kinetic analysis suggests viral, rather than host, factors control viral replication, the following questions are posed: When challenged, how do viruses "sense" the threat and by what mechanism do they modulate replication in response?
Problem 2: Mutation rate
The stability of RNA viral quasispecies poses a major problem: During viral replication the copied genome may either identical to or a variant of parental template (Figure. 1). The probability (ρ) of a mutation occurring during replication is a function of polymerase fidelity; During one replication cycle ρ = (1-(1-Mμ)n), where (Mμ) is mutation rate and (n) genome size. Hepatitis C (a ~9200 bp RNA virus) RNApol introduces mutation at 10-5 substitutions/base, ρ≈0.912. However, for multiple (θ) replications cycles, ρ = (1-(1-Mμ)n)θ. After 20 replication cycles, occurring in <7 days in most patients [52,57], the probability of any original genome remaining un-mutated is ρo≈7.5 × 10-22, meaning effective loss of sequence information, an outcome that should cause quasispecies extinction [16]. Persistence of stable RNA viral quasispecies is, therefore, highly paradoxical [18]. This "theoretical impossibility" of RNA quasispecies stability suggests either a) the consistently reported rates of RNApol infidelity are incorrect (which, even if true, would only delay quasispecies extinction; if Mμ = 10-10, ρo <10-40 within 100 days etc.) or b) that innate viral mechanism(s) control RNApol fidelity and mediate selective replication of consensus sequence genomes. Thus, rates of viral mutation are tightly constrained by the necessity to retain sequence information. On the other hand, overly faithful template replication will restrict antigenic diversity, rendering virus susceptible to immune destruction and unresponsive to ongoing cellular changes. The necessity to retain sequence information by adequate replicative fidelity, and the later requirements (in terms of replicase ⇒ RNApol evolution) of viruses to access cells via evolving cell receptors and evade host defence mechanisms, has placed constraints on replicase (RNApol) function that dictate polymerase fidelity must be tightly, and dynamically, controlled (Figure 3a).
Figure 3 a. Constraints on viral mutation. Inadequate polymerase fidelity will cause loss of sequence information and quasispcies extinction (A, B), while inadequate viral mutation will result in immune recognition and viral clearance (D,E). Viral persistence requires polymerase fidelity responsive to the host environment (C). 3b. Constraints on viral replication. Overly rapid replication will cause cell lysis, tissue injury and premature host death (A,B), while inadequate replication will result viral latency or clearance (D,E). Viral persistence with optimal evolutionary stability requires a polymerase responsive to the host environment (C).
Evolutionary constraints on viral replication
Optimal viral replication is a compromise between maximising host-to-host viral transmission at each host contact versus maximising transmission at sometime during the host's life: Uncontrolled, exponential growth, as might result from the mode of viral replication, would cause rapid cell lysis, host death and a reduced likelihood of stable host-to-host transmission, a prerequisite for viral survival on an evolutionary timescale. While maximising the probability of host-to-host transmission at each contact, high-level viral replication increases the probability of host disease, thus reducing opportunity for transmission long term. Contrariwise, adverse viral outcomes may result from inadequate viral replication causing increased clearance and reduced host-to-host transmission. Viruses that cause premature host death or that are cleared by host mechanisms before transmission to, and infection of, other hosts are biological failures that have strong Darwinian pressures acting against them. Optimal long-term viral stability, therefore, dictates viral replication rates (that is, polymerase processivity) and mutation frequency (that is, polymerase fidelity) must be closely regulated (Figure 3b).
Hypothesis
That viruses capable of chronic persistence auto-regulate replication and mutation rates by replicative homeostasis. Replicative homeostasis results when RNA polymerase end-translation products (envelope and contiguously encoded accessory proteins) interact with RNApol to alter processivity and fidelity.
Evidence for Autoregulation
Substantial clinical and in-vitro evidence, including the kinetic paradox indicate viruses auto-regulate. During successful antiviral treatment levels of virus fall sharply [12,29,52,53,57], often becoming undetectable. However, viral replication rebounds, rapidly and precisely, to pre-treatment levels on drug withdrawal in patients [52,53,57] and in tissue culture [1]. This in-vitro data confirm replication is controlled by factors independent of either cellular or humoral immune function. Auto-regulation of HCV replication was confirmed most emphatically in patients undergoing plasmapharesis in whom 60–90% reduction in levels of virus returned to baseline, but not beyond, within 3–6 hours of plasma exchange [44]. Studies suggesting autoregulation of tobacco mosaic virus replication occurred independent of interferon effects, intrinsic interference or interference by defective virus [34] confirming this phenomenon is not confined to either animal viruses or cells. These data beg the questions: How does the replicative mechanism "choose" any particular level of replication and how does it return, so accurately, to pre-treatment levels?
RNA polymerase control
Most cellular enzymes are under some form of kinetic control, usually by product inhibition. While simple negative-feedback product inhibition is sufficient to control enzyme reaction velocity and the rate of product synthesis, it is inadequate to ensure the functional quality of any complex molecules – including proteins – synthesised. The functionality of RNApol output depends on the functionality of protein(s) translated from any RNA synthesized by RNApol. For viruses, and their polymerase, evolutionary survival – i.e. whether the polymerase, and its viral shell, avoids immune surveillance, gains access to cells, and replicates to infect other hosts – is a function of the properties that the sequence, topological variability and structural integrity of envelope proteins impart. RNA polymerase is responsive to and is influenced by accessory proteins that induce conformational changes to alter both processivity and fidelity [20,31], representing partial "proof of concept" of the mechanism postulated.
Evolutionary stability
Evolutionary stability requires adaptability to changing environmental circumstances. For viruses, an ability to modulate replication and mutation rates dynamically in response to cellular changes is essential. Viruses intrinsically capable of adaptation to environmental changes, including variations in host density, and evolving cell receptor polymorphisms, immune and other host responses, among other variables, will enjoy a competitive advantage over viruses lacking innate responsiveness. Contrariwise, self-replicating molecules, including viruses, that lack innate adaptability, for whom replication is contingent upon a chance confluence of appropriate cellular conditions – including permissive cell receptors, absence of cell defences and so on – are highly vulnerable to extinction by both adverse environmental changes and competition for scarce intracellular resources by molecules capable of adaptation. For viruses, this adaptability requires antigenic and structural diversity be controlled and, in turn, that means the two critical RNApol attributes, fidelity and processivity, be dynamically modifiable, and controllable. These linked functional requirements imply a dynamic nexus between the functional output of RNApol (i.e. envelope proteins) and that polymerase.
Homeostatic systems
Systems capable of homeostatic regulation (auto-regulation) have the following characteristics: i) an efferent arm that effects changes in response to perturbations of an equilibrium; ii) an afferent arm that measures the systems response to those changes; iii) mechanism(s) by which i) and ii) communicate. The mechanism of viral autoregulation – Replicative Homesostasis – described here requires: i) that viral envelope (Env) proteins interact with viral RNA polymerases (RNAPol); ii) that these Env :RNAPol interactions alter both polymerase processivity and fidelity; iii) that wild-type (consensus sequence) Envwt :RNAPol complexes cause more rapid, less faithful RNA replication than variant (variant) Envmt :RNAPol complexes. There is solid evidence for each requirements of replicative homeostasis.
The Envelope-Polymerase relationship: Evidence for mechanism
A large body of literature, for many viruses, establishes an important relationship between envelope and polymerase proteins and documents that Env proteins influence both RNAPol processivity and fidelity.
First, for HIV, overwhelming evidence suggests HIV polymerases properties, and those of related retroviruses – for example, simian immunodeficiency virus (SIV) and the feline immunodeficiency virus (FIV) – are influenced by Env proteins (for example, [9,15,35]. Broadly, these indicate heterologous Env proteins – when administered as live attenuated vaccines [71], adjuvant enhanced protein vaccine [83], or as recombinant Env proteins in cell culture [64] – dramatically alter viral load, and both replication and mutation rates of wild-type virus. Specific examples include data demonstrating HIV Env regions obtained from different patient isolates, when cloned into common HIV-1 backbones, conferred a spectrum of replication kinetics and cytotropisms characteristic of the original Env clone, and independent of either the clones' ability to raise antibody [51], or the replicative characteristics of the 'native' polymerase backbone [51]. Similarly, chimeric HIV-1 viruses expressing heterologous Env, again with a common polymerase backbone, have replication kinetics and cell tropism phenotypes identical to the parental Env clone [39], suggesting the Env is a critical determinant of polymerase function. Similar results obtained with SIV clones [36] strongly support conclusions drawn from feline immunodeficiency virus [37] data. Fine mapping of HIV envelope proteins identified 6 mutations within the V1-V3 loop that increased viral replication in a manner independent of nef [77], confirming other work examining HIV Env recombinants [14], and extending earlier work that demonstrated a single amino acid substitution (at position 32 of the V3 Env domain) was sufficient to change a low replication phenotype into high-replicating phenotype [13]. Finally, for HIV, co-transfection with Env variants at 10 fold excess dramatically inhibited replication of wild-type virus [75], providing direct evidence for both the interaction and differential affinity for wild-type and variant Env for polymerases. Critically, many of these observations are from in-vitro systems, indicating the effects are independent of either cellular or humoral immune influence. Many studies report the effect of Env/polymerase interactions in terms of altered viral tropisms, and did not examine changes to polymerase fidelity explicitly. However, virus replication can alter in only two ways; either there is more or less virus, or the viral genomic sequence may be changed by altered polymerase fidelity. Variant viruses expressing altered envelope proteins will have altered cell receptor affinities and hence, variable cell tropisms.
Second, for HCV, many separate observations document HCV replication and polymerase functionality is dependent on envelope proteins: i) HCV viral genotypes are defined by sequences of either envelope or polymerase regions [43,73,74] and these are necessarily acquired together – a genetic nexus implying a functional relationship. ii) Observations that a) co-infection with multiple HCV genotypes occurs less frequently than predicted by chance and b) certain HCV genotypes become progressively dominant in populations both suggest – at a population level – replicative suppression of some HCV genotypes by others [68]. These observations are supported by observations of both homotypic [33] and heterotypic HCV super-infection [32] documenting genotype-dependent replicative suppression of one HCV genotype by another in individual patients. iii) Functional infectious chimeric viruses with polymerase and Env proteins derived from different genotypes have not been reported. iv) Full-length HCV chimeras, engineered with deletions of p7 envelope proteins, are replication deficient and non-infections, indicating intact genotype-specific HCV envelope sequences are essential for proper HCV replication. Specific replacement of p7 of the 1a clone with p7 from an infectious genotype 2a clone was replication defective, suggesting a genotype-specific interaction between the p7 envelope protein and other genomic regions [66]. v) In two independent chimpanzees studies HCV inoculation resulted in persistent infection only in animals developing anti-envelope (E2) antibodies, whereas failure to produce anti-E2 was associated with viral clearance [4,62], intuitively a highly paradoxical result difficult to rationalize unless E2 proteins are important for sustained HCV replication, as we argued previously [45]. vi) Finally, for HCV, specific motifs within the [polymerase] NS5 region of HCV in chronically infected patients predict response to interferon [19,67] an observation that makes little sense unless interferon interacts directly with NS5 [polymerase] motifs, as in-vitro studies suggest [10].
Third, HBV envelope and polymerase protein genes have overlapping open reading frames and significant alterations in envelope and polymerase gene and protein sequences cannot, therefore, occur independently, a genetic nexus again implying an important functional relationship. Mutations in envelope sequences occurring spontaneously [82] following therapy of HBV with lamuvidine and immunoglobulin prophylaxis [6,72] or after vaccine escape [8] are frequently associated with high level viral replication, although replication-deficient mutations are described [47]. These data are generally interpreted to mean polymerase gene mutation(s) cause altered polymerase protein sequence and, hence, abnormal polymerase function. While this is probably partially true if the functionally relevant HBV RNA polymerase is an envelope/polymerase heterodimer (analogous to the p66/p51heterodimer of HIV RT [30]), then an equally valid interpretation is that mutations in envelope genes may change envelope protein conformation and therefore alter normal envelope/polymerase interactions, thus altering processivity and fidelity of the replication complex. This latter interpretation is convincingly supported by data demonstrating that abnormal polymerase function of HBV envelope variants is reversed by co-transfection of Hep G2 cells with clones expressing wild-type envelope sequences [81] and is further supported by clinical studies demonstrating administration of exogenous HBsAg (protein) to patients with chronic HBV dramatically reduced HBV replication [60].
Fourth, studies of the coliphage Qβ demonstrate phage coat proteins bind to genomic RNA [86]to strongly inhibit (association Kic ≈ 107–8 M-1, inhibition Ki ≈ 109 M-1s-1) [79] RNA replication by direct suppression of polymerase activity by envelope proteins [18]. This interaction is dependent on the binding site conformation, but not RNA sequence[86], suggesting interaction avidity will vary as an inverse function of protein sequence divergence from wild type, an intuitive expectation confirmed experimentally [79]. An impressive body of literature documents similar relationships between envelope and polymerase function in swine fever, tobacco mosaic [34], brome mosaic [2] and other RNA viruses. Importantly, studies of the tobacco mosaic virus confirmed this effect to be host-independent and virus-specific inhibition of viral RNA synthesis and to be quite distinct from any interferon effects, intrinsic interference or interference by defective virus [34]. Thus, there exists solid evidence for each necessary component of replicative homeostasis for HCV, HBV and HIV, and other viruses.
Replicative homeostasis: proposed mechanism
Replicative homeosatsis results from differential interactions of wild-type (Wt) and variant (Mt) envelope proteins on RNApol in a series of feedback epicycles linking RNApol function, RNA replication and protein synthesis (Figure 4, 5). Intracellular accumulation of variant viral proteins causes progressive, direct, inhibition of RNApol and also block EnvWt:RNApol interactions that increase replication and mutation. Progressive blockade of RNApol by variant envelope results in a less processive, more faithful, polymerase, increasing the relative output of wild-type envelope RNAs, and, subsequently, translation of wild-type envelope proteins and, hence, an inexorable progression to stable equilibria. Quasispecies stability, and other consequences (including immune escape and low-level basal replication), are inevitable outcomes that result from equilibria reached because of these interactions (Figure 5). We suggest these interactions, and the resulting equilibria, are important therapeutic targets, and the effective ligands – envelope proteins or topologically homologous molecules – implicit within this hypothesis.
Figure 4 Mechanism of replicative homeostasis. At A, relatively high concentrations of EnvWt (blue, A) favour high affinity Env:RNApol interactions out-competing variant forms (Envmt, red), increasing RNApol processivity but reduced fidelity increasing relative output of variant RNAs. Subsequent ribosomal (R, mauve) translation increases concentrations Envmt (red), relative to EnvWt, returning the system to equilibrium. Relative excess Envmt (B, red) out-compete EnvWt (blue) for interactions with RNApol, favouring Envmt:RNApol, and blocking EnvWt:RNApol interactions. Envmt:RNApol complexes relatively decrease RNApolprocessivity but increase fidelity, increasing output of wild-type RNAs. Subsequent increased translation of EnvWt relative to Envmt restores the equilibrium.
Figure 5 Conseqences of replicative homeostatic cycles. Disturbance to intracellular replicative homeostatic cycles. Events increasing intracellular EnvWt: Envmt ratio (exogenous addition of EnvWt, antibody recognition of Envmt) will favour EnvWt:RNApol interactions, increasing RNApol processivity and reducing fidelity increasing relative output of variant virus. Conversely, events decreasing intracellular EnvWt: Envmt ratios (exogenous addition of Envmt, antibody recognition of EnvWt) will favour Envmt:RNApol interactions, decreasing RNApol processivity and increasing fidelity, thus reducing replication.
Viral polymerases are clearly the effector mechanism – the efferent arm – that determines rate of viral RNA replication and mutation. The afferent arm needs to measure both the rate of viral replication and degree of viral mutation. Intracellular envelope concentrations are a direct function of effective viral replication, while competition between wild-type and variant envelope proteins for interaction with RNApol allows determination of viral mutation rates. Envelope proteins, as opposed to other viral products, are the obvious products to examine for functional variability, and must form part of the afferent arm necessary to "sense" perturbations in the viral equilibrium. While other viral products could be "sensed" to gauge effective viral replication, only functional measurement of envelope protein concentration and topological variability simultaneous measures both the rate of viral replication and envelope functions – properties determined by envelope structure and antigenic diversity – essential for viral survival; immune escape and cell access. Furthermore, envelope and polymerase proteins are typically coded at transcriptionally opposite ends of the viral genome; replication contingent upon a dynamic nexus between envelope and polymerase proteins is, therefore, a functional check of the integrity of the entire viral genome. Importantly, this facet of replicative homeostasis is a direct mechanism of Darwinian selection operating at a molecular level, that ensurs preferential selection and replication of "fit" viral genomes, and maintenance of genotypes (species).
Viruses, notably HIV, produce many accessory proteins (such as HIV Nef, gag, rev and HBeAg) that affect viral replication and mutation rate. However, these proteins are encoded within envelope open reading frames (ORFs) or are contiguous with them and are likely to alter functionally with any mutation affecting envelope sequences (Figure 6). While these accessory proteins may interact with RNApol (with or without Env) to reset replicative equilibrium (by changing replication rate or mutation frequency or both), stable equilibria will still result providing the sum effect of variant proteins encoded within the envelope ORF is to decrease RNApol processivity (v) and mutation (Mu) frequency relative to wild-type protein polymerase interactions.
Figure 6 Phenotypic effects of RNA quasispecies complexity. Two-dimensional representation of multi-dimensional hyperdense sequence-space that define viral quasispecies; vast RNA /proteins populations progressively divergent from consensus sequence (0). As genetic the distance of RNAs increases from consensus sequence the amino acid sequence, conformation, and functional properties of resulting proteins may also change, potentially resulting in proteins that, despite originating from identical [consensus sequence] genetic domains, have diametrically opposed function. As many accessory proteins (for example, HIV rev, tat, nef and HCV HP7) have open reading frames contiguous with Envelope, sequence changes to Env will also affect accessory protein function.
Testing the hypothesis
This hypothesis is simply tested. Manoeuvres that increase intracellular concentrations of variant envelope proteins or decrease wild-type envelope proteins should inhibit viral replication and reduce mutation rates. Conversely, manoeuvres increasing intracellular [EnvWt] or reducing intracellular [Envmt] should accelerate viral replication and mutation. In fact, observations relevant to every aspect of this hypothesis have been reported in a variety of systems and circumstances. All outcomes are completely consistent with those predicted by replicative homeostasis. Replicative homeostasis predicts, for example, HCV E2 proteins derived from genotype 1 HCV sequences would reduce HCV replication when administered to patients with heterologous HCV infection (genotypes 2,3 or 4, for example) and studies examining heterologous envelope proteins as direct RNApol inhibitors are underway.
Discussion
Replicative homeostasis immediately resolves the paradox RNA viral quasispecies stability and explains how these viruses persist and, thereby, cause disease. Replicative homeostasis also explains the initial decline of viral replication, resolving the kinetic paradox, rationalizing the dynamics of chronic viral infection and other enigmatic and unresolved viral behaviours. Most importantly, replicative homeostasis implies a general approach to antiviral therapy.
The equilibria formed by replicative homeostasis are responsive to disturbance of envelope concentrations ensuring viral mutation is neither random nor passive but highly reactive to external influence: Sustained reduction of viral envelope (by immune or other mechanisms) would favour high affinity EnvWt: RNApol interactions that, in turn, increase polymerase processivity but reduce fidelity accelerating synthesis of variant viral RNAs and, consequently, increased translation of antigenically diverse proteins, reactively driving quasispecies expansion and generating the extreme antigenic diversity of RNA quasispecies. Alternatively, in the absence of immunological recognition, variant envelope / polymerase interactions predominate, restricting viral replication and mutation, thus maintaining basal output of consensus viral sequences, thus maintaining genotype. Immune escape and maximal cell tropism are inevitable consequences of the potential antigenic diversity generated by RNA replication mediated by the reactive equilibria of replicative homeostasis.
Potential viral antigenic diversity is numerical superior to any immune response; Theoretically, a small envelope protein of 20 amino acids could assume 2020 (about 1026) possible conformations, greatly exceeding the ~1010 antibody [80] or CTL receptor conformations either humoral and cellular immune responses can generate. A direct consequence of this mismatch and the stable reactive, equilibria resulting from replicative homeostasis is that once infection is established, the clinical outcome is primarily determined by the viruses' ability to maintain control of the quasispecies, rather than the hosts' response to that quasispecies. This sanguine view is supported by both general clinical experience and by kinetic analysis of chronic viral infection (Figure 2); if host responses are unable to clear virus at 105–7 viral equivalents / ml they are not likely to be any more effective at 108–11 eq/ ml.
The varied clinical outcomes of viral infections are explained by replicative homeostasis and its failure: Viral failure to down-regulate replication by RNApol inhibition would cause rapidly progressive or fulminant disease (characterised by massively polyclonal, but ultimately ineffectual, immune responses), while inadequate replication or generation of diversity will result in viral clearance (Figure 3b). Stable, homeostatic replicative equilibria will result in chronic infection with episodic fluctuations in viral replication and host responses (eg ALT; [65]) typical of chronic hepatitis or HIV. The widely varied spectrum and tempo of viral diseases, that for viral hepatitis ranges from asymptomatic healthy chronic carriage to fulminant liver disease and death within days, is far more rationally explained on the basis of a broad spectrum of polymerase properties than highly variable and unpredictable (yet genetically homogeneous) immune responses.
Homeostatic systems functioning without external perturbations – such as thermostatically controlled water tanks – progress rapidly to stasis (Figure 7). In tissue culture, viruses – replicating without immune challenge – are unable (and do not need) to generate antigenic diversity by replicative homeostasis, a phenomenon probably responsible for attenuation of virulence of serially passaged virus cultures. By contrast, in dilute viral culture, where viral envelope and polymerase exist in low concentrations, high affinity EnvWt/polymerase interactions preferentially occur over lower affinity Envmt /polymerase interactions, replicative homeostasis predicts increased viral replication and mutation would occur and this has been confirmed [70]
Figure 7 Homeostatic systems. In absence of external influence, homeostatic systems (A) progress rapidly to stasis (0) while external perturbations (arrows, e.g. immune recognition of virus) cause pseudo-chaotic fluctuating long-term behaviours in complex systems (B).
Perturbations of relative intracellular wild-type and variant envelope concentrations alter RNApol:Env interactions disturbing the replicative equilibria of replicative homeostasis. Antibodies (or CTL) will alter extracellular concentrations of Env proteins, thus changing intracellular envelope concentrations once extracellular /intracellular Env concentrations equilibrate. Therefore, antibodies to heterologous envelope proteins – developing, for example, during immunization against other viruses or heterotypic co-infection – will reduce relative intracellular concentrations of variant envelope, favouring RNApol:EnvWt interactions, thus enhancing replication and increasing mutation rates, a prediction confirmed in practice [38,56]. Contrariwise, antibodies to wild-type surface proteins – for example, during administration of anti-HBsAb following liver transplantation for HBV [63] – would reduce viral replication (Figure 6), as seen in practice. Disturbance of viral replicative equlibria by heterologous extracellular antibodies rationally explains antibody-dependent enhancement (ADE) of HIV [23], Dengue [26], Murray Valley encephalitis[84], Ebola [78] Coxsackie [24] and other viruses. Similarly, increased HIV replication and mutation after influenza [38] or tetanus [56] vaccination; reduced HIV replication during measles [50] and Dengue [85] co-infection; clearance of HBV without hepatocyte lysis or evidence of T cell dependent cytotoxicity[25], are also explained by this mechanism. Previously unexplained and problematic viral behaviours and host responses, including long-term non-progression of HIV [7]; persistence of transcriptionally active HBV despite a robust immune response [48]; long-term antigenic oscillations [54]; spontaneous reactivation of HBV[41] (among many other viruses); and hypermutation of HIV, for example, all rationally resolve within this conceptual framework.
There are clear and quite specific therapeutic implications of replicative homeostasis, as well as more general implications. The envelope/polymerase interactions of replicative homeostasis suggested herein are obvious therapeutic targets, and a site of interferon action: Heterologous envelope proteins from different viruses or genotypes of the same virus, or their structural homologues, are likely to inhibit viral replication, as suppression of HIV replication during measles [50] and Dengue [85] co-infection suggests. Interferon is ineffective for HIV and many patients with HBV, and its efficacy in HCV is highly genotype-dependent, strongly implying a direct, virus-specific action unrelated to "immune enhancement", as in-vitro data [10] and clinical kinetic studies imply [52]. Complexing of interferon to RNApol to reduce processivity and increase fidelity would explain both the genotype specificity of interferon action and the kinetics of action and, incidentally, the apparent "immune enhancement" [59] caused by interferon; if interferon reduces RNApol processivity while increasing its fidelity, viral RNAs synthesized will contain fewer mutations causing synthesis of antigenically restricted proteins, thus presenting a more homogeneous target susceptible to immune attack.
Replicative homeostasis may alter perceptions of strategies underpinning the immune responses. It is possible the primary purpose of the initial polymorphic humoral response to viral infection – typically pentameric IgM – is to push viral replication towards equilibria favouring production of homogeneous virus, thus facilitating a concerted and more focussed humoral and/or cytotoxic T cell response; Strong neutralizing IgG antibodies – antiHBsAg, for example – may develop as a consequence of initially restricted viral replication and mutation permitting effective and specific immune recognition, rather than being the proximate cause of it. The temporal profile of HBsAb, that develops well after HBVreplication falls, strongly supports this view. However, once developed, high-affinity neutralizing antibodies against wild-type virus ensure variant envelope proteins remain dominant within cells, thus maximising polymerase inhibition and inhibiting viral replication.
Replicative homeostasis is an adaptation that facilitates stable viral replication in cells and maximises probability of cell-to-cell (and host-to-host) transmission, a prerequisite for viral survival on an evolutionary time scale (Figure 3). A subtle, more primordial, and evolutionary function of envelope/polymerase interactions may explain the origins of replicative homeostasis; Polymerase function contingent upon recognition of, and response to, complex three-dimensional complementarities between polymerase and envelope proteins constitutes a sophisticated encryption technique, effectively "locking" the polymerase, thereby minimises the likelihood any competing RNA (or DNA) molecules are replicated even if correct 5' transcription initiation sequences are present. This is, again, a powerful mechanism of selection, speciation and genotype preservation. As Spiegleman suggested originally [55], in the fierce competition for finite intracellular resources, reproductive strategies that maximise proliferation of "self" genes, while thwarting propagation of "rival" genes, are strongly selected for, and are highly conserved in evolution. The interferons, and other cytokines, are cellular defence mechanisms that long antedate the immune system. If the interferons are functionally homologous to viral envelope proteins, and interact with viral RNApol to reduce processivity and replication to restrict viral replication and antigenic diversity, increasing their susceptibility to immune clearance, it is possible these genes were acquired as result of positive selection of beneficial virus-cell symbiosis occurring early in eukaryotic cellular evolution, a process responsible for retention of other genes [28].
Although proposed specifically to explain RNA viral quasispecies stability, replicative homeostasis is, fundamentally, a mechanism that regulates RNA transcription and modulates protein expression. If proteins (i.e. phenotype) modulate RNApol properties (in a manner contingent on that proteins functionality) and modulate mutations introduced into the RNA templates RNApol synthesises, a subtle form of "quality control" is exerted over protein synthesis [69]. This mechanism accelerates, and directs, adaptation: While introduction of lethal mutations to most RNA genomes may not adversely influence quasispecies, replicative homeostasis ensures any RNA mutations that do arise, and that result in beneficial phenotype(s), will favour replication of that RNA molecule, ensuring that phenotype is retained within the quasispecies. Minor change to polymerase fidelity will profoundly effect a quasispecies; as Haldane demonstrated [27], a reproductive advantage of only 0.1% is sufficient to increase a gene frequency from 0.1% to 50% over a few thousand generations (~1 year for the average patient with HCV) and this effect, therefore, represents a major moulding force in evolution. Thus, replicative homeostasis provides a powerful counterbalance to Muller's ratchet [17] and, by promoting retention and transmission of acquired phenotype, is a Lamarkian mechanism fully consistent with Darwinian principles and operative at a molecular level.
Finally, accessory proteins that alter the processivity and fidelity of both DNA-dependent RNA polymerases [31] and DNA-dependent DNA polymerases [42] to modulate polymerases activity are strongly conserved in evolution, suggesting a critical cellular function. Control of DNA-dependent RNApol transcription by DNA viruses, cellular micro-organisms (e.g. malaria), and eukaryotic cells, subtly modulating cell-surface protein expression, via replicative homeostasis, to mediate immune escape, control cell division and differentiation, or other functions would not be surprising.
Acknowledgements
I thank Professors WD Reed, MG McCall, RA Joske, Bill Musk, AE Jones and Jay Hoofnagle for critical clinical and scientific guidance and SJ Coleman, Matt and Tim for everything else.
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| 15707489 | PMC552327 | CC BY | 2021-01-04 16:38:59 | no | Virol J. 2005 Feb 11; 2:10 | utf-8 | Virol J | 2,005 | 10.1186/1743-422X-2-10 | oa_comm |
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Virol JVirology Journal1743-422XBioMed Central London 1743-422X-2-111571004810.1186/1743-422X-2-11ResearchSimian immunodeficiency virus (SIV) envelope quasispecies transmission and evolution in infant rhesus macaques after oral challenge with uncloned SIVmac251: increased diversity is associated with neutralizing antibodies and improved survival in previously immunized animals Greenier Jennifer L [email protected] Rompay Koen KA [email protected] David [email protected] Patricia [email protected] Bernard [email protected] Marta L [email protected] California National Primate Research Center, University of California, Davis, CA 95616, USA2 Duke University Medical Center, Durham, NC 27710, USA3 Laboratory of Viral Diseases, National Institutes of Health, Bethesda, MD 20892, USA4 Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA2005 14 2 2005 2 11 11 24 12 2004 14 2 2005 Copyright © 2005 Greenier et al; licensee BioMed Central Ltd.2005Greenier et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Oral infection of infant macaques with simian immunodeficiency virus (SIV) is a useful animal model to test interventions to reduce postnatal HIV transmission via breast-feeding. We previously demonstrated that immunization of infant rhesus macaques with either modified vaccinia virus Ankara (MVA) expressing SIV Gag, Pol and Env, or live-attenuated SIVmac1A11 resulted in lower viremia and longer survival compared to unimmunized controls after oral challenge with virulent SIVmac251 (Van Rompay et al., J. Virology 77:179–190, 2003). Here we evaluate the impact of these vaccines on oral transmission and evolution of SIV envelope variants.
Results
Limiting dilution analysis of SIV RNA followed by heteroduplex mobility assays of the V1–V2 envelope (env) region revealed two major env variants in the uncloned SIVmac251 inoculum. Plasma sampled from all infants 1 week after challenge contained heterogeneous SIV env populations including one or both of the most common env variants in the virus inoculum; no consistent differences in patterns of env variants were found between vaccinated and unvaccinated infants. However, SIV env variant populations diverged in most vaccinated monkeys 3 to 5 months after challenge, in association with the development of neutralizing antibodies.
Conclusions
These patterns of viral envelope diversity, immune responses and disease course in SIV-infected infant macaques are similar to observations in HIV-infected children, and underscore the relevance of this pediatric animal model. The results also support the concept that neonatal immunization with HIV vaccines might modulate disease progression in infants infected with HIV by breast-feeding.
pediatricvaccineHIVHMA
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Background
The continued need for breast-feeding in developing countries due to nutritional or socio-economic reasons poses a considerable risk for postnatal mother-to-child transmission of HIV, and breastfeeding is estimated to account for 33–50% of infant HIV infections worldwide [1-5]. This dilemma underscores the need for a vaccine that, when administered shortly after birth to the infant, could protect against HIV transmission via breast-feeding. The ultimate goal of a neonatal HIV vaccine is to prevent infection; however, vaccination of newborns of HIV-infected women early in life may elicit HIV-specific immune responses that substantially reduce infant disease progression in the event that breast milk transmission occurs.
Advances in the understanding of the mechanisms of oral transmission of HIV variants may aid the development of an effective infant HIV-1 vaccine. Recent studies have demonstrated that infants of HIV-infected women can be infected with single or multiple HIV variants [6,7] shortly before or during the birth process. However, little is known regarding the diversity of HIV transmitted by breastfeeding. These questions are difficult to address in human studies because the characteristics of HIV variants in breast-milk at the time of transmission are unknown. In addition, it is often difficult to obtain virus from infants at early times after HIV infection. Finally, the presence in infants of different levels of transplacentally transferred HIV-specific maternal antibodies with differing anti-viral properties complicates assessments of HIV variant transmission.
Longitudinal studies of HIV-infected adults have shown that the rate of disease progression is inversely related to the rate of evolution of HIV envelope quasispecies [8,9]. Also, without antiviral treatment, virus-specific immune responses are directly related to HIV quasispecies evolution [10]. The reported relationship between HIV envelope variant evolution and disease progression in HIV-infected infants and children is contradictory. Some studies have found greater HIV envelope variant evolution in rapid progressors [11-13] while other investigations have found that slowly progressing HIV-infected children have greater HIV quasispecies divergence or diversity over time [14,15]. However, all of these retrospective studies necessarily evaluated HIV variant evolution in a limited number of serial blood samples during the first months of life from a small number of HIV-infected children (two to six per cohort). More recently, a longitudinal study of 10 perinatally HIV-infected children found that changes in HIV envelope quasispecies during the first year of life were associated with a better clinical outcome [7]. A few reports have described a correlation between nascent HIV-specific immune responses, the evolution of HIV variants and disease progression in HIV-infected infants [16,17].
Simian immunodeficiency virus (SIV) infection of infant macaques is a useful and relevant animal model of pediatric HIV infection for rapidly testing the efficacy of pediatric HIV vaccine and drug interventions [18-20]. This SIV/infant macaque model was previously used to assess the efficacy of two vaccines, (i) modified vaccinia virus Ankara (MVA) expressing SIV Gag, Pol and Env (MVA-SIVgpe) and (ii) live-attenuated SIVmac1A11, against oral challenge with virulent uncloned SIVmac251. We reported an improved clinical outcome (i.e., disease-free survival) for vaccinated compared with unvaccinated infants, which was associated with reduced plasma SIV RNA and sustained SIV-specific humoral immune responses [21]. Here in this report, we used a heteroduplex mobility assay (HMA) to evaluate the genetic diversity in the V1–V2 envelope (env) region of SIV variants present in the SIVmac251 virus inoculum and compare the transmission and evolution of the SIV env quasispecies in plasma following oral inoculation of these vaccinated and unvaccinated infant macaques. Three major questions were addressed: (i) Compared to the SIVmac251 virus inoculum, are few SIV envelope variants transmitted orally?, (ii) Is the lower viremia and better clinical outcome of vaccinated infants related to the initial genetic diversity of SIV env quasispecies?, and, (iii) Is the evolution of SIV envelope quasispecies during the course of infection associated with the development of SIV neutralizing antibody? We demonstrate that while the vaccines did not modulate oral transmission of viral variants, an association was found between vaccine-induced enhanced antiviral immune responses, increased env diversity, and a slower disease course. These findings in vaccinated infant macaques are similar to observations in HIV-infected children with slow disease progression and support the relevance of the SIV infant macaque model for developing neonatal vaccine strategies to prevent pediatric HIV infection and AIDS.
Results
Characterization of variants in SIVmac251-5/98 virus stock
HMA analysis revealed that the undiluted SIVmac251-5/98 virus stock was comprised of a diverse population of V1–V2 env variants. To determine the most common variant(s) in the virus stock, six independent serial dilution experiments were conducted. Viral RNA was isolated from 1 ml of virus stock and 10-fold dilution series (undiluted to 10-9) of the RNA were prepared from 6 separate aliquots of virus stock. The resulting RNA was analyzed by RT-PCR and HMA. Figure 1 shows the results of 4 of these 6 separate virus stock dilution/HMA experiments. The observation that multiple heteroduplex bands were observed through the 10-5 or 10-6 dilutions of viral RNA indicates that the undiluted SIVmac251-5/98 stock contains multiple env variants at high frequency. An RT-PCR endpoint (i.e. dilution to a single variant) was not reached in 3 of the 6 dilution experiments. An example of this is shown in dilution series A (Figure 1). In the other 3 dilution series (Fig. 1, series B, C, and F), the last dilution that yielded an RT-PCR product consisted of a homogeneous population of envelope variants represented by one main variant (homoduplex band). This endpoint variant was designated the virus stock endpoint variant (VSEV). The fact that endpoint variants were reached at different dilutions for each dilution series is probably due to the variability at each step of these independently performed experiments.
Figure 1 Characterization of variants in SIVmac251-5/98 virus stock. HMA analysis of four separate dilution series of viral RNA from the SIVmac251-5/98 virus stock is shown. The presence of multiple bands in the undiluted samples (lane 1 of each gel) reveals the virus stock was comprised of a diverse viral population. The last lane of each gel shows the variants in the highest dilution that yielded an RT-PCR product. Dilution series A shows an example of a dilution experiment that did not result in a virus stock endpoint (homogenous variant population); the 10-6 dilution included more than 1 variant, while the next dilutions (10-7–10-9) dilution did not yield RT-PCR products, and therefore no variant pattern is shown for those dilutions. This dilution pattern was observed in 3 of 6 dilution series (other 2 not shown). For the other 3 dilution series (B, C, and F), the variant (band) remaining in the highest dilution was considered to be the most common variant, and was designated the Virus Stock Endpoint Variant (VSEV). Dilution series B: no product was amplified from the 10-7 dilution (lane 8), but a product was amplified from the 10-8 dilution (lane 9). Dilution series C: lanes 7 and 8 show the presence of 2 different variants (VSEV-1 and VSEV-2) in the endpoint dilutions (10-6 and 10-7) of this series. Dilution series F; the 10-6 dilution in this series harbored an endpoint variant that migrated to the same gel position as VSEV-2 in dilution series C.
The VSEV in dilution series B and F had different mobilities on the HMA gel (Fig. 1). Dilution series C resulted in two endpoint variants, one at 10-6 and the other at 10-7; the positions of these two VSEV corresponded to one of each of the two VSEV in dilution series B and F. Thus, the dilution of the virus stock to an RT-PCR endpoint resulted in 4 independent variants (represented by homoduplex bands) that migrated to two different positions on the HMA gels. Based on these positions, the homoduplex bands that migrated furthest were referred to as VSEV-1 and the variants that migrated a shorter distance were designated VSEV-2 (Fig. 1). To confirm that the four endpoint homoduplexes represented only two variants, an HMA mixture experiment was performed, in which all pairwise combinations of the virus stock endpoint variants were mixed prior to HMA [22]. These experiments demonstrated that the two variants designated VSEV-1 are indeed similar (i.e., = 1–2% difference in nucleotides with no insertion/deletion), as their mixtures resulted in the formation of a single homoduplex band on an HMA gel; similarly, the two variants referred to as VSEV-2 are similar (Fig. 2). In contrast, the formation of heteroduplexes and two main homoduplexes in the mixtures of VSEV-1 and VSEV-2 demonstrate that these 2 variants are significantly different from each other (Fig. 2). Thus, VSEV-1 and VSEV-2 are 2 distinct variants that exist at similar frequencies and represent the most common variants in the undiluted SIVmac251-5/98 virus stock. These results are consistent with observations of the virus stock from which SIVmac251-5/98 was made [22].
Figure 2 Characterization of the dominant variants in SIVmac251-5/98 virus stock. HMA analysis of all four endpoint variants shown in Fig. 1 (lanes 1–4) and all possible pairwise mixtures of those variants (lanes 5-10) are shown. Letters B, C, and F refer to the dilution series shown in Fig. 1. Lane numbers refer to the lane designations of the variants that were mixed in lanes 5–10 (e.g., L1 + L2 indicates that the variants shown in lanes 1 and 2 were mixed). Lane 6 shows that the 2 endpoint variants labeled VSEV-1 (B 10-8 and C 10-7) are similar variants due to the formation of a single homoduplex and no heteroduplexes when these 2 variants were mixed. Lane 9 indicates that the 2 endpoint variants labeled VSEV-2 (C 10-6 and F 10-6) in Fig. 1 are very similar. The formation of heteroduplexes and two main homoduplexes in the mixtures shown in lanes 5, 7, 8, and 10 indicate that VSEV-1 and VSEV-2 do not share the same V1–V2 envelope sequence.
Experimental design of animal experiments and summary of outcome
Nineteen newborn rhesus macaques were divided into 5 experimental vaccine groups (table 1). Group 1 (n = 5) consisted of unimmunized control animals. Group 2 (n = 2), group 3 (n = 4) and group 4 (n = 4) were vaccinated with MVA-SIVgpe at 0 and 3 weeks of age; group 4 had maternally-derived SIV antibodies (due to immunization of their mothers with inactivated SIV). Group 5 (n = 4) was immunized with live-attenuated SIVmac1A11 at 0 and 3 weeks of age. As described elsewhere [21], except for group 2, all other groups were inoculated orally with SIVmac251-5/98 at 4 weeks of age; all these animals became persistently viremic, but the immunized animals had lower virus levels, enhanced antiviral immune responses and a delayed disease course in comparison to the unimmunized animals. Four of the 5 unimmunized infected animals developed AIDS within 14 weeks of age, while the fifth animal needed euthanasia at 28 weeks. Four MVA-SIVgpe-vaccinated SIVmac251-5/98-infected animals developed AIDS by 19 to 27 weeks of age (2 animals of groups 3 and 4 each; table 1). The remaining eight vaccinated SIVmac251-5/98-infected infants, including all four SIVmac1A11-vaccinated animals, were clinically stable at the end of the observation period (28 weeks of age).
Table 1 Experimental design and summary of outcome.
Immunizationa groups and animal numbers sex MHC I allelesb Variant Patternc Week 1 Plasma Viral RNAd Time of euthanasia (wks)e
MamuA*01 MamuB*01
Group 1 Unvaccinated + SIVmac251
31319 M + + A 4.3 × 107 13
31321 M +/- - A 1.7 × 108 28
31322 F +/- +/- A 1.2 × 108 14
31325 M + + B 5.5 × 106 12
31608f F +/- +/- C 7.5 × 105 11
Group 2 MVA-SIVgpe only
31480 M - - na na na
31488 M +/- +/- na na na
Group 3 MVA-SIVgpe + SIVmac251
31378 M - - A 4.8 × 105 28g
31533 M +/- - A 3.7 × 107 26
31540 M +/- - C 2.5 × 107 28g
31542 M - - B 3.3 × 105 26
Group 4 MVA-SIVgpe with Mat. Abs. + SIVmac251
31526 M +/- +/- A 6.9 × 107 27
31732 F - +/- A 1.8 × 107 19
31833 F +/- - A/C 4.5 × 105 28g
31856 F - +/- B 1.4 × 106 28g
Group 5 SIVmac1A11 + SIVmac251
31777 F +/- - A 6.8 × 107 28g
31778 F - - B 4.7 × 105 28g
31779 F - - A 2.3 × 108 28g
31780 F +/- - A 9.9 × 107 28g
a Vaccine administered in 2 doses, at birth and 3 weeks of age. Animals of groups 1, 3, 4 and 5 were challenged orally at 4 weeks of age with SIVmac251-5/98.
b The presence of the MHC type I alleles of MamuA*01 and MamuB*01 is indicated as + (present, but unknown whether homozygous or heterozygous), +/- (heterozygous based on known haplotypes of parents), and - (homozygous for absence of particular allele).
c Variants in plasma at one week post-challenge with SIVmac251-5/98.
d Copies of viral RNA per ml one week after challenge with SIVmac251-5/98, as measured by bDNA assay.
e Age (weeks) at time of euthanasia.
f Infant 31608 was born to an SIVmac251-infected macaque, and thus had maternal anti-SIV antibodies, but no virus was detected in this infant at 4 weeks of age.
g indicates that animal was clinically stable at time of experimental euthanasia at 28 weeks of age; all other SIV-infected animals were euthanized due to life-threatening disease prior to or at 28 weeks of age. The animals of group 2 were not euthanized. na indicates not applicable.
Detection of SIV envelope variants in plasma of neonates early after oral inoculation
The genetic diversity of SIV env variant populations in the plasma of the infant monkeys one week after oral inoculation with SIVmac251 was analyzed by HMA (Fig. 3). Each plasma sample was analyzed in replicates (≥ 2) to assure reproducibility of the gel banding patterns. As indicated by the presence of heteroduplex bands, all infants were infected with multiple SIV env variants, indicating that the SIVmac251-5/98 virus stock contained several variants capable of establishing infection by the oral route. However, there were differences in HMA banding patterns. In each group, some animals had several strong heteroduplex bands; this pattern of variant transmission was referred to as infection pattern A (e.g. Fig. 3, animal 31319). In contrast, one or two infants in each group were infected with a genetically more homogenous variant population, consisting of one major variant (homoduplex band), while heteroduplex bands were less pronounced. These monkeys infected with genetically more homogeneous viral populations harbored one of two main env variants, distinguished by different electrophoretic mobilities of the homoduplexes representing these variants. These more homogeneous variant populations were referred to as infection patterns B and C (e.g. Fig. 3, animals 31325 and 31608, respectively). Infection pattern C contained a homoduplex band that migrated slightly slower than the homoduplex band characterizing infection pattern B. One newborn in each vaccine group was infected with a SIV variant of transmission pattern B. Infection pattern C was detected in one newborn of each group except the SIVmac1A11 vaccinates (table 1, group 5). Therefore, no substantial difference was observed among the different vaccine groups in viral genetic diversity in plasma collected 1 week after virus inoculation. However, all but one infant (31540) infected with more homogenous populations of env variants (infection patterns B or C) had 10- to 100-fold lower virus levels one week after SIVmac251 challenge than all but one infant (31378) infected with more heterogeneous populations of SIV variants (transmission pattern A, Table 1). This association of homogeneous viral variants with reduced SIV RNA in plasma at 1 week after infection was statistically significant (P ≤ 0.05; one-sided Fisher's Exact test) but did not persist. From week 2 after challenge throughout the duration of the study, plasma SIV RNA levels showed no correlation with the initial SIV variant pattern detected in plasma. The rate of disease progression in these animals was also not associated with the initial envelope variant transmission patterns (table 1). Further, there was no correlation between the presence of the MHC type I alleles Mamu-A*01 or Mamu-B*01 and the viral variant infection patterns, levels of SIV RNA in plasma, or disease progression (table 1).
Figure 3 Variant populations present in plasma of infant macaques one week after oral challenge with SIVmac251-5/98. RT-PCR and HMA analysis was performed on replicate samples to confirm reproducibility of the results. Three main transmission patterns were observed, labeled A (multiple variants; diverse virus population), B and C (one major homoduplex (Ho) with a few faint heteroduplexes (He); relatively homogenous virus population). One infant (31833) harbored a plasma virus population that had elements of both transmission patterns A and C. SIV251 V.S. indicates the SIVmac251-5/98 virus stock.
To determine which SIV envelope variant was present in the highest frequency in each infection pattern, serial end-point dilution experiments were performed with RNA isolated from plasma collected one week after SIVmac251 challenge, followed by RT-PCR and HMA. Similar to the methods described above, mixture experiments were then performed, including with VSEV-1 and VSEV-2. These experiments demonstrated that 1 week after infection, the most common variants in animals with the more homogenous transmission patterns B and C were similar (i.e., less than 1–2 % difference based on the absence of heteroduplex bands) to VSEV-1 and VSEV-2, respectively (data not shown). The most common variants by end-point dilution in the 11 monkeys with transmission pattern A and A/C were similar to VSEV-1 (5 animals), or VSEV-2 (5 animals) or both (1 animal).
Greater quasispecies diversity in vaccinated compared to control infants during chronic SIV infection
HMA was used to analyze the evolution of genetic diversity of V1–V2 env populations in plasma of the monkeys during the course of infection (1 week after oral SIVmac251-5/98 challenge until euthanasia) (Fig. 4). Results from two standard measures of the nucleotide sequence heterogeneity of V1–V2 env plasma variants derived from the HMA analyses are shown in Fig. 5: (i) entropy (E), an estimate of the overall viral RNA sequence complexity for each sample and, (ii) median mobility shift (MMS), a measure of the SIV quasispecies sequence divergence reflected by the degree of base-pair mismatch after DNA strand re-annealing of strands of envelope variants [8].
Figure 4 Evolution of plasma variants in SIVmac251-5/98-infected infant macaques. HMA analysis was performed on sequential plasma RNA samples, and each analysis was done at least twice to assure reproducibility. Virus diversification is evidenced by the detection of additional minor heteroduplex bands, the disappearance of major heteroduplex bands, and/or the decrease in density of the homoduplex bands. V.S. indicates the SIVmac251-5/98 virus stock. The lane numbers refer to the number of weeks after SIVmac251-5/98 inoculation (which was performed at 4 weeks of age). The homoduplex band for week 0 for animal 31780 (prior to SIVmac251 challenge) represents the vaccine virus SIVmac1A11; viral RNA levels for the other SIVmac1A11-immunized animals at this time were too low to result in a detectable RT-PCR product.
Figure 5 Evolution of viral diversity and SIV neutralizing antibody response. HMA data for each animal (Fig. 4) were further analyzed by calculating the entropy and the median mobility shift (MMS). Viral RNA levels were measured by bDNA. SIV neutralizing antibodies were determined as described in the Materials and Methods; neutralizing antibody titers below cut-off value (i.e., < 30) were given a value of 10 for presentation on these graphs. Dashed lines indicate a regression line for entropy, MMS or neutralizing antibody titer that is significantly different (P < 0.05) from zero (i.e. significantly increasing or decreasing values from 1 week to 24 weeks pc, with r2 values ≥ 0.45).
The diversity of SIV env quasispecies in plasma varied among animals at the first sample (1 week after challenge) as indicated by the gel banding pattern (Fig. 4) and entropy measures (Fig. 5). Entropy for SIV env populations was high (> 0.9) for all unvaccinated animals (Group 1) and for 7 of the 12 vaccinated animals (Fig. 5). Initial entropy < 0.9 for vaccinated animals was associated with lower SIV RNA in plasma at 1 week after challenge (P < 0.05; Fisher's Exact Test). No consistent pattern of entropy over the 24 week course of infection was observed; in two of the five controls (31321, 31608) and three of the 12 vaccinates (31533, 31732, 31780) entropy decreased near the time of euthanasia. Overall, there was no association of SIV envelope diversity as measured by entropy with either viral RNA levels or virus-specific neutralizing antibodies in plasma (see below and Fig. 5).
The sequence divergence of SIV envelope variants in plasma of each animal over time was estimated by the MMS, shown in Fig. 5. Four of the five unvaccinated animals had initial MMS values ≥ 0.5 which decreased at varying rates until the time of euthanasia; the remaining control animal (31325) had an initial MMS < 0.3 which did not change significantly over the course of infection (Fig. 5). Thus, in unvaccinated infants the population of SIV env variants in plasma exhibited either no sequence divergence or increasing sequence similarity over time; this observation is consistent with the absence of sustained SIV-specific immune responses in these animals ([21]; see below). There was no association between MMS values and SIV RNA plasma levels for these unimmunized animals.
For 3 of the 4 vaccinated animals that developed AIDS within the observation period of 28 weeks (animals 31732, 31533, 31542), we also observed little change or a decrease of genetic divergence (i.e., as measured by stable or decreasing MMS values) of plasma env variant quasispecies. In contrast, diversification in plasma SIV env variant populations (i.e., a significant increase in MSS values) was observed by 3 to 5 months of infection in 4 of the 8 vaccinated monkeys (31540, 31833, 31856 and 31778) that were still relatively healthy at 28 weeks (Fig. 5). This diversification corresponded to the detection of different patterns of heteroduplex bands and/or fainter homoduplex bands over time (Fig. 4). Although increased diversification seemed to correlate with improved disease-free survival, this diversification was not associated with any obvious changes in plasma virus levels.
SIV neutralizing antibodies in vaccinates correlate with evolution of SIV quasispecies diversity
The possibility that SIV envelope-specific immune responses were associated with the observed plasma SIV RNA levels or evolution of SIV env quasispecies was evaluated by measuring levels of plasma antibodies that neutralized the homologous challenge virus, SIVmac251-5/98, during the course of infection (Fig. 5). SIV neutralizing antibodies were detected in none of the unvaccinated control animals, but in all except one (31777) of the 12 vaccinated animals within 16 to 20 weeks after infection (Fig. 5). Although the presence of SIV neutralizing antibodies was associated with increased survival of the vaccinated animals, no obvious relationship was detected between the SIV neutralizing antibody levels and either SIV RNA plasma levels or entropy over time in vaccinated animals. However, in the 5 animals with increasing sequence divergence (i.e., increasing MMS values; animals 31540, 31526, 31833, 31856 and 31778), neutralizing antibodies were detected around the time that MMS values increased, and the neutralizing antibody response was sustained (i.e., detectable in ≥ 3 plasma samples) in these 5 animals (Fig. 5). In contrast, animals with stable or declining MMS values had sustained (31533, 31542, 31779), transiently detected (31378, 31732, 31780) or undetectable (31319, 31321, 31322, 31325, 31608, 31777) anti-SIV neutralizing antibodies. Thus, a sustained SIV neutralizing antibody response was associated with increased divergence of SIV envelope variants in plasma (P = 0.009; Fisher's Exact test).
Discussion
The present study is among the most comprehensive longitudinal studies describing SIV envelope variation in vivo following mucosal SIV infection of infant macaques. In this study, we examined the extent of genetic diversity of the SIV envelope variant pool in the plasma of infant macaques that were inoculated orally at 4 weeks of age with an uncloned, genetically diverse virus stock SIVmac251-5/98. In addition, this is the first study to evaluate whether the transmission and evolution of viral variants was modulated by two different SIV vaccines, MVA-SIVgpe and SIVmac1A11, or the presence of maternally-derived anti-SIV antibodies.
HMA analysis revealed that the animals became infected with multiple SIV envelope variant populations, but which predominantly consisted of one of two single envelope variants that were very similar to the two most common variants in the SIVmac251-5/98 stock. These results are consistent with reports of mother-to-infant HIV transmission of multiple variants [23-25], single variants [14,26,27] or both [6,28-31], but inconsistent with studies reporting vertical transmission of single, minor variants [10,26,32-34] from the mothers' virus population. This discrepancy could be explained by differences in the HIV inoculum regarding dose, virulence and genetic diversity compared to SIV. In the present study, macaques were inoculated with a relatively high dose of SIVmac251-5/98, while infection of human infants is likely to occur due to exposure to lower amounts of virus. An inherent limitation of studies of vertical transmission of HIV is that the exact timing of infection is usually unknown, and therefore the mothers' population of viral variants at the time of transmission and the source (e.g., breast-milk) and dose of virus is unknown. Our observation that oral exposure of 17 infant macaques to the same dose of the same virus stock resulted in different transmission patterns further underscores the complexity of studying variant transmission in humans, and suggests that the different outcomes observed for vertical transmission of HIV may not necessarily reflect "selection" of HIV variants but may be more a stochastic event. In this context, studies looking at the effect of heterogeneity of viral variants in the HIV-1 infected mother and the rate of vertical transmission have also shown conflicting results [6,26,35]. Also, the HIV studies mentioned focused on prenatal or intra-partum transmission, whereas our study modeled postnatal HIV transmission via breastfeeding by oral inoculation of 1-month old infant macaques with SIVmac251-5/98. The route(s) of infection in utero or during birth for individual infants and source of virus (cell-free or cell-associated) is usually unknown, and therefore different mechanisms may be responsible for viral transmission via these routes [6]. Consistent with this view, others have reported that more SIV variants were detected in orally infected newborn macaques than in infants born to SIV-infected female macaques for which transmission occurred in utero [36] or during the late breast-feeding period [37].
Neither of the SIV vaccines used in this experiment (MVA-SIVgpe and SIVmac1A11), nor the presence of maternal antibodies in one of the MVA-SIVgpe immunized groups altered which envelope variants were transmitted because in each group, some monkeys became infected with more heterogeneous and others with more homogeneous virus populations. It is possible that neither MVA-SIVgpe nor SIVmac1A11 elicited immune responses that effectively targeted the predominant SIV env variants in the SIVmac251-5/98 stock, or that anti-envelope immune responses were elicited against regions of the envelope other than V1–V2. It is also possible that vaccine-induced immune mechanisms at the time and/or site(s) of initial infection were not potent enough to modulate the variant transmission patterns.
Viral levels in plasma of monkeys with more homogeneous populations of SIV env variants tended to be lower one week after oral inoculation with SIVmac251-5/98. The higher initial virus levels in infants infected with multiple variants may reflect higher replication capacities of diverse variant populations compared to those comprised of one main variant, especially in the initial target cells during the first days of infection. We have observed this previously for adult macaques inoculated intravaginally [22]. From the second week after SIVmac251-5/98 inoculation onwards, however, there was no correlation between viral genetic complexity (measured by entropy) or divergence (measured by MMS) and plasma SIV RNA levels. Thus, once systemic infection was established, virus replication attained similar levels regardless of the initial diversity, and there was no difference in AIDS-free survival times.
Based on the measurement of MMS values, we observed little change or a decrease in genetic divergence of plasma SIV env variant quasispecies in all unvaccinated and most vaccinated animals that developed AIDS within the observation period of 28 weeks. In contrast, diversification in plasma SIV envelope variant populations was observed in 4 of the 8 vaccinated monkeys that were still relatively healthy at 28 weeks. This increased divergence of plasma viral variants at ~3 to 5 months after infection was generally associated with more sustained levels of SIV-specific neutralizing antibodies, and also of SIV Gag and Env-specific antibodies (measured by ELISA, as shown previously [21]). Similar associations between viral genetic divergence, immune parameters and/or disease progression have been described in HIV-infected adults and children [6,8,12,13,15,16,29,38-44], and recently also in juvenile macaques following intravenous or intra-rectal SIVsm inoculation [45]. Our studies extend these observations by demonstrating that this correlation of more sustained immune responses, enhanced viral divergence and slower disease progression is also observed in infant macaques following oral SIV infection. Together, these results suggest that the rate of virus evolution is determined by a combination of the extent of virus replication (which induces random mutations due to the error-prone reverse transcriptase) and selection pressures such as antiviral immune responses that promote the outgrowth of new variants. The generation of increasingly divergent viral variants ("immune escape mutants") reflects attempts of the immune system, albeit only partially effective, to control virus replication. In contrast, high viremia and little evolution of viral envelope variants is associated with severe immunodeficiency (and thus little immune selection pressure) and rapid disease progression.
Conclusions
The patterns of SIV env variant transmission and evolution in infant macaques that were inoculated orally with the same SIVmac251-5/98 stock reflect the range of results that is observed in mother-to-infant transmission of HIV, where the dose and genetic diversity of the virus at the time of transmission are unknown. While the vaccines tested here did not modulate oral transmission of viral variants, an association was found between vaccination and enhanced antiviral immune responses, increased env diversity, and a slower disease course. These findings are similar to observations in HIV-infected children with slow disease progression and underscore the relevance of the infant macaque model for developing neonatal vaccine strategies to prevent pediatric HIV infection and AIDS [46]. These results also support the concept that neonatal immunization could prevent rapid disease progression in infants who become HIV-infected by breast-feeding.
Materials and Methods
Infant immunizations, virus inoculations, and sample collection
All newborn rhesus macaques (Macaca mulatta) were from the HIV-2, SIV, type D retrovirus, and simian T-cell lymphotropic virus type 1-free colony at the California National Primate Research Center. Newborn monkeys were hand-reared in a primate nursery, and all animals were housed in accordance with American Association for Accreditation of Laboratory Animal Care standards. We adhered to the "Guide for Care and Use of Laboratory Animals" [47]. When necessary, animals were immobilized with 10 mg/kg ketamine hydrochloride (Parke-Davis, Morris Plains, NJ) injected intramuscularly (IM). EDTA-anticoagulated blood samples were collected regularly for monitoring virologic and immunologic parameters as described previously [21].
Four newborn macaques had maternally derived SIV antibodies, because their mothers had been immunized and boosted during three or four consecutive pregnancies with whole-inactivated SIVmac251 plus Montanide ISA 51 adjuvant (Seppic, Fairfield, NJ), administered intramuscularly as previously described [21]. One of two SIV vaccines was administered to newborn monkeys at birth and 3 weeks of age: Modified Vaccinia virus Ankara expressing SIVmac239 gag, pol, and env (MVA-SIVgpe) was given to 8 newborn monkeys, including the 4 with maternal antibodies. SIVmac1A11 was given to 4 newborn monkeys. Details about these vaccines are described elsewhere [21].
At 4 weeks of age, these 17 monkeys were inoculated orally with 2 doses (24 hours apart) of uncloned virulent SIVmac251. Ketamine anesthesia was used for each inoculation. Each dose consisted of 1 ml of undiluted SIVmac251 of a stock designated by lot number -5/98, and was administered atraumatically by dispensing virus slowly into the mouth with a syringe. The SIVmac251-5/98 virus stock used in this study was derived from a previous SIVmac251 stock (lot 8/95) that was serially passaged intravenously in rhesus macaques as described [21]. This SIVmac251-5/98 stock contained 1 × 105 50% tissue culture infective doses (TCID50) and 1.4 × 109 copies of RNA per ml (determined by bDNA assay).
Quantitation of plasma viral RNA
Viral RNA in plasma was quantified using a branched DNA (bDNA) signal amplification assay specific for SIV, with conditions as described previously [22].
RNA isolation and RT-PCR
RNA was extracted from plasma samples (100–140 μl) using a viral RNA isolation kit (Qiagen, Inc., Valencia, CA) following the manufacturer's protocol. A 590 bp fragment encompassing the V1–V2 region of SIV env was then amplified in a nested RT-PCR assay as previously described [22].
Analysis of SIV variants by heteroduplex mobility assay (HMA)
Genetic diversity in viral variant populations was analyzed using a modification of the HMA methods described elsewhere [22,48]. In brief, V1–V2 env fragments were generated by RT-PCR as described above, and the presence of sufficient product was confirmed on a 1.5% agarose gel. The RT-PCR products were then mixed with 1.5 μl of 10× annealing buffer (1 M NaCl, 100 mM Tris, 20 mM EDTA), denatured at 94°C for 2 minutes and placed immediately on wet ice to promote heteroduplex formation. Samples were then run on non-denaturing 5% polyacrylamide gels and stained with ethidium bromide (0.5 μg/ml). Reverse-images of the stained gels were photographed with a digital imaging system (Alpha Innotech Corporation, San Leandro, CA). All gel images were color reversed to enhance visualization of banding patterns (e.g. black bands on white background). The number of heteroduplex bands observed is a measure of SIV envelope diversity in each monkey's virus population (i.e. a large number of bands on a gel corresponds to a large number of V1–V2 variants in the sample). The RT-PCR and HMA analysis on plasma samples was performed in replicates (of at least 2) to assure reproducibility of the gel banding patterns.
To further characterize SIV envelope variants, we used an additional form of HMA analysis that assesses the relative genetic similarity of specific viral variants by comparing the HMA patterns that result from combinations of these variants. Mixtures of SIV RNA from plasma samples were analyzed by HMA to allow estimation of sequence similarity between two different homogeneous virus populations (i.e. the most common inoculum variants and/or plasma variants from infected monkeys). This "mixture analysis" is based on the HMA HIV subtyping protocol developed by Delwart et al. [49] and was performed as described previously [22]. Briefly, mixtures of equal volumes of SIV V1–V2 env PCR product amplified from two different samples were mixed and subjected to HMA analysis as described above. Mixtures that result in a single homoduplex band are 98–100% identical [48] in the nucleotide sequence of the PCR fragment analyzed (V1–V2 env region). Mixtures that result in the formation of heteroduplexes are comprised of variant populations with nucleotide sequences that differ by more than 1–2% or have an insertion/deletion (i.e., a single codon length difference will also cause a gel shift) [48]. We have validated this HMA method for SIVmac251 in a previous study [22].
Calculation of entropy and median mobility shift
All measures of entropy (E) and median mobility shift (MMS) were estimated according to methods described by Delwart et al. [8]. Images from HMA gels were captured with a CCD camera as binary TIFF files and color reversed to enhance visualization of banding patterns (e.g. black bands on white background). Each TIFF gel image file was then opened using Adobe Photoshop Version 6.0 (Adobe Inc., San Jose, CA) and edited to ensure that the lightest inter-lane areas of the gel image had "0" signal intensity (as read by the NIH Image Program and required for the Hdent program described below). Digitized gel lanes were scanned by using the plot profile function of the NIH Image Program (available at ). Lane scans within the same gel were of equal length (i.e. same number of pixels) and were recorded from positions immediately below the single-stranded DNA position to immediately below the homoduplex. The signal intensity at each pixel along the scan was transferred to a Microsoft Excel (Richmond, Wash.) file. Because different numbers of pixels per lane were acquired from different gels, each gel was standardized by partitioning into 191 divisions, the smallest number of pixels in the scans under study. This allowed the maximum distinction of fine banding patterns, while permitting unbiased comparison between gels.
The quasispecies diversity for each sample was estimated by calculating a normalized Shannon entropy, a measure of the breadth or spread of the signal distribution in each HMA gel lane, using the HDent program (available at ) as described by Delwart et al. [8]. The Shannon entropy (S) is defined as: S = - Σ (from i = 1 to N)P(i)ln [P(i)], where N is the number of partitions in a lane, and P(i) is the fraction of the total signal in partition i. The maximum possible entropy is ln(N), and we defined the normalized entropy as S/ln(N). The normalized entropy has a range of 0 to 1, where 0 reflects no diversity (all of the signal is in a single partition), and 1 reflects maximum entropy, in which the signal is evenly distributed throughout all partitions in the lane. Thus, entropy is large for lanes with many, closely spaced or overlapping bands and small for lanes with only one band or a few, narrow bands.
Shannon entropy estimates quasispecies genetic diversity by measuring the pattern of SIV V1–V2 env heteroduplex distribution in an HMA gel lane rather than the specific electrophoretic mobility of heteroduplexes. However, the electrophoretic mobility of heteroduplexes through a polyacrylamide gel is proportional to the sequence differences in reannealed DNA strands [38,48,49]. The degree of SIV quasispecies envelope sequence divergence among the V1–V2 env variants present in plasma samples was estimated by calculating a median mobility shift (MMS) for each HMA gel lane using the HDent program. The MMS is a measure of the midpoint of the total signal in an HMA gel lane that has values between 0 and 1, where 0 corresponds to the bottom of an HMA gel lane (i.e. nearest homoduplex bands) and 1 corresponds to the top of the lane. Thus, a MMS score of 1 reflects maximum sequence diversity (i.e. all heteroduplexes bands have maximum mobility reduction and no visible homoduplexes); a MMS value of 0 reflects maximum sequence similarity (> 98%) where all signal for a lane is in homoduplex bands and there are no visible heteroduplexes.
Assessment of MHC class I alleles
DNA extracted from lymphoid cells (with QIAamp® DNA mini kit, QIAgen, Valencia, CA) was used to screen for the presence of the rhesus macaque major histocompatibility complex (MHC) class I alleles Mamu A*01 and Mamu B*01, using a PCR-based technique [50,51].
Neutralizing antibodies
Neutralizing antibody titers in EDTA-anticoagulated plasma were measured according to methods described previously [52], except that CEM-R5 cells (i.e. CEM×174 cells expressing CCR5 by transfection; generously provided by James Robinson) were used. Neutralizing antibody titers were expressed as the reciprocal of the plasma dilution at which 50% of cells were protected from virus-induced killing as measured by neutral red uptake. The virus consisted of SIVmac251-5/98 briefly propagated in human PBMC.
Statistical analysis
Fisher's exact test, performed with Instat v. 2.03 (GraphPad Software, Inc., San Diego, CA), was used to evaluate possible association of SIV env V1–V2 variants detected by HMA in plasma of monkeys one week after oral SIVmac251 inoculation with the levels of plasma viral RNA at this same time point. To determine potential linear associations of Entropy values or MMS values over time and SIV neutralizing antibody levels over time, linear regression was performed using Prism v.3.0 (GraphPad Software Inc., San Diego CA). For all statistical comparisons, a P value less than 0.05 was considered significant.
Competing interests
The author(s) declare they have no competing interests.
Authors' contributions
JG carried out the HMA studies and drafted the manuscript; KVR participated in the design and coordination of the study, acquisition and analysis of data, and helped draft the manuscript; DM analyzed and interpreted all neutralizing antibody assays; PE and BM designed and provided the MVA constructs, participated in the experimental design and manuscript writing; MM designed and coordinated the study, assisted in the data analyses and helped draft the manuscript.
Acknowledgments
We thank D. Bennett, D. Brandt, I. Bolton, L. Brignolo, K. Christe, L. Hirst, A. Spinner, W. von Morgenland and the California National Primate Research Center Colony Services for expert technical assistance; M. Ma for assistance with image analysis of HMA gels; E. Delwart (Univ. of California, San Francisco) for useful discussions and suggestions. We thank Shilpa Hattangadi and Lynn Frampton for construction of recombinant MVAs. This work was supported by Public Health Science grant RR00169 from the National Center for Research Resources, NIH/NIAID grants AI39109 and AI46320 (MLM), and Elizabeth Glaser Scientist award #8-97 (MLM) from the Elizabeth Glaser Pediatric AIDS Foundation.
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| 15710048 | PMC552328 | CC BY | 2021-01-04 16:38:59 | no | Virol J. 2005 Feb 14; 2:11 | utf-8 | Virol J | 2,005 | 10.1186/1743-422X-2-11 | oa_comm |
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Virol JVirology Journal1743-422XBioMed Central London 1743-422X-2-121572535510.1186/1743-422X-2-12ResearchRecombinant Tula hantavirus shows reduced fitness but is able to survive in the presence of a parental virus: analysis of consecutive passages in a cell culture Plyusnina Angelina [email protected] Alexander [email protected] Haartman Institute, Department of Virology, University of Helsinki POB 21, FIN-00014, Helsinki, Finland2005 22 2 2005 2 12 12 1 2 2005 22 2 2005 Copyright © 2005 Plyusnina and Plyusnin; licensee BioMed Central Ltd.2005Plyusnina and Plyusnin; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Tula hantavirus carrying recombinant S RNA segment (recTULV) grew in a cell culture to the same titers as the original cell adapted variant but presented no real match to the parental virus. Our data showed that the lower competitiveness of recTULV could not be increased by pre-passaging in the cell culture. Nevertheless, the recombinant virus was able to survive in the presence of the parental virus during five consecutive passages. The observed survival time seems to be sufficient for transmission of newly formed recombinant hantaviruses in nature.
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Background
Recombination in RNA viruses serves two main purposes: (i) it generates and spreads advantageous genetic combinations; and (ii) it counters the deleterious effect of mutations that, due to the low fidelity of viral RNA polymerases and lack of proofreading, occur with high frequency [1]. The purging function is, naturally, attributed to the homologous recombination (HRec), i.e. recombination between homologous parental molecules through crossover at homologous sites. HRec was first described for the positive-sense RNA viruses [2,3] and subsequent studies lead to the widely accepted copy-choice model [4]. HRec was later shown to occur in rotaviruses thus adding double-stranded RNA viruses to the list of viruses capable of recombination [5]. Negative-sense RNA viruses that occupy the largest domain in the virus kingdom until recently were known to undergo non-homologous recombination only, forming either defective genomes, like polymerase "mosaics" of influenza A virus DI-particles [6] and "copy-backs" of parainfluenza virus [7] or hybrids between viral and cellular genes [8] or between different viral genes [9]. The first evidence for HRec in a negative-sense RNA virus has been obtained on hantaviruses [10,11].
Hantaviruses (genus Hantavirus, family Bunyaviridae) have a tripartite genome comprising the L segment encoding the RNA-polymerase, the M segment encoding two external glycoproteins, and the S segment encoding the nucleocapsid (N) protein [12]. Hantaviruses are maintained in nature in persistently infected rodents, each hantavirus type being predominantly associated with a distinct rodent host species [13]. When transmitted to humans, some hantaviruses cause hemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome, whereas other hantaviruses are apathogenic [14,15]. Persistent infection in natural hosts allows for the simultaneous presence of more than one genetically distinct hantavirus variant in the same rodent. This may result in hantavirus genome reassortment [16,17] or recombination, as proposed in the above-mentioned study of Sibold et al [10] who showed a mosaic-like structure of the S RNA segment and the N protein of Tula hantavirus (TULV). Most recently, we have shown transfection-mediated rescue of TULV with recombinant S segment, in which nt 1–332 originate from the cell culture isolate Moravia/Ma5302V/94 (or TULV02, for short) [18], nt 369–1853 originate from the strain Tula/Ma23/87 [19], and nt 333–368, that are identical in both variants, can be of either origin. Both M and L segments of the recombinant virus (recTULV) originate from TULV02 [11]. RecTULV was functionally competent but less competitive than TULV02. One reason for the observed lower fitness of the recTULV might be that it was generated in the presence of the wt variant, with which it has to compete, and thus not given enough time to to establish a well balanced, mature quasi-species population. We, therefore, decided to compare fitness of TULV02 with that of recTULV that underwent several passages in cell culture.
Results and discussion
First, we designed RT-PCR primers able to discriminate between non-recombinant (V-type) and recombinant (REC-type) types of TULV S RNA. The resullts presented in Fig. 1 show that the primer pairs designed to generate the 118 bp- long products from either V-type or REC-type S RNA amplified, indeed, homologous sequences only, whether these were taken along (lines 1 and 6) or mixed with the heterologous sequences (lines 3 and 7). Using the two specific RT-PCR conditions, the presence of V-type and REC-type S RNA was monitored on ten sequential passages of the mixture of TULV02 and RecTULV5 variants (Fig. 2). S RNA of V-type was seen on all passages (Fig. 2A, lines 1–10). In contrast, S RNA of REC-type, was detected up to the fifth passage (Fig. 2B, lines 1–5), and then disappeared (Fig. 2B, lines 6–10). An alternative approach to check the presence of the two different types of S RNA using specific primer pairs at the stage of nested PCR gave exactly the same result. The V-type S RNA was detected during all ten passages while the REC-type totally disappeared after the 5th passage (data not shown). These data confirmed our earlier observation [11] that the transfection-mediated HRec yields functionally competent and stable virus, recTULV. The purified and pre-passaged recombinant virus, however, presented no real match to the original cell adapted variant, TUL02, it terms of fitness. Taking into account that the in situ formed recombinant S RNA disappeared from the mixture after four passages [11], one should conclude that the lower competitiveness of the recombinant virus seen earlier did not result from its "immature" status. When, under similar experimental settings, TUL02 has been passaging in the presence of another isolate, TULV/Lodz, none of the two viruses was able to establish a dominance during ten consecutive passages (Plyusnin et al., unpublished data).
Figure 1 Checking of specificity of RT-PCRs for the wt and the recombinant S RNA segments. Lines 1–3: products of RT-PCR with primers VF738 and VR855 on RNA from cells infected with TULV02 (line 1), on RNA from cells infected with the recTULV (line 2) and on the mechanical mixture of both RNA preparations (line 3). Lines 5–7: the corresponding products of RT-PCR with primers RECF738 and RECR855. Lines 4 and 8 show negative controls. M, molecular weight marker; bands of 147 and 110 bp are indicated by arrows.
Figure 2 Monitoring of wt and recS-RNA during sequential passages of the mixture of TUL02 and recTULV. A. PCR-amplicons (118 bp), obtained in RT- PCR with the primers VF738 and VR855 (specific for the wt virus) on RNA from infected cells collected on passages 1 to 10. B. PCR-amplicons (118 bp), obtained in RT- PCR with the primers RECF738 and RECR855 (specific for the recombinant virus) on RNA from infected cells collected on passages 1 to 10. NC, negative controls. M, molecular weight markers; bands of 147 and 110 bp are indicated by arrows.
Although relatively short, the observed survival time of the recTULV in the presence of the original variant TUL02 seems to be sufficient for transmission of a recombinant virus, in a hypothetical in vivo situation, from one rodent to another. If transmission is performed in a sampling-like fashion – and this seems to be the case for hantaviruses [13] – the recombinant would have fair chances to survive. The existence of wt recombinant strains of TULV [10] supports this way of reasoning. Evidence for the recombination in the hantavirus evolution continues to accumulate [20,21].
The genetic swarm of S RNA molecules from the recTULV is represented almost exclusively by the variant with a single break point located between nt332 and nt368. The proportion of the dominant variant is larger in the passaged recTULV (13 of 14 cDNA clones analyzed, or 93%) than in the freshly formed mixture of recS RNAs (12 of 20 cDNA clones, or 60%) [11]. Thus, recTULV already represents a product of a micro-evolutionary play, in which the best-fit variant has been selected from the initial mixture of recS RNA. Whether this resulted from higher frequency of recombination through the "hot-spot" located between nt332 and nt368 or from the swift elimination of all other products of random recombination due to their lower fitness (the situation reported for polio- and coronaviruses [22,23]), or both, remains unclear. We favor the first explanation as the modeling of the S RNA folding suggests formation of a relatively long hairpin-like structure within the recombination "hot-spot" (Fig. 3). Secondary structure elements of this kind, which might present obstacles for sliding of the viral RNA polymerase along the template, were suggested as promoters for the template-switching in the early studies on polioviruses [22] and considered a crucial prerequisite for recombination [25,24]. The hairpin in TULV plus-sense S RNA (Fig. 3) is formed by the almost perfect inverted repeat that includes nt 344 to 374. In the minus-sense RNA, the structure is slightly weaker due to the fact that two non-canonical G:U base pairs presented in the plus-sense RNA occur as non-pairing C/A bases in the minus-sense RNA. Interestingly, in Puumala hantavirus, a hairpin-like structure formed by a highly conserved inverted repeat in the 3'-noncoding region of the S segment seems to be involved in recombination events, leading, however, to the deletion of the hairpin-forming sequences (A. Plyusnin, unpublished observations). The role of RNA folding in hantavirus recombination awaits further investigation.
Figure 3 Hairpin-like structures predicted for the recombination "hot-spot" in the plus- and minus- sense S RNA of TULV.
Conclusion
The data presented in this paper show that the recTULV presents no real match to the original cell adapted variant and that the lower fitness of the recombinant virus can not be increased by pre-passaging in cell culture. The observed survival time of the recTULV in the presence of the parental virus seems to be sufficient for transmission of newly formed recombinant hantaviruses in nature.
Methods
Recombinant TULV
RecTULV (clone 5) was purified from the mixture it formed with the original variant, TULV02, using two consequent passages under terminal dilutions [11]. After the purification, recTULV underwent three more passages, performed under standard conditions, i.e. without dilution. The presence of recS-RNA on the passages was monitored by RT-PCR and the isolate appeared to have a stable genotype (data not shown). RecTULV formed foci similar in size to those of the original variant and grew to the titers 5 × 103 – 104 FFU/ml.
Competition experiments
Vero E6 cells (5 × 106 cells) were infected with the 1:1 mixture of recTULV and TULV02, approximately 104 FFU altogether. After 7–12 days the supernatant (~20 ml) was collected and RNA was extracted from the cells with TriPure™ isolation reagent, Boehringer Mannheim. Aliquots (2 ml) of the supernatant were used to infect fresh cells; the rest was kept at -70°C. The following nine passages were performed in the same way.
Reverse transcription (RT), polymerase chain reaction (PCR) and sequencing
RT was performed with MuLV reverse transcriptase (New England Biolabs); for PCR, AmpliTaq DNA polymerase (Perkin Elmer, Roche Molecular Systems) was used. To monitor the presence of TULV S RNA on passages, RT-PCR was performed with primers VF738 (5'GCCTGAAAAGATTGAGGAGTTCC3'; nt 738–760) and VR855 (5'TTCACGTCCTAAAAGGTAAGCATCA3'; nt 831–855). To monitor the presence of recTULV S RNA, RT-PCR was performed with primers RECF738 (5'GCCAGAGAAGATTGAGGCATTTC3'; nt 738–760) and RECR855 (5'TTCTCTCCCAATTAGGTAAGCATCA3'; nt 831–855). All four primers were perfect matches to the homologous sequences; to the heterologous sequences, the forward primers have five mismatches while the reverse primers have six. Alternatively, complete S segment sequences of both variants of TULV were amplified using a single universal primer [19] and then either of the two pairs of primers was used in nested PCR. Authenticity of the PCR amplicons was confirmed by direct sequencing using the ABI PRISM Dye Terminator Sequencing kit (Perkin Elmer Applied Biosystems Division).
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
AngP participated in the design of the study, carried out the experiments and helped to draft the manuscript. AlexP participated in the design of the study and drafted the manuscript. Both authors read and approved the final manuscript.
Acknowledgements
The authors thank Prof. Åke Lundkvist for fruitful discussion and Prof. Antti Vaheri for general support. This work was supported by the research grants RFA915 and 202012 from the Academy of Finland.
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| 15725355 | PMC552329 | CC BY | 2021-01-04 16:38:59 | no | Virol J. 2005 Feb 22; 2:12 | utf-8 | Virol J | 2,005 | 10.1186/1743-422X-2-12 | oa_comm |
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Virol JVirology Journal1743-422XBioMed Central London 1743-422X-2-91570749210.1186/1743-422X-2-9ResearchInhibition of human immunodeficiency virus type-1 (HIV-1) glycoprotein-mediated cell-cell fusion by immunor (IM28) Mavoungou Donatien [email protected] Virginie [email protected] Marie-Yvonne [email protected] Brice [email protected] Elie [email protected] Centre de recherche sur les pathologies hormonales, Libreville, Gabon2 Department of Parasitology, Institute for Tropical Medicine, University of Tübingen, Tübingen, Germany3 Département de Pharmacologie, Université de Montréal, Montréal, Québec, Canada4 Département de Physiologie, Université de Montréal, Montréal, Québec, Canada2005 11 2 2005 2 9 9 22 12 2004 11 2 2005 Copyright © 2005 Mavoungou et al; licensee BioMed Central Ltd.2005Mavoungou et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Immunor (IM28), an analog of dehydroepiandrosterone (DHEA), inhibits human immunodeficiency virus type-1 (HIV-1) by inhibiting reverse transcriptase. We assessed the ability of IM28 to inhibit the cell-cell fusion mediated by HIV envelope glycoprotein in an in vitro system. For this purpose, we co-cultured TF228.1.16, a T-cell line expressing stably HIV-1 glycoprotein envelopes, with an equal number of 293/CD4+, another T cell line expressing CD4, and with the SupT1 cell line with or without IM28.
Results
In the absence of IM28, TF228.1.16 fused with 293/CD4+, inducing numerous large syncytia. Syncytia appeared more rapidly when TF228.1.16 was co-cultured with SupT1 cells than when it was co-cultured with the 293/CD4+ cell line. IM28 (1.6 – 45 μg/ml) completely inhibits cell-cell fusion. IM28 also prevented the development of new syncytia in infected cells and protected naive SupT1 cells from HIV-1 infection. Evaluation of 50% inhibitory dose (IC50) of IM28 revealed a decrease in HIV-1 replication with an IC50 of 22 mM and 50% cytotoxicity dose (CC50) as determined on MT2 cells was 75 mM giving a selectivity index of 3.4
Conclusions
These findings suggest that IM28 exerts an inhibitory action on the env proteins that mediate cell-cell fusion between infected and healthy cells. They also suggest that IM28 interferes with biochemical processes to stop the progression of existing syncytia. This property may lead to the development of a new class of therapeutic drug.
IM28envelope glycoproteinsyncitiafusion membraneHIV-1
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Background
The human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein is composed of two subunits: a surface glycoprotein (gp120) and a trans-membrane glycoprotein (gp41). These two subunits interact with each other in a non covalent manner. Gp120 is critical for attachment to host cell CD4 receptors, whereas gp41 contains the fusion sequence. HIV and simian immunodeficiency virus (SIV) require a co-receptor in addition to CD4 for entry into cells. Primary HIV can use a broad range of co-receptor molecules, including CCR1, CCR2b, CCR3, CCR4 and CXCR4 [1-3]. However, expression of a co-receptor together with CD4 on some cell types does not confer susceptibility to infection [1]. Not all human cell types that express an appropriate co-receptor support virus replication, indicating that other factors that affect viral tropism are present. HIV-1 viral entry is inhibited in the presence of the ligands to these chemokine receptors. RANTES, MIP-1α and MIP-1β, all of which are ligands for CCR5, inhibit macrophage-tropic isolates, whereas SDF-1, the specific ligand for CXCR4, inhibits entry by T-cell-tropic isolates [4-6]. The ability of HIV-1 envelope glycoproteins to induce cell-cell fusion is an interesting property because molecules that inhibit the fusion process are possible antiviral drugs and may lead to the identification of important functional regions either on the viral glycoprotein or on cell membranes. A hydrophobic, 25-amino acid, conserved segment located at the N-terminus of gp41 and gp120/41 has been shown to be involved in the fusion reaction between the viral envelope and the host cell plasma membrane [7,8]. There is evidence suggesting that this sequence is also involved in the cytopathic process underlying HIV-1 infection of target cells [9,10]. Exposure of this hydrophobic peptide to the aqueous environment in the vicinity of the target cell initially depends on gp120/41 function [11]. This protein is activated after interacting with primary receptor CD4. This activation requires the presence of human co-factors [12,13]. According to this model, further interaction of the fusion peptide to bind membrane lipid with the cell membrane depends mainly on the ability of the peptide to bind membrane lipid components. Hence, drugs that are able to interfere with membrane proteins became relevant for the therapy of HIV, even though it is still important to inhibit virus replication. We have previously shown that IM28 can inhibit HIV-1 reverse transcriptase activity [14]. Here, we assessed its capacity to inhibit the fusion of HIV-1-infected cells to naive cells. We found that IM28 was able to inhibit cell-cell fusion in an in vitro system. We showed that IM28 significantly blocks HIV-1 glycoprotein-mediated cell-cell fusion.
Results
We determined the concentrations of various drugs required to inhibit and to partially inhibit the fusion of TF228.1.16 and 293/CD4+ (Table 1). All these drugs decreased the percentage of surface covered by syncytia. The concentration of IM28 (6.43 μg/ml) that inhibited the formation of syncytia was similar to that of DXSF 500 000 (3.52 μg/ml) (Table 1). There were no statistical differences between the inhibitory concentrations of any of the drugs tested and IM28. To confirm these observations, we used SupT1 cells because fusion takes place more rapidly in these cells. These cells were mixed with TF22.1.16 cells in the presence or absence of dexamethasone or IM28 and fusion was examined by light microscopy after various periods of co-cultivation. In the absence of dexamethasone or IM28, TF228.1.16 cells fused with SupT1 cells, forming aggregates (Figure 1a). Infected cells were spindle-shaped with large syncytia after overnight culture (Figure 1b).
Table 1 Effect of drugs on fusion of TF228.1.16 cells to 293/CD4+ cells
Treatment Effect§
None F F F
IM28 F (0.60) P (1.83) I (6.43)
Dexamethasone F (0.48) P (1.67) I (5.20)
Con A F (0.09) P (0.22) I (0.79)
Heparin F (2.70) P (7.00) I (22.0)
Suramin F (1.57) P (3.90) I (15.0)
Dextran Sulfate 10,000 F (0.02) P (0.06) I (0.20)
Dextran Sulfate 500,000 F (0.37) P (1.15) I (3.52)
§F = 50–60% of the surface is covered by syncytia; P = partial inhibition of fusion: < 10% of the surface is covered by syncytia; I = inhibition of syncytia formation.
TF228.1.16 cells were mixed with 293/CD4+ cells (1:1 cell ratio) and transferred to a 24-well plate (105 cells per well in 200 μl of culture medium). TF228.1.16 cells and 293/CD4+ cells were incubated in the presence or absence of the drug (the final concentration in μg/ml is indicated in parenthesis) for 18 h. Following co-culture, three random fields of cells were photographed (not shown) and percentage fusion was determined as previously described [10].
Figure 1 Photomicrograph of SupT1 cells co-cultivated with TF 228.1.16 cells. Cell forming syncytia are aggregated (A). In the presence of dexamethasone (B) cells are mainly exploded vs. in the presence of IM28.
In the presence of dexamethasone (Figure 2) or IM28 (Figure 3), the fusion of TF228.1.16 and SupT1 cells was completely inhibited in a dose-dependent manner. Indeed, in the presence of 0.5 μg/ml dexamethasone or IM28, time of incubation had no effect on syncytia formation. This concentration of dexamethasone or IM28 did not result in the lysis of existing syncytia but stopped the fusion reaction and the appearance of new syncytia (Figure 3). The time of incubation did not affect the inhibition of syncytia in the presence of dexamethasone, but did have an effect for 0.5 μg/ml IM28. In addition, the highest concentration (> 0.5 μg/ml) of both drugs completely inhibited syncytia formation. At this concentration of dexamethasone, the inhibition of syncytia was accompanied by cell death bursting (Figure 4), whereas the same concentration of IM28 did not lead to the burst (Figure 4).
Figure 2 Effect of dexamethasone on fusion of TF228.1.16 cells with SupT1 cells. TF228.1.16 cells were mixed with SupT11 cells (1:1 cell ratio) and transferred to a 24-well plate (105 cells per well in 200 ml of cultured medium). After 24 h of co-culture in the presence or absence of dexamethasone (10 mg/ml), three random fields of cells were photographed and the percentage fusion was determined as described in Table 1.
Figure 3 Effect of IM28 on fusion of TF228.1.16 cells with SupT1 cells. TF228.1.16 cells were mixed with SupT11 cells (1:1 cell ratio) and transferred to a 24-well plate (105 cells per well in 200 μl of cultured medium). After 24 h of co-culture in the presence or absence of corticosteroids (dexamethasone or IM28) (10 μg/ml), three random fields of cells were photographed and the percentage fusion was determined as described in Table 1.
Figure 4 Effect of IM28 and dexamethasone on SupT1 cells co-cultured with TF228.1.16. Zoom of negative photomicrograph of SupT1 cultures co-cultivated with TF 228.1.16 cells (A) in the presence of dexamethasone (B) and IM28. Note the evident syncytia in (A) with an apparent slender shape of infected cells. Cells treated with dexamethasone were atrophic and sometimes exploded whereas cells incubated with IM28 were round.
To further characterize the biological effect of the drug, the 50% inhibitory dose (IC50) and the cytotoxic dose (CC50) of IM28 were evaluated and the selectivity index which is the CC50/IC50 ratio was determined. The decrease in HIV-1 replication was obtained with an IC50 of 22 mM and the CC50 as determined on MT2 cells was 75 mM giving a selectivity index of 3.4.
Discussion
IM28 is a potent new derivative of DHEA that can stop the replication of HIV-1 by inhibiting its reverse transcriptase activity [14]. Here, we show that IM28 can also prevent and inhibit the fusion of infected cells (TF228.1.16 cells) to naïve cells including 293/CD4+ cells, which are stably transfected with human CD4 and highly susceptible to HIV-1 infection, and SupT1 cells [15,16]. The fusion of 293/CD4+ cells with TF228.1.16 cells was completely inhibited by a lower dose of IM28 than was the fusion of SupT1 cells with TF228.1.16 cells (data not shown). The fusion of TF228.1.16 cells to H4CD4+ (CD4 positive glial cell line) cells obtained by transfection of human neuroglioma cells [17] is also inhibited by IM28 (not shown). Therefore, IM28 and dexamethasone may inhibit cell-cell fusion and recombination-induced fusion mediated by the HIV env protein.
Although the precise site at which IM28 acts to inhibit cell-cell fusion remains unknown, our results suggest that IM28 fights the HIV-1 virus at a new site. It is possible that this drug interacts with phospholipase A2 (PLA2), which plays an important role in the entry of HIV virus in the host cell [18,19]. Indeed, dexamethasone, a glucocorticoid, can inhibit the HIV-1, HIV-2 and SIVmac251 envelope glycoproteins and activate PLA2. PLA2 is activated when the envelope glycoprotein interacts with CD4. Due to its local membrane-destabilizing effect, PLA2 may play an important role in preparing the cell membrane for fusion with the viral particle. Activated PLA2 hydrolyzes membrane phospholipids in the sn-2 position, producing arachidonic acid and lysophospholipids [20]. These biochemical events also have downstream effects; the membrane is destabilized locally [21,22], and arachidonic acid and lysophospholipids are generated. They are potent detergents and may favor fusion [23]. In addition, arachidonic acid is the precursor of eicosanoids, prostanoids, leukotrienes and lipoxins, which may mediate further activation [24] and PLA2-induced hydrolysis of ether lipids gives rise to paf-acether [25]. It is possible that the interaction between gp120 and CD4 specifically modifies the cell membrane locally, preparing it for fusion. We hypothesize that the gp120-CD4-co-receptor complex activates PLA2 through protein kinase C (PKC) and plays a critical role in the fusion of the membrane phospholipids of the host cells and gp41 before viral entry. Indeed, the complex formed by CD4 and p56lck acts as the major receptor for HIV-1, HIV-2 and SIV, delivering intracellular activating signals. This complex binds to the viral envelope glycoprotein gp120. Following this binding, chemokine engagement appears to be required to generate the fusion active form of the envelope protein. This may involve the formation of a gp120-CD4-chemokine receptor complex, in which engagement of the chemokine receptor is dependent on a CD4-induced conformational change in env gp120 [26-28] as previously defined for the number of parameters contributing to fusion, i.e., fusion glycoproteins and the host-cell receptors [29]. However, further investigations are required to determine the real binding site of IM28. It is possible that IM28 acts on virus replication to inhibit existing syncytia, as previously reported [14]. Therefore, although the biochemical basis of this phenomenon remains to be discovered, IM28 prevents and inhibits the cell-cell fusion induced by HIV-1, giving it additional beneficial effects. Since differential ability to incorporate or maintain envelope on the virion might account for the differences in cell-to-cell versus cell-free infections in primary isolates, further studies with a more quantitative assay available for determining fusion inhibition as previously described [33,34] may also provide us with a greater understanding of the HIV-1 envelope structure and the HIV entry process.
Conclusion
In conclusion, our data show that IM28, a potent new analog of DHEA, is able to prevent and to inhibit cell-cell fusion, an important step at the beginning of HIV infection of naive cells, this drug seems to display the required properties for an anti-HIV drug.
Methods
Cell lines
Three cell lines were used: TF228.1.16, which is a BJAB cell line that stably produces functionally active HIV-1 envelope protein (BH-10 clone of HIV-1 LAI) [30]. 293/CD4+ (human embryonic kidney 293 cells which over express human CD4), obtained through the AIDS Research and Reference Reagent Program; and SupT1 cells, purchased from the American Type Culture Collection (Rockville, MD, USA).
Reagents
DHEA, dextran-sulfate (DXSF), dexamethasone, suramin, heparin, the mannose-specific lectin concanavalin A (ConA) and Rowell Park Memorial Institute (RPMI)-1640 medium were purchased from Sigma-Aldrich (St Quentin-Fallavier, France). Cells were cultured in complete medium containing L-gltamine, penicillin, streptomycin and fetal calf serum. All these reagents were purchased from Invitrogen (Eragny, France). IM28 was produced from DHEA as specified in its data sheet (INPI 0990847; Fr2792201; Wo0106666; CRPH, Gabon).
Fusion and syncytia assays
Cultured 293/CD4+ cells in complete medium were harvested by trypsinization. These cells (5 × 104) were combined with an equal number of TF228.1.16 cells in a 24-well plate and incubated overnight at 37°C in a humidified incubator with 5% carbon dioxide as described by Moore et al. 1993 [11]. Adherent cells were fixed and stained with diff-quick (Sigma-Aldrich) and then observed under a Leitz microscope.
To examine the effect of IM28 on HIV-1 envelope glycoprotein-mediated fusion, 293/CD4+ cells were mixed with TF228.1.16 cells in the presence of IM28. As a positive control for fusion inhibition, cells were incubated in parallel with dexamethasone, ConA, heparin, suramin and dextran sulfate 10 000 or 500 000, compounds known to interfere with mannose residues of envelope glycoprotein on HIV infectivity and HIV and measles virus-induced cell fusion [31,32]. The inhibitory activity of IM28 on fusion of 293/CD4+ cells with TF228.1.16 cells is expressed as a function of concentration and was compared with the inhibitory activity of the above mentioned compounds that interact with the HIV envelope protein. Fusion was examined by light microscopy after co-cultivation for 32 h. The percentage fusion is the ratio of cell surface involved in syncytia to the total cell surface. Syncytia were defined as giant cells, with diameters more than four times bigger than those of single cells. Percentage fusion was divided into three classes: 56–00% of the surface covered by syncytial = fusion; partial inhibition of fusion: < 10% of the surface is covered by syncytial = P; inhibition of syncytia formation = I.
Statistical analysis
Data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett' test. All analyses were performed using the Graph-Pad Prism® computer program. Only P < 0.05 was considered significant.
Authors' contributions
D M coordinated and participated in the design of the study, statistical analysis and the drafting of the manuscript. V P-M carried out and participated in the biological tests. M-Y A carried out and participated in the biological tests. B O carried out and participated in the biological tests. E M participated in the design of the study, carried out the biological tests and participated in the drafting of the manuscript.
Acknowledgements
This paper is dedicated to the memory of Professor Wojciech Nowaczynski for his research work on DHEA. We thank the technical and secretarial staff of CRPH for their valuable contributions.
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Aust New Zealand Health PolicyAustralia and New Zealand Health Policy1743-8462BioMed Central London 1743-8462-2-31572072810.1186/1743-8462-2-3Short ReportAustralia's international health relations in 2003 Barraclough Simon [email protected] School of Public Health, La Trobe University, Australia2005 21 2 2005 2 3 3 1 9 2004 21 2 2005 Copyright © 2005 Barraclough; licensee BioMed Central Ltd.2005Barraclough; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
A survey for the year 2003 of significant developments in Australia's official international health relations, and their domestic ramifications, is presented. The discussion is set within the broader context of Australian foreign policy. Sources include official documents, media reports and consultations with officers of the Department of Health and Ageing responsible for international linkages.
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Australia's health relations with other nations in the field of health constitute an important sub-set of health policy not only because of the intrinsic significance of bi-lateral and multilateral linkages, but also because of their ramifications for health policy at the domestic level.
In broad terms, these health relations encompass a range of interactions with consequences for health, including: membership of global and regional bodies; the negotiation of international agreements; action to counter particular external threats to health; assistance to developing countries; and international trade and investment in health-related goods and services. In 2003 there were continuing developments in all these areas within a wider foreign affairs context overshadowed by official policy concerns about global and regional security, the deployment of the Australian armed forces in various theatres of service, and renewed fears of the human and economic costs of infectious diseases. Balancing these concerns with national defence were renewed efforts to forge bi-lateral trade links in global trade environment characterized by the emergence of trade blocs centred in North America, Europe and Southeast Asia.
Although consultation occurs with states and territories, it is the Australian Government that is constitutionally responsible for conducting Australia's international relations. These responsibilities include appointing representatives to international bodies and organizations, such as the United Nations and its various agencies, including the World Health Organization and assenting to agreements and regulations promulgated by international agencies. The formulation and implementation of policy with direct or indirect international health ramifications is not centralized, but is usually the result of consultations between various relevant government departments and statutory authorities.
An important element of the Australian Government's foreign affairs powers relates to international treaties. While a degree of consultation with state and territory governments and with the public occurs, and the national parliament is able to scrutinize and comment upon international treaties, it is the executive that has the final decision on such agreements.
WHO and other international agencies concerned with health
In 2003 Australia continued to play a strategically important and respected role in international organizations concerned with health, especially the World Health Organization. At the World Health Assembly, the governing body of the WHO, the Australian delegation supported resolutions concerned with strengthening nursing and midwifery and child and adolescent health. In the wake of SARS, Australia also supported the review of the International Health Regulations and is likely to subscribe to them [1]. The voluntary nature of WHO standards and regulations, which can be accepted or rejected by member states, is well illustrated by the International Health Regulations since Australia and Papua New Guinea declined to accept them when they were last promulgated. Australia should be better placed to influence developments in WHO in the next three years as a result of being nominated for a term on the Executive Board.
The Department of Health and Ageing was closely involved with international comparative health data projects including WHO's World Health Survey and the health systems performance survey of the Organization for Economic Cooperation and Development. Australia's participation in the health mandate of the Commonwealth of Nations was illustrated by the Therapeutic Goods Administration's provision of a secretariat for the Clearing House of Commonwealth Agencies for Chemical Safety. Australia also participated in the meeting of Commonwealth health ministers on the eve of the annual World Health Assembly of WHO in Geneva [2].
International agreements
In December Australia signed the Framework Convention on Tobacco Control (FCTC) the first multilateral treaty negotiated under the auspices of the World Health Organization. For the first time, nations were invited to implement control measures covering such issues as health warnings, advertising, packaging and labelling, sales, and smuggling. They were also called upon to embrace policy measures designed to counter the global tobacco epidemic [3]. The FCTC provided an impetus to the domestic policies of many countries with limited progress on tobacco control and also allowed for the transnational activities of tobacco corporations to be countered with global policy action. The FCTC has limited potential to further Australian domestic policy, which is in advance of that in most countries. If necessary, the Australian Government could call upon its "external affairs" to assert constitutional primacy over this policy area. However, this is unlikely in the context of close cooperation between various levels of government in Australia in establishing national tobacco control policies. Australian leadership was evident in WHO's formulation of the FCTC, having been nominated by the Western Pacific region as vice-chair of the Bureau for the Negotiating Body.
A reciprocal health care agreement with Norway was signed, further expanding the rights of Australian residents to immediate and necessary treatment in the national health systems of countries with which Australia has reciprocal treaties. These include New Zealand, UK, Italy, Malta, Holland, Sweden, Finland, and the Republic of Ireland. These arrangements are "cost neutral" and do not include costly accounting or administrative procedures. In terms of domestic policy, the continuing "internationalisation" of Medicare (pioneered by the Hawke Labor Party ministry at the time of Medicare's introduction) by the Liberal-National Party Coalition is paradoxical since local citizens are being encouraged to opt out of public hospital treatment through a rebate on private health insurance and penalties for higher income earners who do not insure privately. Whilst these agreements have cemented closer diplomatic ties, their potential benefits to international travellers, especially those subject to punitive insurance premiums or the refusal of insurance due to old age or infirmity, remain inadequately publicized. Treaties are also being negotiated with Denmark and Belgium.
Following years of negotiations and planning, a treaty was signed with New Zealand establishing a single joint therapeutic goods agency. This body, due to commence operations in 2005, will regulate prescription and retail drugs, therapeutic devices and also complementary medicines. It will replace the Australian Therapeutic Goods Administration and its New Zealand counterpart. To a large extent, the two regulatory systems will have been integrated, although there are still areas of disagreement (e.g. policies on the advertising of PBS medicines) which will need to be negotiated. This joint agency creates a model in international health relations which other states could profitably emulate where they share common concerns and have similar health systems. In December 2002 the two countries finalized treaty arrangements establishing both a joint standards code and a joint statutory authority, Food Standards Australia New Zealand [4]. These arrangements parallel bi-lateral developments for the joint regulation of food standards.
These developments have furthered Australian foreign policy concerned with establishing trans-Tasman free trade, commenced some two decades ago with the negotiation of the Closer Economic Relations agreement with New Zealand. The new regulatory arrangements have created a virtual trans-Tasman free market in food (subject to plant and animal quarantine considerations) and therapeutic drugs.
While not having the legal status of a treaty, for some years the Department of Health and Ageing has had memoranda of understanding with its counterparts in China, Indonesia, Thailand and Japan. In 2003 further activities were undertaken under the auspices of these agreements. During the state visit of China's president Hu Jinta, a plan of action was signed between the two health ministries. The Indonesian relationship continued with the inclusion of a health delegation to the Sixth Australia Indonesia Ministerial Forum in Jakarta in March, preceded by two rounds of meetings between officials of the Indonesian and Australian health departments. The Australia-Japan Partnership in Health and Family Services formed the basis for negotiations for joint research on mental health and an international conference on suicide prevention [5]. In a related development, the Department of Foreign Affairs and Trade promoted aged care expertise as an export service through the Australia Japan Conference.
In the course of 2003 Australia finalized free trade agreements (in reality, preferential trade agreements) with Singapore and Thailand and continued negotiations with the USA [6]. From the perspective of the Australian health industry, the agreement with Singapore offered tariff-free trade in pharmaceuticals and other therapeutic goods and the gradual removal of tariffs in the case of Thailand. All countries imposed reservations on free trade in the sensitive areas of health services, although traditional Thai massage exponents will be permitted to operate in Australia. Domestically, these agreements required intersectoral policy collaboration in the interests of health. Policy makers in the Department of Health needed to intensify their understanding of the dynamics of international trade, while those making foreign policy had to consider the health dimensions of ostensibly commercial arrangements.
The free trade agreement with the USA raised controversies about attempts to include the Pharmaceutical Benefits Scheme (PBS) in concessions demanded by US negotiators. These issues have been outlined in the account of developments in the PBS elsewhere in this series of review articles.
SARS
The emergence and rapid spread of Severe Acute Respiratory Syndrome (SARS) to several countries in East and Southeast Asia and to Canada revived popular atavistic fears of pandemics and damaged the tourism and travel industry, as well as some Australian suppliers of goods and services to Asia. WHO issued a global alert on the disease in March, and the last reported case of international occurred in July. So serious was the threat of SARS to the economies of some countries that a special meeting of health ministers, attended by Australia, was organized by Asia-Pacific Economic Cooperation (APEC) to discuss the situation. A task force was subsequently established by APEC to deal with SARS. An example of the economic costs of the disease was the decision by the Governments of Singapore and Australia to postpone negotiations on a greater share of the Sydney-Los Angeles air route (dominated by QANTAS) for the Singapore carrier, due to uncertainty about demand.
In Australia SARS was declared a quarantinable disease under the Quarantine Act 1908 and policy guidelines for health professionals, airline and border control staff and the general public were developed by the Department of Health and Ageing, which also led an inter-departmental task force to monitor world developments. Until July 2003, when the WHO announced that no country was still considered SARS-affected, international aircraft arriving at Australian airports were required to obtain "SARS-free" clearance, nurses were posted at airports and restrictions on elective surgery were placed on travellers returning from affected countries [7]. During the period of WHO's alert, Australia had reported only five probable, and one laboratory-confirmed, case of SARS.
Health and foreign aid
Australia's official international development assistance programme is an important foreign policy tool, especially in the Asia-Pacific region. Some $225 m. (of a total of $ 1.8 b.) was allocated to health-related international development assistance in 2003–4 budget of the Australian Agency for International Development (AusAID). However, while Australia's contribution to HIV/AIDS control and its regional advisory role associated with SARS were acknowledged by the Foreign Affairs Minister in his report to Parliament, health assistance received little prominence. Security, good governance and counter-terrorism were emphasized as the focus for the official foreign aid programme. Support for essential services in Papua New Guinea continued as a major imperative [8]. The lower priority of health was further underscored by a decision to no longer appoint designated health advisors to the permanent staff of AusAID. It should be noted, however, that the emergence of SARS served to reinforce health as an important element on the international assistance agenda.
Global health workforce mobility
The fact that the domestic health workforce is now part of a global market for skilled workers was further demonstrated by continuing efforts to recruit nurses from overseas, the decision of the Australian Health Ministers Conference to sanction dentists from selected Commonwealth countries to work in public clinics. In addition, a scheme to recruit overseas-trained medical practitioners was included in the Australian Government's Medicare Plus policy initiatives [9]. It is intended that these doctors will work in rural and remote areas officially designated as having medical workforce shortages and also in positions within Aboriginal Controlled Community Health Services. Yet, metropolitan hospitals are have also become reliant upon overseas-trained doctors for their staffing. This policy, accompanied by the liberalization of immigration arrangements for medical doctors, has represented a volte face from previous policies deliberately designed to discourage foreign doctors from immigrating in the belief that controlling the number of doctors would contribute to cost-containments of Medicare. It also continues to raise the ethical danger of Australia contributing to a "brain drain" of medical staff from countries that are themselves short of such expertise. In 2002 the Commonwealth of Nations had agreed to a code of practice for the international recruitment of health workers to help safeguard the interests of developing nations. Australia has endorsed the code.
The Australian Government will need to handle policies associated with the recruitment of overseas-trained health personnel with care due to professional sensitivities and the need for legislation at the state level to regularize the status of some professions.
Concluding observations
This brief review of Australia's international health relations in 2003 has demonstrated that health must be seen as an integral part of trade and security within the wider foreign policy context. The protection of health in free trade arrangements is important for their domestic legitimacy. It is vital that those involved in health policy are aware of its potential international dimensions, while those responsible for foreign affairs include health in their approach. Official health linkages have served to promote good will in some otherwise difficult relationships, as has been the case with Indonesia. They have also helped to promote a positive international image for Australia.
Note
The opinions expressed in this article are the sole responsibility of the author.
==== Refs
Department of Health and Ageing (2003) Annual Report 2002–03
International Health, Issue 11, Winter 2003
World Health Organization, WHO Framework Convention on Tobacco Control, Geneva, 2003
International Health, Issue 10, Summer 2003
International Health, Issue 10, Summer 2003
Department of Foreign Affairs and Trade (2003), WTO and Free Trade Agreements accessed 26.2.04
Department of Health and Ageing (2003) Media Release, 29 July 2003
Australian Agency for International Development (2003) Aid Budget Summary 2003–04
Department of Health and Ageing (2003) Medicare Factsheet 3
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Cell Commun SignalCell communication and signaling : CCS1478-811XBioMed Central London 1478-811X-3-21568353910.1186/1478-811X-3-2ResearchPlatelet-derived growth factor modulates rat vascular smooth muscle cell responses on laminin-5 via mitogen-activated protein kinase-sensitive pathways Kingsley Karl [email protected] George E [email protected] Department of Biomedical Sciences, University of Nevada, Las Vegas, School of Dental Medicine, 1001 Shadow Lane B-234, Las Vegas, Nevada, 89106-4124, USA2 Department of Biology, Rensselear Polytechnic Institute, 110 8th Street, Troy, New York, 12180-3596, USA3 (previous institutional affiliation) Department of Biological Sciences, University of Nevada, Las Vegas, 4505 Maryland Parkway, Box 454004, Las Vegas, Nevada, 89154-4004, USA2005 31 1 2005 3 2 2 27 7 2004 31 1 2005 Copyright © 2005 Kingsley and Plopper; licensee BioMed Central Ltd.2005Kingsley and Plopper; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
A treatment to remove vascular blockages, angioplasty, can cause damage to the vessel wall and a subsequent abnormal wound healing response, known as restenosis. Vascular smooth muscle cells (VSMC) lining the vessel wall respond to growth factors and other stimuli released by injured cells. However, the extracellular matrix (ECM) may differentially modulate VSMC responses to these growth factors, such as proliferation, migration and adhesion. Our previous reports of low-level expression of one ECM molecule, laminin-5, in normal and injured vessels suggest that laminin-5, in addition to growth factors, may mediate VSMC response following vascular injury. To elucidate VSMC response on laminin-5 we investigated-the role of platelet-derived growth factor (PDGF-BB) in activating the mitogen-activated protein kinase (MAPK) signaling cascade as a possible link between growth-factor initiated phenotypic changes in vitro and the ECM.
Results
Using a system of in vitro assays we assessed rat vascular smooth muscle cell (rVSMC) responses plated on laminin-5 to the addition of exogenous, soluble PDGF-BB. Our results indicate that although laminin-5 induces haptotactic migration of rVSMC, the addition of PDGF-BB significantly increases rVSMC migration on laminin-5, which is inhibited in a dose-dependent manner by the MAPK inhibitor, PD98059, and transforming growth factor (TGF-β1). In addition, PDGF-BB greatly reduces rVSMC adhesion to laminin-5, an effect that is reversible by MAPK inhibition or the addition of TGF-β1. In addition, this reduction in adhesion is less significant on another ECM substrate, fibronectin and is reversible using TGF-β1 but not MAPK inhibition. PDGF-BB also strongly increased rVSMC proliferation on laminin-5, but had no effect on rVSMC plated on fibronectin. Finally, plating rVSMC on laminin-5 did not induce an increase in MAPK activation, while plating on fibronectin or the addition of soluble PDGF-BB did.
Conclusion
These results suggest that rVSMC binding to laminin-5 activates integrin-dependent intracellular signaling cascades that are different from those of fibronectin or PDGF-BB, causing rVSMC to respond more acutely to the inhibition of MAPK. In contrast, our results suggest that fibronectin and PDGF-BB may activate parallel, reinforcing intracellular signaling cascades that converge in the activation of MAPK and are therefore less sensitive to MAPK inhibition. These results suggest a partial mechanism to explain the regulation of rVSMC behaviors, including migration, adhesion, and proliferation that may be responsible for the progression of restenosis.
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Background
Angioplasty is a procedure designed to treat vascular stenosis, blockage(s), or atherosclerotic lesions, but it may also, simultaneously, cause damage to the integrity of the blood vessel wall. Restenosis is the subsequent narrowing and occlusion of the blood vessel in response to the injury or damage sustained during angioplastic procedures such as balloon dilation [1]. During restenosis, vascular smooth muscle cells (VSMC) from the injured blood vessel wall migrate into the lumen of the vessel, creating a new or neointima. The subsequent proliferation of these neointimal VSMC can lead to a thickening of this neointimal layer and re-occlusion of the vessel [1].
The characteristic response of VSMC, endothelial cells, platelets, and macrophages at the site of injury is the release of specific soluble growth factors which include platelet-derived growth factor (PDGF), transforming growth factor (TGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) [2-4]. VSMC of the vessel wall respond to these factors by secreting proteolytic matrix metalloproteinases that degrade the extracellular matrix (ECM) and stimulate deposition of new ECM proteins such as collagen, elastin, fibronectin, and laminin in the neointima [5-7]. These ECM modulate VSMC integrin-dependent behaviors such as transluminal migration, adhesion, and proliferation [8-10].
To date, the precise molecular mechanisms that link growth factor-initiated intracellular signaling to ECM-mediated adhesion, migration, and proliferation of VSMC are still unknown. Our previous reports of low-level laminin-5 expression in the intima of normal vasculature and an increased expression of laminin-5 in the neointima of injured vessels suggest that laminin-5, in addition to PDGF and TGF, may mediate VSMC responsiveness following vascular injury [11-13].
To further elucidate VSMC response to growth factors and the intracellular signaling cascades that may be linked to ECM-mediated adhesion, we used in vitro assays to study the role of laminin-5 in modulating these behaviors in rat vascular smooth muscle cells (rVSMC). We report here that PDGF induces differential responses in rVSMC behaviors on laminin-5, but not on fibronectin. In addition, we find that the PDGF-induced responses on laminin-5 are inhibited in a dose-dependent manner by an inhibitor of the mitogen-activated protein kinase (MAPK) pathway, PD98059, but not on fibronectin.
These differences in MAPK-sensitive rVSMC responses in vitro may be the result of different signaling pathways that are initiated by integrin-mediated adhesion to laminin-5. We suggest that rVSMC binding to laminin-5 may initially activate a MAPK-independent signaling cascade that may make these cells more responsive to MAPK inhibition. In contrast, rVSMC binding to fibronectin may activate a signaling pathway shared by PDGF that ultimately converges in MAPK activation. These results suggest that a complex interaction of ECM and growth factors may closely regulate VSMC behavior following vascular injury and these studies may directly identify define molecular targets that may reduce the incidence of restenosis following angioplasty.
Results
Migration
We have previously reported that laminin-5 expression by rat vascular smooth muscle cells (rVSMC) is upregulated by platelet-derived growth factor (PDGF-BB) and that laminin-5 specifically enhances PDGF-BB-stimulated rVSMC migration [12]. In addition, our previous results suggested that PD98059 (a specific inhibitor of MEK and a general inhibitor of the mitogen-activated protein kinase ERK1/2 pathway) blocked PDGF-BB-stimulated migration on laminin-5 [13]. The current study characterizes this synergistic relationship between PDGF-BB and laminin-5 in the modulation of rVSMC cell behaviors, including migration.
The level of rVSMC migration was observed over 18 hours in transwell migration filters in the presence or absence of laminin-5, with and without PDGF-BB or serum. Maximal cell migration (chemotaxis) was obtained using cell migration media containing ten (10) percent fetal calf serum (FCS). Similar to our previous reports, laminin-5-coated wells induced a greater than four-fold increase in rVSMC migration over that measured in naked plastic controls in three independent experiments as shown in Figure 1 (n = 24, p < 0.05). Furthermore, PDGF-BB, tested over a biologically relevant range of 5 – 50 ng/mL, stimulated a dose-dependent increase in rVSMC migration on laminin-5 (between 5 – 25 ng/mL), increasing migration fifteen (15) to sixty (60) percent over laminin-5-stimulated migration alone (n = 24, p < 0.005). This PDGF-BB-stimulated increase in migration peaked at 25 ng/mL and did not significantly increase over the range of 25 – 50 ng/mL (data not shown).
Figure 1 PDGF-BB increased rVSMC migration on laminin-5 in vitro. The addition of PDGF-BB increased rVSMC migration on laminin-5, in a dose-dependent manner, over rVSMC haptotactic migration induced by the presence of laminin-5. rVSMC migration on laminin-5 was inhibited by both PD98059 (MEK1 inhibitor) and TGF-β1, in a dose-dependent manner.
The mitogen-activated protein kinase (MAPK) pathway is known to be activated by PDGF-BB-stimulation in VSMC [14]. To explore the mechanism of increased rVSMC migration on laminin-5 via PDGF-BB-stimulation, we used PD98059 which is a specific inhibitor of MEK1 and MEK2, and a general inhibitor of MAPK activation. The addition of PD98059 in concentrations between 10 – 50 ng/mL reduced PDGF-BB-stimulated migration on laminin-5 (25 ng/mL) in a dose-dependent manner, reaching a maximal reduction of sixty (60) to seventy (70) percent over the range of 20 – 40 ng/mL, as shown in Figure 1 (n = 24, p < 0.005). This reduction in PDGF-BB-stimulated migration on laminin-5 was maintained at all concentrations of PDGF-BB tested (5 – 25 ng/mL).
To determine if this modulation is restricted to MEK/MAPK-inhibition, we tested the effects of adding exogenous transforming growth factor (TGF-β1) for its ability to modulate the PDGF-stimulated increase in rVSMC migration on laminin-5. Our results indicated that the addition of TGF-β1 was able to reduce laminin-5-stimulated rVSMC migration to levels approximating the levels observed in naked plastic controls over all tested ranges (5 – 50 ng/mL), although the most statistically distinct reduction was found over the range of 5 – 25 ng/mL, as shown in Figure 1. In addition, TGF-β1 was able to reduce maximal PDGF-BB-stimulated migration of rVSMC (25 ng/mL) on laminin-5 by approximately fifty (50) to sixty (60) to percent, over the range of concentrations from 20 to 40 ng/mL.
Adhesion
To determine whether or not the PDGF-BB-stimulated increase in rVSMC migration correlates with a reduction in rVSMC adhesion to laminin-5, thirty-minute in vitro adhesion assays were performed. In the absence of exogenous growth factor stimulation, laminin-5- and fibronectin-coated wells (20 μg/mL) sustained an approximate two and a half-fold increase in rVSMC adhesion compared with negative controls, as shown in Figure 2 (n = 24, p < 0.05). The addition of PDGF-BB, at the maximal migration-stimulating dose of 25 ng/mL, decreased the adhesion of rVSMC on laminin-5 by more than sixty-five (65) percent (n = 24, p < 0.005). The addition of exogenous PDGF-BB (25 ng/mL), however, decreased rVSMC adhesion on fibronectin by less than thirty (30) percent.
Figure 2 PDGF-BB reduces rVSMC adhesion on laminin-5 adhesion in vitro. The presence of laminin-5 supported greater adhesion of rVSMC over naked plastic, and this increase in adhesion was reduced by the addition of PDGF-BB. Inhibition of MEK1, using PD98059 restored rVSMC adhesion to laminin-5 in the presence of PDGF-BB. Although fibronectin also supported rVSMC adhesion, the effect of adding PDGF-BB was less pronounced and was not restored using PD98059. The addition of TGF-β1, however, completely restored rVSMC adhesion to both fibronectin and laminin-5 in the presence of PDGF-BB.
To investigate if the relationship between the PDGF-BB-stimulated increase in migration and corresponding decrease in adhesion of rVSMC on laminin-5 may be related to MAPK activation, cells were pre-treated with PD98059 (40 ng/mL) for twenty (20) minutes prior to assay and the adhesion media was supplemented with PD98059 (40 ng/mL). The addition of exogenous PD98059 (40 ng/mL) restored the PDGF-BB-stimulated reduction (25 ng/mL PDGF-BB) in rVSMC adhesion to laminin-5, to approximately eighty-five (85) percent of laminin-5 controls (n = 24, p < 0.05). The addition of PD98059, however, did not restore the thirty (30) percent PDGF-BB-stimulated reduction in rVSMC adhesion on fibronectin.
To determine if this modulation is restricted to MEK/MAPK-inhibition, we tested the effects of adding exogenous transforming growth factor (TGF-β1). Our results indicated that the addition of TGF-β1 (25 ng/mL) was able to restore the PDGF-BB-stimulated reduction in rVSMC adhesion on laminin-5 by roughly the same level as PD98059, eighty two (82) percent versus eighty five (85) percent, respectively. In contrast to PD98059, the addition of TGF-β1 (25 ng/mL) was sufficient to restore the PDGF-BB-stimulated reduction in rVSMC adhesion on fibronectin.
Proliferation
Based upon our observations of rVSMC migration and adhesion, we performed in vitro proliferation assays to determine the relative effects of the extracellular matrix (ECM) and exogenous growth factors described above. First, to test the effects of the ECM substrate on rVSMC proliferation in the absence of exogenous growth factors, quiescent cells were plated in cell-culture plates either coated with or without laminin-5 or fibronectin, in serum-free Dulbecco's Modified Eagle's Medium (DMEM) for one (1) to six (6) days.
To induce quiescence, rVSMC were incubated for forty eight (48) hours without mitogen (0% FCS, FBS) at 37°C and quiescence was verified by proliferation controls, cultured for forty eight (48) hours at 37°C with mitogen stimulus as shown in Figure 3a and 3b. Previous studies with VSMC report that greater than 95% of cells incubated in low-serum media (0.4% FCS or less) were arrested in G0(G1) between forty eight (48) and seventy two (72) hours as determined by flow cytometry and determination of [3H] thymidine-labeled nuclei [15].
Figure 3 The effect of growth factors on rVSMC proliferation in vitro. The addition of PDGF-BB stimulated proliferation of rVSMC on laminin-5 and to some extent on naked plastic, but not on fibronectin. The presence of fibronectin alone was able to stimulate proliferation of rVSMC. The proliferative response of rVSMC to presence of laminin-5 with PDGF-BB or to fibronectin was suppressed by the addition of TGF-β1. Figure 3b The ECM induces differential rVSMC proliferation responses in vitro. The plating of rVSMC on fibronectin, but not laminin, induced increases in proliferation over four days. The addition of PDGF-BB, however, increased rVSMC migration on laminin-5, but not on fibronectin. The addition of TGF-β1 was sufficient to suppress rVSMC proliferation on both laminin-5 and fibronectin.
Our results suggest that culture of rVSMC plated on exogenous laminin-5 (coated at a concentration of 20 μg/mL) did not significantly increase cellular proliferation compared with naked plastic controls over a period of four (4) days, as shown in Figure 3a (n = 24, p < 0.02). Culturing of rVSMC plated on exogenous fibronectin (coated at a concentration of 20 μg/mL) however, did significantly increase cellular proliferation over a period of four (4) days by nearly two-fold, as shown in Figures 3a and 3b.
Next, to test the modulating effects of exogenous growth factors on rVSMC proliferation when cultured on these ECM substrates, quiescent cells were plated in cell-culture plates either naked or coated with laminin-5 or fibronectin, in the presence of TGF-β1 or PDGF-BB, both at a concentration of 25 ng/mL, from one (1) to six (6) days. Our results indicated that the addition of exogenous TGF-β1 (25 ng/mL) to the cell culture medium was sufficient to induce a suppressing effect on rVSMC proliferation on naked plastic, as well as laminin-5 and fibronectin, to levels approximating the quiescent growth controls.
The addition of PDGF-BB was sufficient to induce an increase in rVSMC proliferation on laminin-5 of nearly one-half (46%) over laminin-5 DMEM (no serum, no mitogen) controls (n = 24, p < 0.01). However, our results indicate that the addition of exogenous PDGF-BB (25 ng/mL) did not have a statistically significant effect on cells plated on fibronectin.
Mitogen Activated Protein Kinase (MAPK) Western Blot
To examine MAPK activation, rVSMC were plated onto laminin-5- or fibronectin-coated plates, then lysed after thirty minutes and prepared for immunoblotting. Our results indicated that laminin-5 did not induce a detectable increase in MAPK activation over a thirty (30) minute time interval, as shown in Figure 4. rVSMC plated on fibronectin did exhibit a significant increase in MAPK levels after thirty (30) minutes.
Figure 4 The MAPK pathway in rVSMC is activated by different stimuli. The addition of FCS or PDGF-BB or the plating of rVSMC on fibronectin was sufficient to induce measurable increases in p44/p42 activation over 30 minutes. However, the addition of TGF-β1 or the plating of rVSMC on laminin-5 was not sufficient to induce MAPK activation.
The addition of growth factors, such as PDGF-BB and TGF-β1, have been associated with increases in p44/p42 (ERK1/2) or MAPK activation [16-18]. To determine the effects of PDGF-BB and TGF-β1 on rVSMC, subconfluent cell cultures were pre-treated with FCS (10%), PDGF-BB (25 ng/mL) or TGF-β1 (25 ng/mL) for thirty (30) minutes, then lysed and prepared for immunoblotting. p44/p42 phosphorylation levels were not detectable in DMEM-treated control cells, as shown in Figure 4. However, the addition of PDGF-BB, but not TGF-β1, was sufficient to induce measurable increases in MAPK activation levels.
Discussion
PDGF-BB is an in vitro VSMC mitogen and may be responsible for initiating the phenotypic changes in VSMC migration and proliferation during restenosis in vivo [19,20]. Our recent reports of low-level laminin-5 expression in the intima of normal arteries and increased expression in the neointima of injured arteries suggest that laminin-5, in conjunction with soluble growth factors and mitogens, may determine VSMC phenotype during restenosis [11-13].
The current study augments the body of evidence that suggests VSMC growth is influenced by an ECM-VSMC interaction [21] and that VSMC proliferation in different species respond differently to growth factor stimulus [15]. More specifically, this study explores evidence that laminin-5 and PDGF-BB may have combined and synergistic effects in determining rVSMC phenotype in vitro [6,8,10]. More specifically, our studies suggest that PDGF-BB strongly influences rVSMC behaviors such as migration. In addition, PDGF-BB significantly alters other cellular behaviors such as adhesion and proliferation on laminin-5, but only to a lesser extent on fibronectin.
Because PDGF-BB stimulation increases the overall levels of intracellular MAPK, as well as MAPK phosphorylation, we sought to explore this signaling cascade to determine its role in modulating these rVSMC behaviors on laminin-5 [22]. Specifically, the ERK1/2 form of MAPK mediates signaling by PDGF-AA, PDGF-AB, and PDGF-BB in VSMC, as well as signaling through laminin-5 binding integrins, and is therefore the most likely signaling molecule to modulate these cellular behaviors [23-25]. Our results suggest that rVSMC behaviors in vitro, driven by PDGF-BB responsiveness, can be blocked by MAPK inhibition (via MEK1 inhibitor: PD98059) on laminin-5, but not on fibronectin.
An additional signaling regulator, TGF-β1, has been implicated in the negative regulation and decreased rate of proliferation of VSMC stimulated with serum or PDGF [26-28]. Our results from this study indicate that TGF-β1, unlike the MEK1-inhibitor PD98059, was sufficient to block rVSMC behaviors on both laminin-5 and fibronectin. Specifically, the addition of TGF-β1 was able to reduce PDGF-BB-stimulated migration and proliferation of rVSMC on both ECM substrates and was also able to restore PDGF-BB-stimulated reductions in VSMC adhesion on these ECM.
Several lines of evidence now suggest that the anti-mitogenic effects of TGF-β1 may be dissociated from inhibition of ERK1/2 signaling pathways [17,18]. These reports suggest that TGF-β1 inhibition of PDGF-BB may be temporally independent of other early signaling pathways, such as MAPK, and is more likely to block VSMC behaviors, such as proliferation, by inhibiting events later in the G1 phase of mitosis.
Although our previous reports linked ERK1/2 to rVSMC adhesion and migration, these studies did not examine the possibility for differential signaling initiated by rVSMC binding to laminin-5 or fibronectin [12,13]. Expanding our original analysis of growth factor stimulation of MAPK to include ECM binding reveals that integrin binding of rVSMC to fibronectin strongly increases detectable MAPK activation levels, as does FCS and PDGF-BB stimulation, whereas binding to laminin-5 does not. These differences may help to explain the differing effects on cellular behaviors of binding to these ECM ligands, as fibronectin and PDGF-BB may act in unison to activate intracellular signaling cascades that converge in MAPK activation, while laminin-5 may not.
Conclusions
Our results indicate that laminin-5 activates different intracellular signaling pathways from those of fibronectin and PDGF-BB in rVSMC and that binding to laminin-5 may modulate rVSMC behaviors that are distinctive from those modulated by fibronectin. Although binding of rVSMC to laminin-5 may not cause an initial increase in MAPK activation levels or proliferation, laminin-5 can augment PDGF-BB-stimulated proliferation and migration of rVSMC in vitro. Based upon these findings, we postulate that PDGF-BB and laminin-5 binding may initially activate different intracellular signaling cascades, causing rVSMC to be more responsive to the inhibition of MEK1 and MAPK on laminin-5 than those activated on fibronectin, as outlined in Figure 5.
Figure 5 Integrins and growth factors activate intracellular signaling cascades in rVSMC. Intracellular signaling pathways that converge through MEK and ERK activation, may be initiated in rVSMC by the addition of PDGF-BB, as well as binding to laminin-5 or fibronectin. The MEK1 inhibitor PD98059 blocks MEK1 activation and alters rVSMC responses to PDGF-BB on laminin-5, but not fibronectin. The addition of TGF-β1, which may block later events in the cell cycle, is sufficient to block PDGF-BB induced responses of rVSMC on both laminin-5 and fibronectin.
In contrast to laminin-5, fibonectin and PDGF-BB may have parallel, reinforcing roles in MAPK activation. Our analysis of the effects of TGF-β1 demonstrated that TGF-β1 does not strongly activate MAPK in rVSMC, but rather strongly inhibits the effects of fibronectin and PDGF-BB-stimulation on laminin-5. Our results support the previous findings that TGF-β1 may inhibit mitogenesis and other VSMC behaviors via mechanisms independent of MAPK activation.
These results suggest that a clear understanding of the roles and contributions of each ECM may provide new insights into the mechanisms of regulating rVSMC cell behaviors, including migration, adhesion, and proliferation. Further analysis of the events that trigger and sustain the underlying cellular mechanisms of rVSMC behaviors may help aid in the design of more effective therapies for the treatment of restenosis.
Methods
Cell culture
Cells were maintained in 100 mm × 20 mm Corning tissue-culture dishes (Plainfield, NJ) at 37°C and 5% CO2 in humidified chambers. Cells were maintained in DMEM High Glucose, supplemented with 10% fetal bovine serum and 1% L-glutamine (29.2 mg/mL), penicillin G (10,000 U/mL), and streptomycin sulfate (10,000 mcg/mL) (GPS) from Irvine Scientific (Santa Ana, CA). Rat aortic smooth muscle cell explants were a gift from RC Smith and were isolated and passaged as previously described [29]. Although greater than 95% quiescence, G0(G1) arrest can routinely be induced by incubation of cells for 72 h. in low-mitogen (0.5% FBS) medium [15,30], these authors suggest that incubation of VSMC for 48 h. without mitogen (0% FBS) is sufficient to induce quiescence. This was verified by proliferation control cells, cultured for 48 h. at 37°C with and without mitogen stimulus.
Migration assays
Cell migration assays were performed in Costar transwell filter plates either coated with purified matrix (laminin-5 or fibronectin) at a protein concentration of 20 μg/mL for one hour (60 min.) at room temperature, 25°C, and washed twice with phosphate-buffered saline 0.2% Tween-20 and 5% skim milk (PBST) prior to assay as previously described [31,32]. Cells were seeded at a concentration of 1.2 × 105 in each of 96-transwell chamber filters (100 μL of 1.2 × 106 cells/mL solution) with and without ECM in the presence or absence of PDGF-BB at the indicated concentrations (5–25 ng/mL) and allowed to migrate for 18 hours at 37°C. Where applicable, the medium was supplemented with PD98059 (MEK1-inhibitor) at the indicated concentration. Cells were counted at the end of an 18-hour interval as indicated, quantified with the following modification. 30 minutes prior to measuring migration, 5 μM calcein AM from Molecular Probes (Eugene, OR) was added to the migration wells at 37°C. To quantitate migration, cells were removed from the top of the filter with cotton-tipped applicators and fluorescence of the incorporated calcein was measured from the bottom of the filter with a fluorescence plate reader. Relative fluorescence values for each experimental condition are expressed relative to controls and untreated samples.
Adhesion assays
Cell adhesion assays were performed as previously described [31,32] using Costar 96-well cell culture cluster plates, coated with either laminin-5 or fibronectin solution at a protein concentration of 20 μg/mL for 1 hour (60 min.) at room temperature, 25°C. Wells were then washed twice with PBST prior to assay. Cells were seeded at a concentration of 1.2 × 105 in each of 96-transwell chamber filters (100 μL of 1.2 × 106cells/mL solution) with and without ECM-coating (described above) in the presence or absence of PDGF-BB (25 ng/mL), TGF-β1 (25 ng/mL), or both, and allowed to attach for 30 minutes at 37°C. Where applicable, cells were first incubated for 20 minutes with PD98059 (40 ng/mL), a MEK1 inhibitor from New England Biolabs (Beverly, MA) at 37°C and the adhesion assay culture medium was supplemented with PD98059 at 40 ng/mL. Following adhesion, non-adherent cells were removed by suspending plates upside down in a rotating tank of PBS for 10 minutes at room temperature, 25°C. Adherent cells were then fixed and stained and the relative absorbance was measured using a TECAN-SPECTRAFluor spectrophotometer (TECAN, Durham, NC) at 595 nm.
Proliferation assays
Tissue culture plates were coated with purified fibronectin from Calbiochem (La Jolla, CA) or laminin-5 from Demos (La Jolla, CA) at a 20 μg/mL protein concentration for 1 hour (60 min.) at room temperature, 25°C as previously described [31]. Cells were seeded at a concentration of 1.2 × 105 in 100 mm2 cell culture plates with and without fibronectin or laminin-5 and allowed to attach overnight (12 h.) at 37°C. Cells were then starved in serum-free DMEM for 48 hours to induce quiescence at 37°C, as outlined in Cell Culture Methods below. The medium was then replaced with fresh medium containing 25 ng/mL of TGF-β1 or PDGF-BB obtained from Calbiochem (La Jolla, CA) and incubated at 37°C. Cells were removed from culture wells with trypsin/EDTA and counted using trypan blue stain from Gibco Life Technologies (Rockville, MD) and a VWR Scientific Counting Chamber (Plainfield, NJ) at 24 hour intervals, from 1 – 6 days.
Western Blot analysis
Quiescent VSMC were pre-treated with culture medium containing 10% FCS, PDGF-BB (25 ng/mL), TGF-β1 (25 ng/mL), or plated on laminin-5- or fibronectin-coated tissue culture plates with DMEM for 30 minutes at 37°C. Subconfluent cell cultures were then lysed using ice-cold, 1X lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1.0% Triton X-100, pH 7.5) and boiled in SDS Boiling Buffer prior to analysis as previously described [33]. Proteins were separated by 7.5% SDS-PAGE and transferred to Immobilon-P transfer membranes from Millipore (Bedford, MA), incubated with primary antibody (1:200 dilution at 4°C overnight, 12 h.), secondary antibody (1:2000 dilution at 25°C for 1 h.), and then exposed to NEN CDP-Star chemiluminescence reagent (5–7 min.) and developed on Kodak X-OMAT LS scientific imaging film for subsequent analysis. Antibodies used for this analysis were anti-phospho ERK (p42/p44) rabbit polyclonal IgG primary antibody and goat anti-rabbit IgG-AP secondary antibody from Santa Cruz Biotechnology (Santa Cruz, CA).
Statistics
The differences between untreated and treated cell populations were measured using a t distribution. All samples were measured using two-tailed t tests as departure from normality can make more of a difference in a one-tailed than in a two-tailed t test. So long as the sample size is even moderate (>20) for each group, quite severe departures from normality make little practical difference in the conclusions reached from these analyses [34].
Competing Interests
The author(s) declare that they have no competing interests.
Authors' contributions
KK carried out the migration, adhesion, and proliferation assays, the Western Blot analysis and assisted with experimental design. GEP conceived, monitored, and coordinated the experimental design. Both KK and GEP contributed equally to the writing of this manuscript.
Acknowledgements
KK and GEP thank Janice L. Huff and William L Rust for their assistance in reviewing experimental data, as well as Autumn Martinez and Christina Brown for their technical assistance in maintaining cell cultures and preparing in vitro assays.
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| 15683539 | PMC552332 | CC BY | 2021-01-04 16:39:15 | no | Cell Commun Signal. 2005 Jan 31; 3:2 | utf-8 | Cell Commun Signal | 2,005 | 10.1186/1478-811X-3-2 | oa_comm |
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Cell Commun SignalCell communication and signaling : CCS1478-811XBioMed Central London 1478-811X-3-21568353910.1186/1478-811X-3-2ResearchPlatelet-derived growth factor modulates rat vascular smooth muscle cell responses on laminin-5 via mitogen-activated protein kinase-sensitive pathways Kingsley Karl [email protected] George E [email protected] Department of Biomedical Sciences, University of Nevada, Las Vegas, School of Dental Medicine, 1001 Shadow Lane B-234, Las Vegas, Nevada, 89106-4124, USA2 Department of Biology, Rensselear Polytechnic Institute, 110 8th Street, Troy, New York, 12180-3596, USA3 (previous institutional affiliation) Department of Biological Sciences, University of Nevada, Las Vegas, 4505 Maryland Parkway, Box 454004, Las Vegas, Nevada, 89154-4004, USA2005 31 1 2005 3 2 2 27 7 2004 31 1 2005 Copyright © 2005 Kingsley and Plopper; licensee BioMed Central Ltd.2005Kingsley and Plopper; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
A treatment to remove vascular blockages, angioplasty, can cause damage to the vessel wall and a subsequent abnormal wound healing response, known as restenosis. Vascular smooth muscle cells (VSMC) lining the vessel wall respond to growth factors and other stimuli released by injured cells. However, the extracellular matrix (ECM) may differentially modulate VSMC responses to these growth factors, such as proliferation, migration and adhesion. Our previous reports of low-level expression of one ECM molecule, laminin-5, in normal and injured vessels suggest that laminin-5, in addition to growth factors, may mediate VSMC response following vascular injury. To elucidate VSMC response on laminin-5 we investigated-the role of platelet-derived growth factor (PDGF-BB) in activating the mitogen-activated protein kinase (MAPK) signaling cascade as a possible link between growth-factor initiated phenotypic changes in vitro and the ECM.
Results
Using a system of in vitro assays we assessed rat vascular smooth muscle cell (rVSMC) responses plated on laminin-5 to the addition of exogenous, soluble PDGF-BB. Our results indicate that although laminin-5 induces haptotactic migration of rVSMC, the addition of PDGF-BB significantly increases rVSMC migration on laminin-5, which is inhibited in a dose-dependent manner by the MAPK inhibitor, PD98059, and transforming growth factor (TGF-β1). In addition, PDGF-BB greatly reduces rVSMC adhesion to laminin-5, an effect that is reversible by MAPK inhibition or the addition of TGF-β1. In addition, this reduction in adhesion is less significant on another ECM substrate, fibronectin and is reversible using TGF-β1 but not MAPK inhibition. PDGF-BB also strongly increased rVSMC proliferation on laminin-5, but had no effect on rVSMC plated on fibronectin. Finally, plating rVSMC on laminin-5 did not induce an increase in MAPK activation, while plating on fibronectin or the addition of soluble PDGF-BB did.
Conclusion
These results suggest that rVSMC binding to laminin-5 activates integrin-dependent intracellular signaling cascades that are different from those of fibronectin or PDGF-BB, causing rVSMC to respond more acutely to the inhibition of MAPK. In contrast, our results suggest that fibronectin and PDGF-BB may activate parallel, reinforcing intracellular signaling cascades that converge in the activation of MAPK and are therefore less sensitive to MAPK inhibition. These results suggest a partial mechanism to explain the regulation of rVSMC behaviors, including migration, adhesion, and proliferation that may be responsible for the progression of restenosis.
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Background
Angioplasty is a procedure designed to treat vascular stenosis, blockage(s), or atherosclerotic lesions, but it may also, simultaneously, cause damage to the integrity of the blood vessel wall. Restenosis is the subsequent narrowing and occlusion of the blood vessel in response to the injury or damage sustained during angioplastic procedures such as balloon dilation [1]. During restenosis, vascular smooth muscle cells (VSMC) from the injured blood vessel wall migrate into the lumen of the vessel, creating a new or neointima. The subsequent proliferation of these neointimal VSMC can lead to a thickening of this neointimal layer and re-occlusion of the vessel [1].
The characteristic response of VSMC, endothelial cells, platelets, and macrophages at the site of injury is the release of specific soluble growth factors which include platelet-derived growth factor (PDGF), transforming growth factor (TGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) [2-4]. VSMC of the vessel wall respond to these factors by secreting proteolytic matrix metalloproteinases that degrade the extracellular matrix (ECM) and stimulate deposition of new ECM proteins such as collagen, elastin, fibronectin, and laminin in the neointima [5-7]. These ECM modulate VSMC integrin-dependent behaviors such as transluminal migration, adhesion, and proliferation [8-10].
To date, the precise molecular mechanisms that link growth factor-initiated intracellular signaling to ECM-mediated adhesion, migration, and proliferation of VSMC are still unknown. Our previous reports of low-level laminin-5 expression in the intima of normal vasculature and an increased expression of laminin-5 in the neointima of injured vessels suggest that laminin-5, in addition to PDGF and TGF, may mediate VSMC responsiveness following vascular injury [11-13].
To further elucidate VSMC response to growth factors and the intracellular signaling cascades that may be linked to ECM-mediated adhesion, we used in vitro assays to study the role of laminin-5 in modulating these behaviors in rat vascular smooth muscle cells (rVSMC). We report here that PDGF induces differential responses in rVSMC behaviors on laminin-5, but not on fibronectin. In addition, we find that the PDGF-induced responses on laminin-5 are inhibited in a dose-dependent manner by an inhibitor of the mitogen-activated protein kinase (MAPK) pathway, PD98059, but not on fibronectin.
These differences in MAPK-sensitive rVSMC responses in vitro may be the result of different signaling pathways that are initiated by integrin-mediated adhesion to laminin-5. We suggest that rVSMC binding to laminin-5 may initially activate a MAPK-independent signaling cascade that may make these cells more responsive to MAPK inhibition. In contrast, rVSMC binding to fibronectin may activate a signaling pathway shared by PDGF that ultimately converges in MAPK activation. These results suggest that a complex interaction of ECM and growth factors may closely regulate VSMC behavior following vascular injury and these studies may directly identify define molecular targets that may reduce the incidence of restenosis following angioplasty.
Results
Migration
We have previously reported that laminin-5 expression by rat vascular smooth muscle cells (rVSMC) is upregulated by platelet-derived growth factor (PDGF-BB) and that laminin-5 specifically enhances PDGF-BB-stimulated rVSMC migration [12]. In addition, our previous results suggested that PD98059 (a specific inhibitor of MEK and a general inhibitor of the mitogen-activated protein kinase ERK1/2 pathway) blocked PDGF-BB-stimulated migration on laminin-5 [13]. The current study characterizes this synergistic relationship between PDGF-BB and laminin-5 in the modulation of rVSMC cell behaviors, including migration.
The level of rVSMC migration was observed over 18 hours in transwell migration filters in the presence or absence of laminin-5, with and without PDGF-BB or serum. Maximal cell migration (chemotaxis) was obtained using cell migration media containing ten (10) percent fetal calf serum (FCS). Similar to our previous reports, laminin-5-coated wells induced a greater than four-fold increase in rVSMC migration over that measured in naked plastic controls in three independent experiments as shown in Figure 1 (n = 24, p < 0.05). Furthermore, PDGF-BB, tested over a biologically relevant range of 5 – 50 ng/mL, stimulated a dose-dependent increase in rVSMC migration on laminin-5 (between 5 – 25 ng/mL), increasing migration fifteen (15) to sixty (60) percent over laminin-5-stimulated migration alone (n = 24, p < 0.005). This PDGF-BB-stimulated increase in migration peaked at 25 ng/mL and did not significantly increase over the range of 25 – 50 ng/mL (data not shown).
Figure 1 PDGF-BB increased rVSMC migration on laminin-5 in vitro. The addition of PDGF-BB increased rVSMC migration on laminin-5, in a dose-dependent manner, over rVSMC haptotactic migration induced by the presence of laminin-5. rVSMC migration on laminin-5 was inhibited by both PD98059 (MEK1 inhibitor) and TGF-β1, in a dose-dependent manner.
The mitogen-activated protein kinase (MAPK) pathway is known to be activated by PDGF-BB-stimulation in VSMC [14]. To explore the mechanism of increased rVSMC migration on laminin-5 via PDGF-BB-stimulation, we used PD98059 which is a specific inhibitor of MEK1 and MEK2, and a general inhibitor of MAPK activation. The addition of PD98059 in concentrations between 10 – 50 ng/mL reduced PDGF-BB-stimulated migration on laminin-5 (25 ng/mL) in a dose-dependent manner, reaching a maximal reduction of sixty (60) to seventy (70) percent over the range of 20 – 40 ng/mL, as shown in Figure 1 (n = 24, p < 0.005). This reduction in PDGF-BB-stimulated migration on laminin-5 was maintained at all concentrations of PDGF-BB tested (5 – 25 ng/mL).
To determine if this modulation is restricted to MEK/MAPK-inhibition, we tested the effects of adding exogenous transforming growth factor (TGF-β1) for its ability to modulate the PDGF-stimulated increase in rVSMC migration on laminin-5. Our results indicated that the addition of TGF-β1 was able to reduce laminin-5-stimulated rVSMC migration to levels approximating the levels observed in naked plastic controls over all tested ranges (5 – 50 ng/mL), although the most statistically distinct reduction was found over the range of 5 – 25 ng/mL, as shown in Figure 1. In addition, TGF-β1 was able to reduce maximal PDGF-BB-stimulated migration of rVSMC (25 ng/mL) on laminin-5 by approximately fifty (50) to sixty (60) to percent, over the range of concentrations from 20 to 40 ng/mL.
Adhesion
To determine whether or not the PDGF-BB-stimulated increase in rVSMC migration correlates with a reduction in rVSMC adhesion to laminin-5, thirty-minute in vitro adhesion assays were performed. In the absence of exogenous growth factor stimulation, laminin-5- and fibronectin-coated wells (20 μg/mL) sustained an approximate two and a half-fold increase in rVSMC adhesion compared with negative controls, as shown in Figure 2 (n = 24, p < 0.05). The addition of PDGF-BB, at the maximal migration-stimulating dose of 25 ng/mL, decreased the adhesion of rVSMC on laminin-5 by more than sixty-five (65) percent (n = 24, p < 0.005). The addition of exogenous PDGF-BB (25 ng/mL), however, decreased rVSMC adhesion on fibronectin by less than thirty (30) percent.
Figure 2 PDGF-BB reduces rVSMC adhesion on laminin-5 adhesion in vitro. The presence of laminin-5 supported greater adhesion of rVSMC over naked plastic, and this increase in adhesion was reduced by the addition of PDGF-BB. Inhibition of MEK1, using PD98059 restored rVSMC adhesion to laminin-5 in the presence of PDGF-BB. Although fibronectin also supported rVSMC adhesion, the effect of adding PDGF-BB was less pronounced and was not restored using PD98059. The addition of TGF-β1, however, completely restored rVSMC adhesion to both fibronectin and laminin-5 in the presence of PDGF-BB.
To investigate if the relationship between the PDGF-BB-stimulated increase in migration and corresponding decrease in adhesion of rVSMC on laminin-5 may be related to MAPK activation, cells were pre-treated with PD98059 (40 ng/mL) for twenty (20) minutes prior to assay and the adhesion media was supplemented with PD98059 (40 ng/mL). The addition of exogenous PD98059 (40 ng/mL) restored the PDGF-BB-stimulated reduction (25 ng/mL PDGF-BB) in rVSMC adhesion to laminin-5, to approximately eighty-five (85) percent of laminin-5 controls (n = 24, p < 0.05). The addition of PD98059, however, did not restore the thirty (30) percent PDGF-BB-stimulated reduction in rVSMC adhesion on fibronectin.
To determine if this modulation is restricted to MEK/MAPK-inhibition, we tested the effects of adding exogenous transforming growth factor (TGF-β1). Our results indicated that the addition of TGF-β1 (25 ng/mL) was able to restore the PDGF-BB-stimulated reduction in rVSMC adhesion on laminin-5 by roughly the same level as PD98059, eighty two (82) percent versus eighty five (85) percent, respectively. In contrast to PD98059, the addition of TGF-β1 (25 ng/mL) was sufficient to restore the PDGF-BB-stimulated reduction in rVSMC adhesion on fibronectin.
Proliferation
Based upon our observations of rVSMC migration and adhesion, we performed in vitro proliferation assays to determine the relative effects of the extracellular matrix (ECM) and exogenous growth factors described above. First, to test the effects of the ECM substrate on rVSMC proliferation in the absence of exogenous growth factors, quiescent cells were plated in cell-culture plates either coated with or without laminin-5 or fibronectin, in serum-free Dulbecco's Modified Eagle's Medium (DMEM) for one (1) to six (6) days.
To induce quiescence, rVSMC were incubated for forty eight (48) hours without mitogen (0% FCS, FBS) at 37°C and quiescence was verified by proliferation controls, cultured for forty eight (48) hours at 37°C with mitogen stimulus as shown in Figure 3a and 3b. Previous studies with VSMC report that greater than 95% of cells incubated in low-serum media (0.4% FCS or less) were arrested in G0(G1) between forty eight (48) and seventy two (72) hours as determined by flow cytometry and determination of [3H] thymidine-labeled nuclei [15].
Figure 3 The effect of growth factors on rVSMC proliferation in vitro. The addition of PDGF-BB stimulated proliferation of rVSMC on laminin-5 and to some extent on naked plastic, but not on fibronectin. The presence of fibronectin alone was able to stimulate proliferation of rVSMC. The proliferative response of rVSMC to presence of laminin-5 with PDGF-BB or to fibronectin was suppressed by the addition of TGF-β1. Figure 3b The ECM induces differential rVSMC proliferation responses in vitro. The plating of rVSMC on fibronectin, but not laminin, induced increases in proliferation over four days. The addition of PDGF-BB, however, increased rVSMC migration on laminin-5, but not on fibronectin. The addition of TGF-β1 was sufficient to suppress rVSMC proliferation on both laminin-5 and fibronectin.
Our results suggest that culture of rVSMC plated on exogenous laminin-5 (coated at a concentration of 20 μg/mL) did not significantly increase cellular proliferation compared with naked plastic controls over a period of four (4) days, as shown in Figure 3a (n = 24, p < 0.02). Culturing of rVSMC plated on exogenous fibronectin (coated at a concentration of 20 μg/mL) however, did significantly increase cellular proliferation over a period of four (4) days by nearly two-fold, as shown in Figures 3a and 3b.
Next, to test the modulating effects of exogenous growth factors on rVSMC proliferation when cultured on these ECM substrates, quiescent cells were plated in cell-culture plates either naked or coated with laminin-5 or fibronectin, in the presence of TGF-β1 or PDGF-BB, both at a concentration of 25 ng/mL, from one (1) to six (6) days. Our results indicated that the addition of exogenous TGF-β1 (25 ng/mL) to the cell culture medium was sufficient to induce a suppressing effect on rVSMC proliferation on naked plastic, as well as laminin-5 and fibronectin, to levels approximating the quiescent growth controls.
The addition of PDGF-BB was sufficient to induce an increase in rVSMC proliferation on laminin-5 of nearly one-half (46%) over laminin-5 DMEM (no serum, no mitogen) controls (n = 24, p < 0.01). However, our results indicate that the addition of exogenous PDGF-BB (25 ng/mL) did not have a statistically significant effect on cells plated on fibronectin.
Mitogen Activated Protein Kinase (MAPK) Western Blot
To examine MAPK activation, rVSMC were plated onto laminin-5- or fibronectin-coated plates, then lysed after thirty minutes and prepared for immunoblotting. Our results indicated that laminin-5 did not induce a detectable increase in MAPK activation over a thirty (30) minute time interval, as shown in Figure 4. rVSMC plated on fibronectin did exhibit a significant increase in MAPK levels after thirty (30) minutes.
Figure 4 The MAPK pathway in rVSMC is activated by different stimuli. The addition of FCS or PDGF-BB or the plating of rVSMC on fibronectin was sufficient to induce measurable increases in p44/p42 activation over 30 minutes. However, the addition of TGF-β1 or the plating of rVSMC on laminin-5 was not sufficient to induce MAPK activation.
The addition of growth factors, such as PDGF-BB and TGF-β1, have been associated with increases in p44/p42 (ERK1/2) or MAPK activation [16-18]. To determine the effects of PDGF-BB and TGF-β1 on rVSMC, subconfluent cell cultures were pre-treated with FCS (10%), PDGF-BB (25 ng/mL) or TGF-β1 (25 ng/mL) for thirty (30) minutes, then lysed and prepared for immunoblotting. p44/p42 phosphorylation levels were not detectable in DMEM-treated control cells, as shown in Figure 4. However, the addition of PDGF-BB, but not TGF-β1, was sufficient to induce measurable increases in MAPK activation levels.
Discussion
PDGF-BB is an in vitro VSMC mitogen and may be responsible for initiating the phenotypic changes in VSMC migration and proliferation during restenosis in vivo [19,20]. Our recent reports of low-level laminin-5 expression in the intima of normal arteries and increased expression in the neointima of injured arteries suggest that laminin-5, in conjunction with soluble growth factors and mitogens, may determine VSMC phenotype during restenosis [11-13].
The current study augments the body of evidence that suggests VSMC growth is influenced by an ECM-VSMC interaction [21] and that VSMC proliferation in different species respond differently to growth factor stimulus [15]. More specifically, this study explores evidence that laminin-5 and PDGF-BB may have combined and synergistic effects in determining rVSMC phenotype in vitro [6,8,10]. More specifically, our studies suggest that PDGF-BB strongly influences rVSMC behaviors such as migration. In addition, PDGF-BB significantly alters other cellular behaviors such as adhesion and proliferation on laminin-5, but only to a lesser extent on fibronectin.
Because PDGF-BB stimulation increases the overall levels of intracellular MAPK, as well as MAPK phosphorylation, we sought to explore this signaling cascade to determine its role in modulating these rVSMC behaviors on laminin-5 [22]. Specifically, the ERK1/2 form of MAPK mediates signaling by PDGF-AA, PDGF-AB, and PDGF-BB in VSMC, as well as signaling through laminin-5 binding integrins, and is therefore the most likely signaling molecule to modulate these cellular behaviors [23-25]. Our results suggest that rVSMC behaviors in vitro, driven by PDGF-BB responsiveness, can be blocked by MAPK inhibition (via MEK1 inhibitor: PD98059) on laminin-5, but not on fibronectin.
An additional signaling regulator, TGF-β1, has been implicated in the negative regulation and decreased rate of proliferation of VSMC stimulated with serum or PDGF [26-28]. Our results from this study indicate that TGF-β1, unlike the MEK1-inhibitor PD98059, was sufficient to block rVSMC behaviors on both laminin-5 and fibronectin. Specifically, the addition of TGF-β1 was able to reduce PDGF-BB-stimulated migration and proliferation of rVSMC on both ECM substrates and was also able to restore PDGF-BB-stimulated reductions in VSMC adhesion on these ECM.
Several lines of evidence now suggest that the anti-mitogenic effects of TGF-β1 may be dissociated from inhibition of ERK1/2 signaling pathways [17,18]. These reports suggest that TGF-β1 inhibition of PDGF-BB may be temporally independent of other early signaling pathways, such as MAPK, and is more likely to block VSMC behaviors, such as proliferation, by inhibiting events later in the G1 phase of mitosis.
Although our previous reports linked ERK1/2 to rVSMC adhesion and migration, these studies did not examine the possibility for differential signaling initiated by rVSMC binding to laminin-5 or fibronectin [12,13]. Expanding our original analysis of growth factor stimulation of MAPK to include ECM binding reveals that integrin binding of rVSMC to fibronectin strongly increases detectable MAPK activation levels, as does FCS and PDGF-BB stimulation, whereas binding to laminin-5 does not. These differences may help to explain the differing effects on cellular behaviors of binding to these ECM ligands, as fibronectin and PDGF-BB may act in unison to activate intracellular signaling cascades that converge in MAPK activation, while laminin-5 may not.
Conclusions
Our results indicate that laminin-5 activates different intracellular signaling pathways from those of fibronectin and PDGF-BB in rVSMC and that binding to laminin-5 may modulate rVSMC behaviors that are distinctive from those modulated by fibronectin. Although binding of rVSMC to laminin-5 may not cause an initial increase in MAPK activation levels or proliferation, laminin-5 can augment PDGF-BB-stimulated proliferation and migration of rVSMC in vitro. Based upon these findings, we postulate that PDGF-BB and laminin-5 binding may initially activate different intracellular signaling cascades, causing rVSMC to be more responsive to the inhibition of MEK1 and MAPK on laminin-5 than those activated on fibronectin, as outlined in Figure 5.
Figure 5 Integrins and growth factors activate intracellular signaling cascades in rVSMC. Intracellular signaling pathways that converge through MEK and ERK activation, may be initiated in rVSMC by the addition of PDGF-BB, as well as binding to laminin-5 or fibronectin. The MEK1 inhibitor PD98059 blocks MEK1 activation and alters rVSMC responses to PDGF-BB on laminin-5, but not fibronectin. The addition of TGF-β1, which may block later events in the cell cycle, is sufficient to block PDGF-BB induced responses of rVSMC on both laminin-5 and fibronectin.
In contrast to laminin-5, fibonectin and PDGF-BB may have parallel, reinforcing roles in MAPK activation. Our analysis of the effects of TGF-β1 demonstrated that TGF-β1 does not strongly activate MAPK in rVSMC, but rather strongly inhibits the effects of fibronectin and PDGF-BB-stimulation on laminin-5. Our results support the previous findings that TGF-β1 may inhibit mitogenesis and other VSMC behaviors via mechanisms independent of MAPK activation.
These results suggest that a clear understanding of the roles and contributions of each ECM may provide new insights into the mechanisms of regulating rVSMC cell behaviors, including migration, adhesion, and proliferation. Further analysis of the events that trigger and sustain the underlying cellular mechanisms of rVSMC behaviors may help aid in the design of more effective therapies for the treatment of restenosis.
Methods
Cell culture
Cells were maintained in 100 mm × 20 mm Corning tissue-culture dishes (Plainfield, NJ) at 37°C and 5% CO2 in humidified chambers. Cells were maintained in DMEM High Glucose, supplemented with 10% fetal bovine serum and 1% L-glutamine (29.2 mg/mL), penicillin G (10,000 U/mL), and streptomycin sulfate (10,000 mcg/mL) (GPS) from Irvine Scientific (Santa Ana, CA). Rat aortic smooth muscle cell explants were a gift from RC Smith and were isolated and passaged as previously described [29]. Although greater than 95% quiescence, G0(G1) arrest can routinely be induced by incubation of cells for 72 h. in low-mitogen (0.5% FBS) medium [15,30], these authors suggest that incubation of VSMC for 48 h. without mitogen (0% FBS) is sufficient to induce quiescence. This was verified by proliferation control cells, cultured for 48 h. at 37°C with and without mitogen stimulus.
Migration assays
Cell migration assays were performed in Costar transwell filter plates either coated with purified matrix (laminin-5 or fibronectin) at a protein concentration of 20 μg/mL for one hour (60 min.) at room temperature, 25°C, and washed twice with phosphate-buffered saline 0.2% Tween-20 and 5% skim milk (PBST) prior to assay as previously described [31,32]. Cells were seeded at a concentration of 1.2 × 105 in each of 96-transwell chamber filters (100 μL of 1.2 × 106 cells/mL solution) with and without ECM in the presence or absence of PDGF-BB at the indicated concentrations (5–25 ng/mL) and allowed to migrate for 18 hours at 37°C. Where applicable, the medium was supplemented with PD98059 (MEK1-inhibitor) at the indicated concentration. Cells were counted at the end of an 18-hour interval as indicated, quantified with the following modification. 30 minutes prior to measuring migration, 5 μM calcein AM from Molecular Probes (Eugene, OR) was added to the migration wells at 37°C. To quantitate migration, cells were removed from the top of the filter with cotton-tipped applicators and fluorescence of the incorporated calcein was measured from the bottom of the filter with a fluorescence plate reader. Relative fluorescence values for each experimental condition are expressed relative to controls and untreated samples.
Adhesion assays
Cell adhesion assays were performed as previously described [31,32] using Costar 96-well cell culture cluster plates, coated with either laminin-5 or fibronectin solution at a protein concentration of 20 μg/mL for 1 hour (60 min.) at room temperature, 25°C. Wells were then washed twice with PBST prior to assay. Cells were seeded at a concentration of 1.2 × 105 in each of 96-transwell chamber filters (100 μL of 1.2 × 106cells/mL solution) with and without ECM-coating (described above) in the presence or absence of PDGF-BB (25 ng/mL), TGF-β1 (25 ng/mL), or both, and allowed to attach for 30 minutes at 37°C. Where applicable, cells were first incubated for 20 minutes with PD98059 (40 ng/mL), a MEK1 inhibitor from New England Biolabs (Beverly, MA) at 37°C and the adhesion assay culture medium was supplemented with PD98059 at 40 ng/mL. Following adhesion, non-adherent cells were removed by suspending plates upside down in a rotating tank of PBS for 10 minutes at room temperature, 25°C. Adherent cells were then fixed and stained and the relative absorbance was measured using a TECAN-SPECTRAFluor spectrophotometer (TECAN, Durham, NC) at 595 nm.
Proliferation assays
Tissue culture plates were coated with purified fibronectin from Calbiochem (La Jolla, CA) or laminin-5 from Demos (La Jolla, CA) at a 20 μg/mL protein concentration for 1 hour (60 min.) at room temperature, 25°C as previously described [31]. Cells were seeded at a concentration of 1.2 × 105 in 100 mm2 cell culture plates with and without fibronectin or laminin-5 and allowed to attach overnight (12 h.) at 37°C. Cells were then starved in serum-free DMEM for 48 hours to induce quiescence at 37°C, as outlined in Cell Culture Methods below. The medium was then replaced with fresh medium containing 25 ng/mL of TGF-β1 or PDGF-BB obtained from Calbiochem (La Jolla, CA) and incubated at 37°C. Cells were removed from culture wells with trypsin/EDTA and counted using trypan blue stain from Gibco Life Technologies (Rockville, MD) and a VWR Scientific Counting Chamber (Plainfield, NJ) at 24 hour intervals, from 1 – 6 days.
Western Blot analysis
Quiescent VSMC were pre-treated with culture medium containing 10% FCS, PDGF-BB (25 ng/mL), TGF-β1 (25 ng/mL), or plated on laminin-5- or fibronectin-coated tissue culture plates with DMEM for 30 minutes at 37°C. Subconfluent cell cultures were then lysed using ice-cold, 1X lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1.0% Triton X-100, pH 7.5) and boiled in SDS Boiling Buffer prior to analysis as previously described [33]. Proteins were separated by 7.5% SDS-PAGE and transferred to Immobilon-P transfer membranes from Millipore (Bedford, MA), incubated with primary antibody (1:200 dilution at 4°C overnight, 12 h.), secondary antibody (1:2000 dilution at 25°C for 1 h.), and then exposed to NEN CDP-Star chemiluminescence reagent (5–7 min.) and developed on Kodak X-OMAT LS scientific imaging film for subsequent analysis. Antibodies used for this analysis were anti-phospho ERK (p42/p44) rabbit polyclonal IgG primary antibody and goat anti-rabbit IgG-AP secondary antibody from Santa Cruz Biotechnology (Santa Cruz, CA).
Statistics
The differences between untreated and treated cell populations were measured using a t distribution. All samples were measured using two-tailed t tests as departure from normality can make more of a difference in a one-tailed than in a two-tailed t test. So long as the sample size is even moderate (>20) for each group, quite severe departures from normality make little practical difference in the conclusions reached from these analyses [34].
Competing Interests
The author(s) declare that they have no competing interests.
Authors' contributions
KK carried out the migration, adhesion, and proliferation assays, the Western Blot analysis and assisted with experimental design. GEP conceived, monitored, and coordinated the experimental design. Both KK and GEP contributed equally to the writing of this manuscript.
Acknowledgements
KK and GEP thank Janice L. Huff and William L Rust for their assistance in reviewing experimental data, as well as Autumn Martinez and Christina Brown for their technical assistance in maintaining cell cultures and preparing in vitro assays.
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CytojournalCytoJournal1742-6413BioMed Central London 1742-6413-2-51571591110.1186/1742-6413-2-5EditorialAnal screening cytology Leiman Gladwyn [email protected] University of Vermont, 111 Colchester Avenue, Burlington, VT 05446, USA2005 16 2 2005 2 5 5 14 2 2005 16 2 2005 Copyright © 2005 Leiman; licensee BioMed Central Ltd.2005Leiman; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This issue of CytoJournal contains an article on screening for anal intraepithelial neoplasia in high-risk male patients. This accompanying Editorial focuses on current understanding of this relatively new disease entity, with insights as to the potential role of screening cytopathology in the epidemiology, pathophysiology and clinical management of this HIV and HPV related anal lesion, which predominates in male patients living long-term with AIDS. Mention is made of techniques of obtaining samples, methods of preparation, and morphologic classification. Issues of anoscopic confirmation, as well as topical and surgical management are emphasized. The similarity of initial experiences in anal screening to problems encountered early in cervical cancer screening programs several decades ago, are highlighted.
Anal intraepithelial neoplasiaanal PapHIV-positiveanal cytology screeningASIL cytomorphologyanoscopy
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Note
For corresponding research article please see Arian et al, 2005 [15]
The subject of anal screening cytology has entered the epidemiologic and cytopathologic literature as a topic of interest over the last decade, and is highlighted in this issue of CytoJournal in an article by Arain and colleagues. Until recently, anal cancer was not considered to be a neoplasm of major public health concern [1]. It occurred infrequently, usually in older people, affecting women more often than men; further, being a neoplasm of low incidence, it remained under the radar in terms of screening potential. In some respects, anal cancer mirrored cervical cancer in unscreened women, presenting late in the course of the disease with a variety of pelvic symptoms, and having a protracted, ultimately fatal, course. Patients invariably became social outcasts, suffering from intolerable fecal complications, which presented major nursing challenges. Two aspects of this scenario have changed. First was the fairly recent introduction of effective modern treatment regimes for invasive anal squamous cancers utilizing chemo-radiation, leading to improvement in morbidity and long term survival [2,3]. The second significant alteration was seen in epidemiology, and this is the area which has come to involve screening cytopathology.
During the 1990s, in several European and North American cancer centers, an initially unaccountable increase in anal cancers was seen in younger people. It soon became apparent that HIV-positive homosexual males were affected in numbers greatly in excess of those expected. This was first noted in those urban areas in which large concentrations of homosexual men had been treated since the outbreak of the HIV epidemic. The term "males having sex with males" (MSM) was coined to cover these high-risk individuals. Preliminary information gleaned from screening programs in high HIV-positive incidence areas amongst MSM, showed detection rates of intraepithelial and early invasive neoplasia higher than any incidence ever recorded for cervical cancer screening. Further, HIV-negative homosexual MSMs, and HIV positive non-homosexual men (eg drug users) also exhibited an increased incidence of pre-malignant and invasive anal carcinomas. More recently, the syndrome of early onset anal cancer has been extended to include HIV-positive female patients, as well as females who are not HIV-positive, but who have genital HPV. All these groups show a higher incidence of abnormal anal cytopathology, though none quite as high as that found in HIV positive MSM. The unifying factor in most instances is ano-receptive intercourse, or extension of HPV infection from the genital tract to the anal mucosa, most obvious in the face of deficient immunity, or high viral load [4-6].
Epidemiologic studies have indicated that a prolonged preclinical phase precedes the onset of anal cancer in these high-risk groups. Just as in the cervix, there is a transitional zone (although not an abrupt squamocolumnar junction) in the anus. Rectal mucosa, with goblet cells, ends about one inch proximal to the external sphincter, giving way to a transitional epithelium, which in turn blends into a stratified squamous epithelium at the level of the anus. (External to the anus, any lesions which arise are considered to be primary skin lesions rather than anal lesions.) Almost all anal cancers develop in the transitional zone, where atypical, dysplastic and in-situ lesions are identifiable histologically. The cytologic counterparts are those of atypical cells of uncertain significance (ASCUS), low grade squamous intraepithelial lesions (LSIL) and high grade squamous intraepithelial lesions (HSIL). Once this natural history had been demonstrated and confirmed, it seemed natural that exfoliative cytology could be investigated as an "anal Pap smear" [7,8].
As with the cervix, the technique of obtaining the anal sample is critical to the success of screening. Standard colonic preparation is not required, but the rectum should be emptied prior to obtaining the anal sample. Brushes, brooms and Dacron swabs have all been used, the type of spatula probably being less important than the skill of the operator. The instrument is inserted to a depth of one and a half inches beyond the external sphincter, and subsequently withdrawn in a firm downward spiral movement incorporating 10–12 rotations, to ensure the device has made contact with the full surface area of the transformation zone. Some have used direct smears onto glass slides with immediate wet fixation; most centers, however, employ immediate insertion of the scraping device into liquid fixative for thin layer preparation. This appears both to improve adequacy and preservation, and also to eliminate any fecal contamination; residual material in the vial can be used for HPV studies if required, or for the creation of a bank of teaching slides.
There has been a relative dearth of literature on the cytomorphology of anal samples [9-11]. Classification according to Bethesda guidelines has been advised, implying similarity of the exfoliative cytology of atypical squamous cells of undetermined significance (ASC-US), anal intraepithelial lesions (A-SIL) and invasive squamous-cell neoplasms to those encountered in the cervix. This early in the development of the science of anal screening, and evident also in the current article in CytoJournal, there are indications that cytologic evaluation may not fully anticipate the severity of some lesions, when compared with biopsies taken simultaneously. Several groups have advised that all abnormal anal Paps should be followed by anoscopic evaluation, with biopsy confirmation when necessary. Anoscopy essentially mirrors colposcopy, enabling the viewer to see vascular abnormalities at magnification, and direct biopsies to the most abnormal-appearing areas. Despite these basic similarities, it is strongly stated in the literature that expertise in colposcopy does not equate to immediate competence in anoscopy; a significant learning curve exists for those wishing to acquire excellence in anoscopic technique, with associated accurate biopsy sampling. It is important that the anoscopist not be sidelined by visible condylomata, which may merely be sentinels of deeper flat lesions of higher grade. At the present time, lack of available expertise in this interventional follow-up of abnormal anal Paps may be the single limiting factor in any new screening program. It thus behooves those interpreting anal samples to be as proficient as they can be in pre-interventional assessment of the anal transformation zone.
Experience over the last decade suggests that anal cancer may be as highly appropriate a target for screening as is the cervix, in selected populations. The neoplasm is frequently encountered in well-defined high-risk groups. It has a detectable pre-malignant phase, and is amenable to easy cytologic sampling. Cytodiagnosis is reasonably sensitive and highly specific, and histologic confirmation is relatively easily obtained by well-trained personnel. If there is a current area of deficiency in such programs, it may well be in the treatment of intraepithelial lesions, which, as yet, has not been adequately assessed in large numbers of patients. It is known that highly active anti-retroviral therapy (HAART) does not appear to alter the pathophysiology of anal lesions once initiated. A variety of topical agents such as podophyllotoxin and imiquimod have been tried, as has intralesional interferon; superficial ablative therapies including liquid nitrogen, electrocautery, laser and LEEP have been attempted with varying success rates. Circumferential surgical resection almost inevitably results in unacceptable loss of sphincter control and soiling, but anoscopy-directed limited excision may prove less morbid. The fact that so many options are available implies perhaps that no single modality is yet considered sufficiently effective, with minimal complication [12,13]. This, too, is reminiscent of the early years of management of cervical pre-neoplasia, when a host of methods was pursued in attempted elimination of focal lesions of the squamocolumnar junction. As in the cervix, human ingenuity will undoubtedly prevail, and one or two forms of extirpation will emerge as both efficacious and uncomplicated.
An interesting consideration is whether or not anal cancer, and thereby anal cytopathology screening programs, would be of value in the developing world, particularly in Africa, India and China, where the bulk of the global incidence of HIV resides. This will depend on two very different factors. First is the nature of transmission. Unlike the situation in the developed world, AIDS in these regions is not essentially a disease of homosexual males; thus, without anal intercourse predominating, an upward trend in the incidence of anal cancer would seem unlikely. Anal screening programs would be unnecessary or cost-ineffective in these communities. The second feature dictating the institution of screening programs in developing countries relates to antiretroviral therapy. Anal intraepithelial neoplasia and cancer are not encountered early in the progression of HIV/AIDS. Rather, they are late complications of patients living long-term with AIDS, usually implying patients living long-term on HAART [14]. Unless affordable very low cost antiretroviral drugs could be manufactured and distributed widely, it seems unlikely that patients in developing countries would survive into the time zone in which delayed neoplasms such as anal cancer become a public health priority.
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Johnson LG Madeleine MM Newcomer LM Anal cancer incidence and survival: the surveillance, epidemiology, and end results experience, 1973–2000 Cancer 2004 101 281 288 15241824 10.1002/cncr.20364
Cleator S Fife K Nelson M Treatment of HIV-associated invasive anal cancer with combined chemoradiation Eur J Cancer 2000 36 754 758 10762748 10.1016/S0959-8049(00)00009-5
Whiteford MH Stevens KR JrOh S The evolving treatment of anal cancer: How are we doing? Arch Surg 2001 136 886 891 11485523 10.1001/archsurg.136.8.886
Goldstone SE Winkler B Ufford LJ High prevalence of anal squamous intraepithelial lesions and squamous cell carcinoma in men who have sex with men as seen in a surgical practice Dis Colon Rectum 2001 44 690 698 11357031
Holly EA Ralson ML Darragh TM Prevalence and risk factors for anal squamous intraepithelial lesions in women J Natl Cancer Inst 2001 93 843 849 11390533 10.1093/jnci/93.11.843
Daling JR Madeleine MM Johnson LG Human papillomavirus, smoking, and sexual practices in the etiology of anal cancer Cancer 2004 101 270 280 15241823 10.1002/cncr.20365
Goldie SJ Kuntz KM Weinstein MC The clinical effectiveness and cost-effectiveness of screening for anal squamous intraepithelial lesions in homosexual and bisexual HIV-positive men JAMA 1999 281 1822 1829 10340370 10.1001/jama.281.19.1822
Palefsky JM Holly EA Hogeboom CJ Virologic, immunologic, and clinical parameters in the incidence and progression of anal squamous intraepithelial lesions in HIV-positive and HIV-negative homosexual men J Acquir Immune Defic Syndr Hum Retrovirol 1998 17 314 9525431
Scholefield JH Johnson J Hitchcock A Guidelines for anal cytology – to make cytological diagnosis and follow up much more reliable Cytopathology 1998 9 15 22 9523124 10.1046/j.1365-2303.1998.00134.x
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Arian S Walts AE Thomas P Bose S The Anal Pap Smear: Cytomorphology of squamous intraepithelial lesions Cytojournal 2005 2 4 15715910 10.1186/1742-6413-2-4
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J Neuroengineering RehabilJournal of NeuroEngineering and Rehabilitation1743-0003BioMed Central London 1743-0003-2-21573332210.1186/1743-0003-2-2EditorialAdvances in wearable technology and applications in physical medicine and rehabilitation Bonato Paolo [email protected] Department of Physical Medicine and Rehabilitation, Harvard Medical School and The Harvard-MIT Division of Health Sciences and Technology, Spaulding Rehabilitation Hospital, 125 Nashua Street, Boston MA 02114, USA2005 25 2 2005 2 2 2 24 2 2005 25 2 2005 Copyright © 2005 Bonato; licensee BioMed Central Ltd.2005Bonato; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The development of miniature sensors that can be unobtrusively attached to the body or can be part of clothing items, such as sensing elements embedded in the fabric of garments, have opened countless possibilities of monitoring patients in the field over extended periods of time. This is of particular relevance to the practice of physical medicine and rehabilitation. Wearable technology addresses a major question in the management of patients undergoing rehabilitation, i.e. have clinical interventions a significant impact on the real life of patients? Wearable technology allows clinicians to gather data where it matters the most to answer this question, i.e. the home and community settings. Direct observations concerning the impact of clinical interventions on mobility, level of independence, and quality of life can be performed by means of wearable systems. Researchers have focused on three main areas of work to develop tools of clinical interest: 1)the design and implementation of sensors that are minimally obtrusive and reliably record movement or physiological signals, 2)the development of systems that unobtrusively gather data from multiple wearable sensors and deliver this information to clinicians in the way that is most appropriate for each application, and 3)the design and implementation of algorithms to extract clinically relevant information from data recorded using wearable technology. Journal of NeuroEngineering and Rehabilitation has devoted a series of articles to this topic with the objective of offering a description of the state of the art in this research field and pointing to emerging applications that are relevant to the clinical practice in physical medicine and rehabilitation.
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The potential impact of wearable technology on physical medicine and rehabilitation
Understanding the impact of clinical interventions on the real life of individuals is an essential component of physical medicine and rehabilitation. While assessments performed in the clinical setting have value, it is difficult to perform thorough, costly evaluations of impairment and functional limitation within the time constraints and limited resources available in outpatient units of rehabilitation hospitals. Furthermore, it is often questioned whether assessments performed in the clinical setting are truly representative of how a given clinical intervention affects the real life of patients. While this observation has fostered a great deal of interest for the development and validation of outcome measures that largely rely on the use of questionnaires [1], researchers and clinicians have looked at recent advances in wearable technology intrigued by the possibility offered by this technology of gathering sensor data in the field [2,3]. Likely to be complementary to outcome measures, the use of wearable systems in the clinical management of individuals undergoing rehabilitation is very attractive because it provides the opportunity of recording quantitative data in the settings that matter the most, i.e. the home and the community.
A number of clinical applications of wearable systems in physical medicine and rehabilitation emerged in the past few years. They range from simple monitoring of daily activities, for the purpose of assessing mobility and level of independence in individuals, to integrating miniature sensors to enhance the function of devices utilized by patients to perform motor tasks that they would be otherwise unable to accomplish.
Monitoring functional motor activities was one of the first goals of research teams interested in clinical applications of wearable technology. The focus was initially on using accelerometers [4-8] or a combination of accelerometers and electromyographic sensors [9] to capture movement and muscle activity patterns associated with a given set of functional motor tasks. The set of tasks to be identified varied according to the clinical application. Their study was combined with monitoring systemic responses when the clinical assessment required combining motor activities and cardio-respiratory data such as in the clinical management of patients with chronic obstructive pulmonary disease [10].
A level of complexity was added when researchers started investigating motor disorders and the possibility of utilizing wearable technology to assess the effect of clinical interventions on the quality of movement observed while patients performed functional tasks. Two applications worth mentioning are the one to assess symptoms and motor complications in patients with Parkinson's disease [11-14] and the study of motor recovery in post-stroke individuals [15-17]. This shift from identifying functional motor activities to studying motor patterns associated with motor disorders generated significant interest for more complex ways to monitor movement, i.e. utilizing not only accelerometers but also gyroscopes and magnetometers or inclinometers. The combination of multiple sensors allows one to estimate the kinematics of movement [18-21] with a reliability that cannot be obtained by solely relying on accelerometers [22].
Finally, recent studies have been focused on integrating wearable, miniature sensor technology with orthoses, prostheses, and mobility assistive devices. Sensor technology is particularly appealing in these applications because it allows implementing closed-loop strategies that take advantage of the increased complexity and flexibility that robotics is contributing to the design of orthoses, prostheses, and mobility assistive devices. Namely, the characteristics of such devices can be constantly modified as a function of the task individuals are engaged into and environmental disturbances [23,24].
In all the emerging applications summarized above, either continuous recording of sensor data or at least monitoring over extended periods of time are necessary to design and implement an effective clinical intervention. Unobtrusive, wearable systems providing ease of data gathering and some processing capabilities are essential to achieve the objective of making the leap between the preliminary results obtained as part of the research carried on so far and the daily clinical practice of physical medicine and rehabilitation. Three areas of work are essential to achieve this objective: 1)the development of wearable sensors that unobtrusively and reliably record movement and other physiological data relevant to rehabilitation; 2)the design and implementation of systems that integrate multiple sensors, record data simultaneously from wearable sensors of different types, and relay sensor data to a remote location at the time and in the way that is most appropriate for the clinical application of interest; and 3)the development of methodologies to manipulate wearable sensor data to extract information in a clinically relevant manner to perform clinical assessments or control devices aimed at enhancing mobility in individuals with conditions that limit their level of independence. A series of papers have been assembled to provide the readership of Journal of NeuroEngineering and Rehabilitation with a description of the state of the art of the application of wearable technology in physical medicine and rehabilitation.
Wearable sensors to measure movement and physiological signals
A first set of the papers that have been assembled for publication on Journal of NeuroEngineering and Rehabilitation on the topic of wearable technology in physical medicine and rehabilitation has the objective of describing recent advances in wearable sensor technology. Two manuscripts describe attempts by different groups of measuring angular displacements for upper and lower extremity joints by embedding conductive fibers into the fabric of undergarments. The paper by Gibbs and Asada, entitled "Wearable conductive fiber sensors for multi-axis human joint angle measurements", reports encouraging preliminary results concerning monitoring lower limb joint displacements during ambulation by utilizing such technology. The manuscript by Tognetti et al, entitled "Wearable kinesthetic system for capturing and classifying upper limb gesture in post-stroke rehabilitation", describes the design and implementation of a system similar to the one proposed by Gibbs and Asada but geared toward monitoring movements of the upper extremities. The authors also explore the application of these wearable sensors to monitoring motor recovery in post-stroke individuals. Simone and Kamper focus their contribution on unobtrusively measuring finger movements in patients undergoing rehabilitation. Their manuscript "Design considerations for a wearable monitor to measure finger posture" summarizes the authors' recent work toward developing ways to record fine motor control tasks involving manipulation of objects requiring fine motor control of the hand and fingers. This technology has immediate application in patients such as post-stroke individuals undergoing rehabilitation that targets fine motor control skills. While initial research in the area of wearable technology was aimed at combining existing, miniature sensors with special fabrics or wireless technology, recent advances in this field have been focused on the development of sensing elements that can be even more easily embedded in clothing items. An example of such effort is reported in the paper by Dunne et al entitled "Initial development and testing of a novel foam-based pressure sensor for wearable sensing". This paper summarizes positive preliminary results by the research team aimed at measuring shoulder movements, neck movements, and scapular pressure. The sensing elements can also be used to monitor respiratory rate. Devoted to monitoring systemic responses is the last of the papers focused on wearable sensors. In this manuscript, Yan et al describe a new method to reliably measure heart rate and oxygen saturation. The paper is entitled "Reduction of motion artifacts in pulse oximetry by smoothed pseudo Wigner-Ville distribution" and demonstrates how advanced processing techniques may be necessary to derive reliable data when recordings are performed in the field.
Wearable systems to gather data unobtrusively and reliably over extended periods of time
A second area of research relevant to the application of wearable technology in physical medicine and rehabilitation concerns the integration of wearable sensors into systems. Following the seminal work by Park and Jayaraman [25], several researchers relied on conductive fabrics to deliver sensor data to a data-logger and then integrated it into a system that allowed remote access to the data. Other researchers explored the use of wireless technology as a means to relay wearable sensor data to a base station for data recording and remote access to clinically relevant information. Jovanov et al summarize recent advances by their research team toward developing body area networks in the manuscript entitled "A wireless body area network of intelligent motion sensors for computer assisted physical rehabilitation". Key points concerning the use of wireless technology in field monitoring of patients undergoing rehabilitation are the design of low-power transmission devices, the integration of multiple sensors, and the ability of providing processing capability that may reduce the amount of information to be transmitted. These issues are addressed in the above-referenced paper as well as in the manuscript by Sung et al entitled "Wearable feedback systems for rehabilitation". Sung et al describe a platform of wearable sensors recently developed by their team as well as potential applications currently under investigation.
Clinical applications of wearable technology in physical medicine and rehabilitation
A final set of papers is focused on applications that are relevant to physical medicine and rehabilitation. Sherrill et al describe in their paper entitled "A clustering technique to assess feasibility of motor activity identification in COPD patients via analysis of wearable-sensor data" a method to design classifiers of motor activities such as walking and stair climbing. The proposed technique relies on the examination of small datasets via clustering methods. Measures are derived from clusters associated with different motor activities to evaluate whether the set of wearable sensors and features derived from the recorded data are suitable to reliably identify the motor tasks of interest. Wang and Winters put the information gathered via wearable systems into a clinical context via processing that relies on neuro-fuzzy models. Their paper entitled "A dynamic neuro-fuzzy model providing bio-state estimation and prognosis prediction for wearable intelligent assistants" presents encouraging results indicating that the proposed method can put in the correct context dynamic changes observed in post-stroke individuals undergoing rehabilitation. Wang and Kiryu in their manuscript entitled "Personal customizing exercise with a wearable measurement and control unit" summarize their results on customizing machine-based exercise routines on the basis of physiological data that are continuously gathered from individuals performing such routines. Their results demonstrate the feasibility of a closed-loop system that optimally adapts workload. Dozza et al describe a wearable system designed to reduce body sway in individuals with severe vestibular problems. Their manuscript entitled "Influence of a portable audio-feedback device on structural properties of postural sway" summarizes positive results obtained with a prototype wearable system that utilizes audio-feedback to improve balance. Finally, Mavroidis et al describe how miniature sensor technology can be used to design a new generation of smart rehabilitation devices. Three devices are described in their paper entitled "Smart portable rehabilitation devices": a passive motion elbow device, a knee brace that provides variable resistance by controlling damping via the use of an electro-rheological fluid, and a portable knee device that combines electrical stimulation and biofeedback. These devices combine sensing technology and control strategies to enhance rehabilitation.
Conclusion
This collection of papers provides an up-to-date description of the state of the art in the field of wearable technology applied to physical medicine and rehabilitation. The field is rapidly advancing and numerous research groups have already demonstrated applications of great clinical relevance. The potential impact of this technology on the clinical practice of physical medicine and rehabilitation is remarkable. A significant shift in focus is possible thanks to wearable technology. While the main focus of clinical assessment techniques is currently on methods that are implemented in the clinical setting, wearable technology has the potential to redirect such focus on field recordings. This is expected to allow clinicians to eventually benefit from both data gathered in the home and the community settings during the performance of activities of daily living and data recorded in the clinical setting under controlled conditions. Complementarities are expected between field and clinical evaluations. Future research will surely address optimal ways to combine these two types of assessment to optimize the design of rehabilitation interventions.
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Keijsers NL Horstink MW Gielen SC Online monitoring of dyskinesia in patients with Parkinson's disease IEEE Eng Med Biol Mag 2003 22 96 103 12845825 10.1109/MEMB.2003.1213632
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Nutr Metab (Lond)Nutrition & Metabolism1743-7075BioMed Central London 1743-7075-2-51572370210.1186/1743-7075-2-5ReviewFructose, insulin resistance, and metabolic dyslipidemia Basciano Heather [email protected] Lisa [email protected] Khosrow [email protected] Clinical Biochemistry Division, Department of Laboratory Medicine and Pathobiology, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada2005 21 2 2005 2 5 5 15 2 2005 21 2 2005 Copyright © 2005 Basciano et al; licensee BioMed Central Ltd.2005Basciano et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Obesity and type 2 diabetes are occurring at epidemic rates in the United States and many parts of the world. The "obesity epidemic" appears to have emerged largely from changes in our diet and reduced physical activity. An important but not well-appreciated dietary change has been the substantial increase in the amount of dietary fructose consumption from high intake of sucrose and high fructose corn syrup, a common sweetener used in the food industry. A high flux of fructose to the liver, the main organ capable of metabolizing this simple carbohydrate, perturbs glucose metabolism and glucose uptake pathways, and leads to a significantly enhanced rate of de novo lipogenesis and triglyceride (TG) synthesis, driven by the high flux of glycerol and acyl portions of TG molecules from fructose catabolism. These metabolic disturbances appear to underlie the induction of insulin resistance commonly observed with high fructose feeding in both humans and animal models. Fructose-induced insulin resistant states are commonly characterized by a profound metabolic dyslipidemia, which appears to result from hepatic and intestinal overproduction of atherogenic lipoprotein particles. Thus, emerging evidence from recent epidemiological and biochemical studies clearly suggests that the high dietary intake of fructose has rapidly become an important causative factor in the development of the metabolic syndrome. There is an urgent need for increased public awareness of the risks associated with high fructose consumption and greater efforts should be made to curb the supplementation of packaged foods with high fructose additives. The present review will discuss the trends in fructose consumption, the metabolic consequences of increased fructose intake, and the molecular mechanisms leading to fructose-induced lipogenesis, insulin resistance and metabolic dyslipidemia.
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Emerging epidemic of the Metabolic Syndrome
The new millennium has witnessed the emergence of a modern epidemic, the metabolic syndrome, with frightful consequences to the health of humans worldwide. The metabolic syndrome, also referred to as "Diabesity" [1] describes the increasing incidence of diabetes in combination with obesity as a result of changes in human behaviour, available nutrition, and the adoption of more sedentary lifestyles. Obesity and type 2 diabetes are occurring at epidemic rates in the United States [2-4] and developing countries including China [5] and India [6]. From 1935 to 1996, the prevalence of diagnosed type 2 diabetes climbed nearly 765% [7]. The global figures are predicted to rise 46% from 150 million cases in 2000 to 221 million in 2010 [8]. This epidemic of type 2 diabetes is complicated by the fact that it is a multi-factorial disease, frequently associated with a cluster of pathologies including obesity, hypertriglyceridemia, impaired glucose tolerance, and insulin resistance, collectively referred to as the metabolic syndrome (formerly known as syndrome X and insulin resistance syndrome). Although there is no universally accepted definition of the metabolic syndrome, most would agree that the syndrome includes a cluster of common pathologies: obesity, insulin resistance, dyslipidemia, and hypertension. It is present in 25–50% of the United States population [9]. There has been a heightened awareness of the metabolic syndrome and a subsequent increase in clinical attention directed towards prevention, due to its strong association with premature morbidity and mortality [8,10]. In particular, these risk factors predispose the individual to greater risk for developing cardiovascular disease and Type 2 diabetes. Recently, the National Cholesterol Education Panel (NCEP) has officially described and identified a number of these risk factors for cardiovascular diseases [11]. These include: 1) abdominal obesity, 2) elevated TG levels, 3) low high density lipoprotein (HDL)-cholesterol levels, 4) increased blood pressure, and 5) impaired fasting glucose [12]. There is also now consensus that insulin resistance and obesity are actually part of one common pathologic mechanism of the metabolic syndrome [13,14]. Evidence shows that the metabolic syndrome process begins early in life and persistence from childhood to adolescent/adult life produces type 2 diabetes and cardiovascular disease [15,16]. The symptoms of metabolic syndrome are not necessarily manifestations of age, but develop over a predisposed background established at a young age [17,18]. This is a dangerous predisposition, with trends in modern diet and habit likely influencing health and behaviour in increasingly younger populations.
The main driving forces for the increased prevalence of insulin resistance are modern Westernized diets and patterns of eating associated with the dramatic rises in obesity. Insulin resistance is often linked to the macronutrient content in the diet. In the past, diets high in saturated fats have been shown to induce weight gain, insulin resistance, and hyperlipidemia in humans and animals [19-22]. Recent research suggests that a high intake of refined carbohydrates may also increase the risk of insulin resistance [23-26]. In addition, diets specifically high in fructose have been shown to contribute to a metabolic disturbance in animal models resulting in weight gain, hyperlipidemia [27], and hypertension [28].
Nutritional factors influencing the development of the Metabolic Syndrome
Nutrition represents a lifestyle element that can be controlled, and that can directly influence health; therefore preventative nutrition and weight control should become a main focus of consumers and prepared-food providers [29]. The Westernization of diets, with an increase in availability of high calorie foods certainly contributes to the epidemic of metabolic syndrome. In the past, physicians and scientists have made an association between dietary energy from fat and body fat. A large market has developed for the popularity and promotion of low fat diets. Interestingly, however, the decline in dietary fat consumption has not corresponded to a decrease in obesity – in fact, the opposite trend has emerged [30]. Certainly, diets high in saturated fats have been shown to induce weight gain, insulin resistance, and hyperlipidemia in humans and animals [19-22,31], but the emphasis on fat reductions has had no significant benefits relative to the obesity epidemic. More importantly, the focus on dietary fat is more likely a distraction to more significant causes of metabolic syndrome [30]. If fat is not the culprit in metabolic disorders, then what is? Increasing evidence now suggests that the rise in consumption of carbohydrates, particularly refined sugars high in fructose, appears to be at least one very important contributing factor.
Carbohydrates and the link to the Metabolic Syndrome
The general increases in consumption of calories, and specifically of refined carbohydrates and fructose, is clear and correlates positively with an alarming increases in metabolic syndrome. Can these seemingly harmless nutrients actually be directly associated with metabolic syndrome? Recent studies appear to support this link. In a 2004 study, Gross et al examined nutrient consumption in the United States between 1909 and 1997, and discovered there was a significant correlation in the prevalence of diabetes with fat, carbohydrate, corn syrup, and total energy intakes. Most striking was the fact that when total energy intake was accounted for, corn syrup was positively associated with type 2 diabetes, while protein and fat were not [32]. High fructose corn syrups (HFCS) are quite commonly found in soft drinks and juice beverages, and are incorporated into many convenient pre-packaged foods, such as breakfast cereals and baked goods. Fructose consumption has thus largely increased over the past few decades most likely as a result of this increased use of HFCS, which contains between 55–90% fructose. The use of HFCS has increased an alarming 1000% between 1970 and 1990 [33]. In 1970, individual consumption of fructose was only 0.5 lb/year. However, in 1997, this figure rose to an alarming 62.4 lb/year [34]. The type of common, general use sweeteners represent as large an impact as the dramatic increase in the use of these caloric sweeteners. Between 1909 and 1997, sweetener use increased by 86%; and specifically, corn syrup sweeteners now represent over 20% of total daily carbohydrate intake, at an increase of 2100% [32].
These documented trends have inspired a number of consumption studies and recommendations towards HFCS intake. In 1992, the USDA recommended that only 40 g of extra sugars should be added to a standard 2000 calorie a day diet [35]. The amount of HFCS found in only one 12-oz soft drink equals this total proportion of daily intake. HFCS consumption trends are further exacerbated by the fact that soft drink and fruit juice consumption itself has increased dramatically, adding even more extraneous calories and fructose to the diet. From 1965 to 1996, a food consumption study involving 11 to 18 year olds revealed that total energy and fat intakes were decreasing. There were significant decreases in milk consumption but large increases in the consumption of soft drinks and non-citrus juices [36]. Increasingly, children seem to be choosing mass-produced, 'tasty' artificial juices and sodas over healthier alternatives. In a recent letter to the editor, Jacobson [37] illustrates some important factors that contribute to increased consumption of soft drinks, and the link to obesity; a) Society is constantly bombarded by huge million-dollar advertising campaigns for soft drinks, offered extra-extra-large serving sizes with free refills, and surrounded by ubiquitous access to soft drink vending machines even in schools, and b) children's standard drinks to accompany meals, and especially fast food, have become soft drinks. The increased use of HFCS in soft drinks and food products are thus exacerbated by increased exposure, and consumption of these products. HFCS are the main caloric sweeteners utilized in soft drinks in the United States, with fructose representing over 40% of sweeteners added to prepared foods and beverages [33]. In a study of females aged 12 to 19 years milk intake decreased by 36%, whereas sodas and fruit drink consumption increased to nearly double from the 1970s to the mid 1990s. From 1994 to 1996, it was found that even though intake of soda, juices, tea, and alcoholic beverages remained constant, the steady decrease of milk intake continued [38]. This becomes a major problem, because while these high-calorie beverages are being consumed, calories from the rest of the diet are not subsequently reduced. The reality is that people do not eliminate or reduce their food portions because they drank a can of soda that day. Data indicate that energy from beverages generally does not displace or decrease energy from other foods consumed, leading to energy imbalances [39]. The main diet issues involve a general lack of education and/or understanding of the implications with recent consumption patterns. Despite education programs to prevent obesity and diabetes worldwide, there has been little focus on the reduction of fructose and HFCS in beverages.
Fructose metabolism
Fructose is readily absorbed and rapidly metabolized by human liver. For thousands of years humans consumed fructose amounting to 16–20 grams per day, largely from fresh fruits. Westernization of diets has resulted in significant increases in added fructose, leading to typical daily consumptions amounting to 85–100 grams of fructose per day. The exposure of the liver to such large quantities of fructose leads to rapid stimulation of lipogenesis and TG accumulation, which in turn contributes to reduced insulin sensitivity and hepatic insulin resistance/glucose intolerance. These negative effects of fructose are the reason that fructose metabolism has gained recent research attention. Interestingly, small catalytic quantities of fructose can have positive effects, and actually decrease the glycemic response to glucose loads, and improve glucose tolerance. These effects are also observed without any changes in insulin responses and non-esterified fatty acid (NEFA) and TG levels [40,41]. In 1976, sugar substitutes such as fructose had been found to offer the 'advantage' of a 'better' utilization in conditions of limited insulin production. Fructose had a smaller influence on serum insulin concentrations than glucose, and no influence on plasma glucose levels. At that time, this evidence was considered to support fructose as a positive treatment for diabetic control [40]. In 1986 HFCS were even proposed as a low-cost substitute for fructose in diabetic management. Based on these early observations, nutritive sweeteners were considered safe by the Food and Drug Administration, although, it has now been found that intakes above 25% of total energy consumed will cause hypertriglyceridemia and gastrointestinal symptoms [42]. Even with the early positive results, researchers noticed accompanying "unfavorable" influences of these so-called diabetic sugars on obesity and weight gain. Certain metabolic differences exist between glucose and fructose, and the results that were once thought favorable, proved exacerbating to insulin resistance and obesity. In a study comparing normal and diabetic patients, glycemic effects of HFCS were compared to glucose. The negative results of HFCS on immunoreactive insulin, glycemic effect, and immunoreactive C-peptide did not support its use as a substitute for glucose in diabetic patients [43].
Unfortunately, one out of every four children in the United States consumes above the recommended 25% of total energy intake from sweeteners [42] and the harmful effects of fructose have been extensively studied in healthy, non-diabetic patients. Studies involving commonly consumed fruit juices showed that natural fructose carbohydrates can alter lipid and protein oxidation biomarkers in the blood, and mediate oxidative stress responses in vivo [44]. A comparative study by Raben et al. examined overweight men and women who consumed fructose-containing sucrose, as opposed to artificial sweeteners as supplements to their diet. Weight, fat mass, and blood pressure were found to be lower in the artificial sweetener-consuming group compared to the sucrose-consuming group, and the sucrose group did not decrease intake of other nutrients to compensate for their increased calorie consumption from the sucrose. Subjects consuming the sweetener did not exhibit increases in energy intake, weight, and blood pressure that seen in the sucrose-consuming subjects [45]. Research in the metabolism of fructose has left more questions about the difference between short-term positive effects, and the negative effects of chronic, long-term use of fructose sugars [46]. The long-term negative effects can include changes in digestion, absorption, plasma hormone levels, appetite, and hepatic metabolism, leading to development of insulin resistance, diabetes, obesity, and inevitably cardiovascular disease.
When the metabolic pathways and characteristics of fructose are examined more closely, many of the questions about its positive and negative effects can be answered. Fructose is a potent regulator of glycogen synthesis and liver glucose uptake. Therefore any catalytic improvements are due to hepatic glucokinase and glucose uptake facilitation. However, as mentioned, the beneficial effects do not continue with chronic fructose utilization [47]. Because of its lipogenic properties, excess fructose in the diet can cause glucose and fructose malabsorption, and greater elevations in TG and cholesterol compared to other carbohydrates [48]. There are key differences in the metabolic pathways that glucose and fructose follow. Upon gastric absorption both fructose and glucose are delivered via the portal vein to the liver. It is believed that the ability of the liver to metabolize high doses of fructose is responsible for the disruption in energy stores and fuel metabolism observed [49-52]. In the liver, fructose is metabolized into glyceraldehyde and dihydroxyacetone phosphate. These particular fructose end products can then readily converge with the glycolytic pathway. Of key importance is the ability of fructose to by-pass the main regulatory step of glycolysis, the conversion of glucose-6-phosphate to fructose 1,6-bisphosphate, controlled by phosphofructokinase. Thus, while glucose metabolism is negatively regulated by phosphofructokinase, fructose can continuously enter the glycolytic pathway. Therefore, fructose can uncontrollably produce glucose, glycogen, lactate, and pyruvate, providing both the glycerol and acyl portions of acyl-glycerol molecules. These particular substrates, and the resultant excess energy flux due to unregulated fructose metabolism, will promote the over-production of TG (reviewed in [53]).
The glycemic index (GI) has been commonly used to differentiate and compare various nutrients, as well as to describe how different foods produce different plasma glucose levels after ingestion. The GI can range from 100 for glucose and baked potato compared to approximately 20 for fructose and whole barley [54]. Foods with varying GIs have different time courses associated with satiety. High GI carbohydrates have been reported to reduce appetite in the short term, whereas low GI carbohydrates possess a more delayed effect on energy intake controls [55]. Fructose appears to have differing effects on appetite compared to glucose, contributing to its negative properties. Anderson et al. determined the association between food intake and blood glucose, comparing glucose and a fructose mixture. Glucose was administered as a high GI preload, which resulted in lower mealtime energy intakes compared to the low GI preload of the glucose-fructose mixture. An inverse relationship was seen between GI (and blood glucose concentrations), and appetite with consequent increased food intakes seen with fructose [56]. In 2002, Vozzo et al. studied the comparative effects of glucose and fructose on blood glucose, insulin, and acute food intake. When subjects drank equienergetic preloads of glucose or fructose before an ad libidum buffet lunch, glucose concentrations were lower in the fructose group compared to glucose, and insulin concentrations were 50% higher in the fructose group in type 2 diabetics than in non-diabetics. The authors concluded that fructose may be a suitable replacement for glucose in diabetic patients – although it was found that satiating efficiencies of fructose certainly offered no advantages [57]. This study differs from others with regards to insulin secretion, but the trend is clear between GI, glucose concentrations, and appetite. An explanation for the variation in glucose and fructose glycemic responses appears to be dependent on rates of hydrolysis and absorption of glucose, and gastric emptying [58]. The variations observed in GI and appetite control of glucose and fructose can also be explained by differences in stimulation of insulin and leptin, important players in the long-term regulation of energy homeostasis. Fructose will generally produce smaller insulin excursions upon consumption because it does not stimulate the secretion of insulin from pancreatic beta cells, whereas glucose does. Insulin-regulated leptin will also have a reduced concentration and a decreased net effect on reducing appetite. Limited effects on appetite suppression, combined with the fact that fructose is favoured by the liver to be metabolized into lipid, will subsequently lead to weight gain, hyperinsulinemia, and the associated insulin resistance [59]. Glucose and fructose comparison studies continued examining new hormonal targets. In 2004, Teff et al. showed that subjects served meals with either 30% glucose beverages, or 30% fructose beverages, had differing hormonal and metabolic responses. Glycemic excursions and insulin responses were reduced by 66% and 65%, respectively, in the fructose-consuming subjects. There was a concomitant reduction in circulating leptin both in the short and long-term as well as a 30% reduction in ghrelin (an orexigenic gastroenteric hormone) in the fructose group compared to the glucose group. A prolonged elevation of TG was also seen in the high fructose subjects [60]. Both fat and fructose consumption usually results in low leptin concentrations which, in turn, leads to overeating in populations consuming energy from these particular macronutrients. An adipocyte hormone, adiponectin, also plays an important role in lipid homeostasis and insulin action [61]. The insulin sensitizer agonist, peroxisome proliferator-activated receptor-gamma, stimulates adiponectin production and adiponectin is in fact thought to be part of this agonist's mechanism lowering circulating fatty acids and increasing fat oxidation. The net effect is to decrease liver TG and increase insulin sensitivity [62]. Chronic fructose consumption reduces adiponectin responses, contributing to insulin resistance [63].
Animal studies have illustrated various differences between glucose and fructose metabolism. In 2002, Miller et al. injected fructose into the cerebroventricles of rats, and observed enhanced food intake, whereas similar concentrations of injected glucose suppressed appetite-agonist stimulated food intake [64]. Feeding rats either 32% glucose, fructose, or sucrose solutions, resulted in increased weight gain, and energy consumption compared to chow fed controls. Rats given the fructose and sucrose solutions also had a decreased ability to tolerate a glucose load, and fructose animals had greater serum TG levels over all other conditions ([65]. This is likely because the hepatic metabolism of fructose favours de novo lipogenesis. In combination with alterations in insulin signaling and leptin regulation, weight gain and unregulated energy intake can occur [33]. In 1986, Levine et al. found that fructose, administered in the form of the disaccharide sucrose, promotes obesity more than glucose because fructose does not stimulate thermogenesis [58]. These hormonal and physiological changes illustrate the important connections between energy intake, appetite control, weight gain, and insulin resistance.
Fructose and insulin resistance
Increasingly, questions have been raised as to whether dietary carbohydrate and fructose intake are directly related to the development of type 2 diabetes. As insulin resistance is often associated with circulating C-peptide concentrations, a cross-sectional study was performed to assess dietary fructose and carbohydrate, and glycemic loads related to C-peptide concentrations. It was found that the highest quintile of fructose intake had 13.9% higher C-peptide concentrations than the lowest quintile. Of note, subjects with high intakes of cereal fiber had 15.6% lower C-peptide concentrations, indicating that these types of nutrients may have opposing roles in the development of insulin resistance [66]. A definite relationship has also been found between metabolic syndrome and hyperhomocysteinemia, which is associated with cardiovascular and cerebrovascular diseases. Rats fed a fructose-enriched diet had a 72% higher homocysteine levels after 5 weeks compared to chow-fed controls [67]. Elevated homocysteine levels are an important risk factor for vascular disease. Homocysteine was found to be higher in patients with stenotic vessels and coronary artery disease scores, and was in fact highest in diabetic patients [68]. This is consistent with the increased TG, very low density lipoprotein (VLDL) secretion, and atherosclerosis associated with chronic fructose feeding.
Although fructose does not appear to acutely increase insulin levels, chronic exposure seems to indirectly cause hyperinsulinemia and obesity through other mechanisms. One proposed mechanism involves GLUT5, a fructose transporter that is found to have significantly higher expression levels in young Zucker obese rats compared to lean controls. As the rats age and become diabetic, GLUT5 abundance and activity is compromised, causing an even more marked insulin resistance over lean rats, implying a possible role of GLUT5 receptors in the pathology of metabolic syndrome associated with fructose feeding and insulin resistance [69]. In rats fed 66% fructose for 2 weeks, insulin receptor mRNA, and subsequent insulin receptor numbers in skeletal muscle and liver were significantly lower compared to rats fed a standard chow diet. Also, blood pressure and plasma TG increased in the fructose-fed rats, even though there was no change in plasma insulin, glucose, or body weight [70]. Evidence shows these early steps in insulin signaling are important for insulin's metabolic effects. In a different study, it was found that after 28 days of fructose feeding there were no changes in insulin receptor concentration, but, insulin stimulated autophosphorylation, a mechanism necessary for insulin action, was reduced to 72% in the liver. Insulin receptor substrate (IRS) protein levels were similar, but there were significant decreases in insulin induced IRS (1/2) phosphorylation in both the liver and muscle of the fructose fed rats [71]. These changes are important, because it has been shown that the products of these insulin independent metabolic pathways lead to polyol formation and advanced glycation end products, which can contribute to the numerous complications and premature atherosclerosis seen in diabetic patients [58]. It is also known that such inflammations can lead to the pathogenesis of diabetes, and there is strong evidence suggesting that increased free fatty acids (FFA) in diabetic subjects and fructose fed models play a role in the inflammatory state of insulin resistance. If FFA are not removed from tissues, as occurs in fructose fed insulin resistant models, there is an increased energy and FFA flux that leads to the increased secretion of TG. Insulin resistance has also been correlated with intracellular TG stores, which are involved in lipotoxicity and beta cell failure leading to diabetes [72]. Another theory explaining how chronic fructose overnutrition can lead to type 2 diabetes is the hexosamine hypothesis, where hexosamine flux is thought to regulate glucose and satiety-sensing pathways. With overexpression of glutamine:fructose-6-phosphate amidotransferase, the key regulatory enzyme in hexosamine synthesis, the liver produces excess fatty acids, skeletal muscle becomes insulin resistant, and hyperinsulinemia results. This pathway of excess hexosamine flux leads to long-term storage of energy, and eventually obesity and type 2 diabetes [73].
Fructose: a highly lipogenic nutrient
There is considerable evidence supporting the ability of high fructose diets to upregulate the lipogenesis pathway, leading to increased TG production [74]. Insulin and glucose are known to directly regulate lipid synthesis and secretion. Insulin controls hepatic sterol regulatory element binding protein (SREBP) expression, which is a key transcription factor responsible for regulating fatty acid and cholesterol biosynthesis. SREBP binds to sterol responsive elements (SRE) found on multiple genes, and can activate a cascade of enzymes involved in cholesterol biosynthetic pathways, such as HMG-CoA reductase [75] and fatty acid synthase (FAS) [76]. Miyazaki et al. reported an induction of the hepatic SREBP-1 isoform and lipogenic gene expression including FAS, acetyl-CoA carboxylase (ACC), and stearoyl-CoA desaturase (SCD) in mice following 7 days on a 60% fructose diet [77]. It is known that SREBPs are regulated by intracellular sterol concentrations. However, more recently, it has been established that hormones such as insulin and platelet derived growth factor play a role in regulating these transcription factors. Expression of SREBP is enhanced by insulin in all three major insulin target tissues, liver, fat, and skeletal muscle [78-81]. Similarly, levels of SREBP are enhanced in the presence of hyperinsulinemia [82,83]. There is evidence that the insulin-mediated stimulation of SREBP occurs through the MAP kinase pathway [84], with ERK1/2 being shown to activate the SREBP-1a isoform by phosphorylating serine 117) [85]. Despite the fact that SREBP-1 is directly stimulated via insulin signaling, the depletion of insulin and insulin signaling through streptozotocin (STZ) treatment paradoxically induces SREBP-1c expression upon glucose, fructose, or sucrose feeding. It would have been expected that SREBP-1c would be downregulated concomitantly along with the reduced insulin availability, but this is not the case. Glucose feeding causes a short-term peak induction, whereas fructose caused a gradual extended increase in SREBP-1c activity, providing evidence that lipogenesis can be independent of insulin signaling, given carbohydrate, and particularly fructose, availability [86].
Emerging evidence suggests that a protein phosphatase, known as PTP-1B, may link high carbohydrate feeding, insulin resistance, and lipogenesis. Recently, PTP-1B has been linked to lipogenesis and SREBP regulation. Shimizu et al. found that overexpression of protein tyrosine phosphatase 1B (PTP-1B), which is associated with dysfunctional insulin signaling, leads to increased mRNA and promoter activity of SREBP-1c, and subsequent increases in the expression of FAS. PTP-1B may therefore regulate the lipogenesis and hypertriglyceridemia associated with insulin resistance syndrome [87]. In insulin resistant fructose fed rats, it has been reported that the increase of hepatic SREBP-1 mRNA [88] occurs in correlation with an increased PTP-1B expression [87]. The authors established a role for PTP-1B in enhancing SREBP-1 gene expression through upregulation of Sp1 transcriptional activity, via an increase in protein phosphatase 2A activity [87]. FAS, an important downstream component of lipid synthesis, was extensively studied in rat livers. Dietary carbohydrates increased the transcriptional rate of FAS in comparison to proteins. Specifically, fructose feeding increased FAS mRNA concentrations, and somewhat increased transcriptional rate. This suggests that fructose may increase the stability of FAS mRNA, while carbohydrates stimulate FAS through increased transcriptional rate [89]. Other studies using animal models of insulin resistance, for example, the Wistar fatty rats, showed the effects of dietary carbohydrates on TG production. Feeding rats fructose stimulated FAS, and created a 56% increase in TG secretion rate, and an 86% increase in plasma TG. Feeding glucose, however, did not have this effect on TG production, nor did it affect induction of FAS. This is likely because glucose stimulates both TG production, and TG removal, maintaining homeostasis. Fructose stimulates TG production, but impairs removal, creating the known dyslipidemic profile [90]. The human liver possesses a large capacity to metabolize fructose to lipids because of its ability to shunt metabolism toward serum TG production. TG stores supply an energy 'sink', providing an almost unlimited TG production capacity. Conversely, glucose as opposed to fructose would decrease serum TG [91]. As discussed earlier, the effects of fructose in promoting TG synthesis are independent of insulinemia. Hirsch argued that carbohydrate overload results in elevated TG because the large amounts of sugar that need to be absorbed so rapidly from the intestine lead to the involvement of other metabolic pathways, such as the hexose monophosphate shunt, that that favour the synthesis of FFA [92]. Again, the liver takes up dietary fructose rapidly where it can be converted to glycerol-3-phosphate. This substrate favours esterification of unbound FFA to form the TG [93]. It has also been found that increases of 1,2-sn-diacylglycerol and elevated expression of a PKC isoenzyme are associated with the enhanced synthesis of TG observed with high fructose diet models [94]. In these scenarios, where there is excess hepatic fatty acid uptake, synthesis and secretion, 'input' of fats in the liver exceed 'outputs', and hepatic steatosis occurs [95]. The mechanisms of steatosis and liver enlargement due to fructose intake are not well understood, but it is believed to be related to microsomal enzyme induction, increased storage of lipids, peroxisome proliferation, and hyperfunction due to excessive hepatic 'workloads' [96]. All of these factors contribute to fructose being a highly lipogenic nutrient, and to the resultant hepatic steatosis.
Mechanisms of fructose induced lipoprotein overproduction
There is growing evidence that the insulin resistant state developed upon fructose feeding is also associated with stimulated hepatic VLDL secretion. Several animal models have been employed to examine the mechanisms of this induction of VLDL, and the subsequent increases in plasma TG observed. Mechanistic studies based on carbohydrate versus lipid metabolism have recently become important because carbohydrate induced hypertriglyceridemia shares a metabolic basis with high fat diet induced endogenous hypertriglycerolemia. The similarly induced dyslipidemias would therefore have the same or similar atherogenic risks [97]. Carbohydrate induced hypertriglycerolemia results from a combination of both TG overproduction, and inadequate TG clearance [97,98]. These disease processes and the hepatic steatosis caused by stimulated lipogenesis have been illustrated by fructose fed animal models showing how aberrant leptin signaling, hyperinsulinemia, and dyslipidemia are related to TG induction [95]. Animals maintained on a chronic high fructose diet develop elevated NEFA and hyperinsulinemia at the expense of glycemic control [99]. This is not surprising, as fructose-induced metabolic dyslipidemia is usually accompanied by whole body insulin resistance [100] and reduced hepatic insulin sensitivity [101]. In the fructose fed hamster model, animals showed decreased glucose disappearance rates, increased plasma NEFA and increased plasma and liver TG [27]. Figure 1 (adapted from ref. 100) shows clear in vivo evidence of fructose-induced insulin resistance as assessed by euglycemic hyperinsulinemic clamp studies. Taghibiglou et al. further characterized the fructose fed hamster model demonstrating the development of a metabolic dyslipidemic state characterized by high plasma levels of VLDL-TG and apolipoprotein B (apoB) due to hepatic lipoprotein overproduction [100]. Serum TGs are elevated via both an increased secretion, and decreased clearance of VLDL [102]. Also, high rates of lipolysis in visceral adipose depots can increase availability of NEFAs and promote hepatic TG synthesis. The TG is then packaged with apoB, and secreted as VLDL particles [93]. Evidence has shown that there is a complex interplay of cellular enzymes regulating lipid synthesis and uptake, as well as export and oxidation. Observations of the actions of insulin affecting lipid secretion as well as inhibition of TG has brought research interests towards the effects of chronic insulin stimulation on VLDL secretion and transport. Excess VLDL secretion has been shown to deliver increased fatty acids and TG to muscle and other tissues, further inducing insulin resistance [103]. Induced cellular changes include alterations in hepatic pyruvate dehydrogenase, changes in insulin signaling phosphorylation, and increases of inflammatory cytokines [104,105]. It is evident that the metabolic effects of fructose occur through rapid utilization in the liver due to the bypassing of the regulatory phosphofructokinase step in glycolysis. This in turn causes activation of pyruvate dehydrogenase, and subsequent modifications favoring esterification of fatty acids, again leading to increased VLDL secretion [53]. Increases in VLDL secretion can then lead to chain reactions in other lipoproteins and lipids, such as low density lipoprotein (LDL). Resultant LDL cholesterol levels induced by high fructose intake are illustrated by comparison of a diet including 20% fructose, contrasted to a starch diet of less than 3% fructose. The 20% fructose diet initiated a cycle of increased fasting serum total and LDL cholesterol of 9% and 11%, respectively, over the starch feeding [106]. Increased evidence was shown in transgenic apo AI-CIII-AIV mice, fed a fructose solution for 9 months, where differential expressions of the apo AI and apo AIV genes were found. This indicated general perturbations in response to dietary intakes, causing long-term adverse effects in this hyperlipidemia mouse model [107]. The male Wistar fatty rat model of obese type 2 diabetes has also shown hyperglycemia. Remarkably, the female Wistar rats only develop this hyperglycemia when given sucrose, containing the responsible element of fructose, which causes increases in gluconeogenic enzymes and decreases in glucokinase. A hypertriglyceridemic effect is seen, presumably due to hepatic overproductions of VLDL and induction of lipogenic enzymes via dietary fructose [108].
Figure 1 Fructose-induced insulin resistance: evidence from euglycemic hyperinsulinemic clamp studies. Mean glucose levels (A) were slightly but significantly higher in fructose-fed vs. control animals during the last 30 mins of the clamp period (p < 0.01). Mean insulin levels (B) were slightly but not significantly higher in the fructose-fed vs. control hamsters during the clamp period. The glucose infusion rate (Ginf) (C) during the clamp period was significantly lower in fructose-fed vs. control animals (p < 0.01). The calculated insulin sensitivity index (SI – see methods) (D) was also significantly lower in the fructose-fed vs. control hamsters (p = 0.03). Fructose-fed (n = 9), control hamsters (n = 10). (adapted from Taghibiglou et al. [100]).
Another contributing factor to VLDL overproduction includes fructose effects on lipid peroxidation. High fructose diets can have a hypertriglyceridemic and pro-oxidant effect, and fructose fed rats have shown less protection from lipid peroxidation. Replacing the fructose in these diets with a more natural source of high fructose, honey, reduces this susceptibility and lowers plasma nitrite and nitrate levels [109]. In 2004, Kelley et al. hypothesized that pro-oxidant stress response pathways may mediate hepatic increases in VLDL secretion and delayed clearance upon fructose feeding. Hypertriglyceridemic fructose fed rats were treated with lipoxygenase inhibitors, which reversed the inflammatory protein activity response, and the lipid dysregulation observed [102]. Recent findings have also shown that the hyperlipidemic and pro-oxidant effect induced by a high fructose diet can be decreased by oligofructose consumption. Oligofructose administered to fructose fed rats did not alter insulin concentrations, and lowered plasma leptin by 50% compared to control groups. Oligofructose prevented TG changes induced by fructose feeding, and decreased hepatic TG accumulation. The peroxidation effect of fructose was also decreased by oligofructose, and had beneficial protective effects [110]. Oxidative stress has often been implicated in the pathology of insulin resistance induced by fructose feeding, and lipid peroxides, diene conjugates, and reactive substances are undeniably elevated in fructose fed animals, especially accompanying a deficient antioxidant system. Administration of alpha-lipoic acid (LA) has been shown to prevent these changes, and improve insulin sensitivity [111]. LA treatment also prevents several deleterious effects of fructose feeding: the increases in cholesterol, TG, activity of lipogenic enzymes, and VLDL secretion, the reductions in lipoprotein lipase and HDL cholesterol and may even normalize a dyslipidemic cholesterol distribution of plasma lipoproteins [112]. Taken together, this evidence shows a clear role of peroxidative stress pathways involved in VLDL oversecretion.
Observations made in our own laboratory have also shown aberrant lipogenesis activity. In primary hepatocytes isolated from fructose fed hamsters, there were significant increases in LXRα, SREBP-1, FAS and SCD, which indicate increased activity of the lipogenic pathways (unpublished observations). Fructose has also been implicated in reducing PPARα levels in rat hepatocytes. PPARα is a ligand activated nuclear hormone receptor that is responsible for inducing mitochondrial and peroxisomal β-oxidation. Nagai et al. found that following 8 weeks of a high fructose diet, rats showed decreased PPARα mRNA and protein levels [88]. In addition, primary rat hepatocytes treated with fructose also showed decreased PPARα expression, suggesting that fructose or its metabolites can directly regulate lipid oxidation. We have also recently detected decreased mRNA levels of PPARα in both liver and intestine of the fructose fed hamster (unpublished observations). Hence, decreased PPARα expression can result in reduced oxidation, leading to cellular lipid accumulation. For example, PPARα null mice have extensive hepatic steatosis because of diminished β-oxidation capacity, such as seen in the insulin resistant state [113]. Other mechanisms have been illustrated by Taghibiglou et al., who found evidence for enhanced lipoprotein assembly, reduced intracellular apoB degradation, and increased microsomal triglyceride transfer protein (MTP) mass, mRNA and activity in the fructose fed hamster [100]. These metabolic changes also coincided with a decrease in ER-60, a cysteine protease that may play a role in apoB degradation, and an increase in synthesis and secretion of apoB [101]. It appears that a complex relationship exists in the fructose fed animal model that links insulin resistance and dyslipidemia through NEFA flux, SREBP-1 expression, de novo lipogenesis and MTP expression. Amplified MTP activity and expression would be expected to stimulate the assembly and secretion of apoB-lipoproteins, as an association has been demonstrated between MTP levels and VLDL production [114]. As insulin is a negative regulator of MTP gene expression [115], the upregulation of MTP that has been observed in insulin resistance states is predictable. MTP is also negatively regulated by SREBP through sterol response element (SRE) regions located within -124 and -116 of the 5' MTP gene promoter [116]. However, in fructose fed animals [87] as well as other models of insulin resistance [117] where increased levels of MTP and SREBP have been established, the regulatory effects of SREBP may play a minor role in regulating MTP expression. Rather increased hepatic NEFA and increased TG stores might stimulate MTP expression [118]. Recent observations in our laboratory show that oleic acid can stimulate the MTP promoter and the stimulation occurs independently of SRE activity (unpublished observations). Thus, in insulin resistance states, increased MTP may occur through another mechanism that may block SREBP-mediated inhibition of the promoter. These phenomena help explain the increased assembly and secretion of apoB in fructose fed models. In addition, increased levels of small dense LDL particles have been observed in insulin resistant states [119]. Early studies by Verschoor et al. showed that fructose diets altered the structure and function of VLDL particles causing and increase in the TG: protein ratio, and an increased total cholesterol and phospholipid content [120]. LDL particle size has been found to be inversely related to TG concentration [121] and therefore the higher TG results in a smaller, denser, more atherogenic LDL particle, which contributes to the morbidity of the metabolic disorders associated with insulin resistance. Several theories are proposed for the overproduction of VLDL: more TG per VLDL particle, increases in particle number, changes in the production rates of VLDL TG or apoB, decreased TG clearance, increased lipoprotein lipase activity, and increased de novo lipogenesis. It is likely a combination of some or all of these factors that contribute to the elevated TG seen in a fructose rich carbohydrate fed model of metabolic disorder. High fructose, which stimulates VLDL secretion, may initiate the cycle that results in metabolic syndrome long before type 2 diabetes and obesity develop [103].
More recently, our studies have identified an interesting link between the development of insulin resistance and deregulation of intestinal lipoprotein metabolism [122]. Chronic fructose feeding stimulated intestinal secretion of apolipoprotein B48-containing lipoprotein particles accompanied by enhanced intestinal lipid synthesis in the form of free cholesterol, cholesterol ester, and triglyceride, as well as increases in both MTP mass and activity. These results suggest that in insulin resistant or diabetic animals, there may be a mechanism causing enhanced intestinal secretion of lipoproteins in the fasting state. Fructose feeding may enhance this basal level of lipoprotein secretion through increased de novo lipogenesis and increased MTP availability. Comparison of plasma lipoproteins from fructose-fed animals showed a significant shift toward secretion of larger, less dense, chylomicrons in the insulin resistant animals [123].
Concluding remarks
The alarming increase in fructose consumption may be an important contributor to the epidemic of obesity and insulin resistant diabetes in both pediatric and adult populations. For thousands of years, the human diet contained a relatively small amount of naturally occurring fructose from fruits and other complex foods. Adaptation of humans to a high glucose/low fructose diet has meant that hepatic carbohydrate metabolism is designed to actively metabolize glucose with a limited capacity for metabolizing a small daily intake of fructose. The increasing application of high fructose sweeteners over the past few decades has resulted in a considerable rise in the dietary intake of fructose. A high flux of fructose to the liver, the main organ capable of metabolizing this simple carbohydrate, disturbs normal hepatic carbohydrate metabolism leading to two major consequences (Figure 2): perturbations in glucose metabolism and glucose uptake pathways, and a significantly enhanced rate of de novo lipogenesis and TG synthesis, driven by the high flux of glycerol and acyl portions of TG molecules coming from fructose catabolism. These metabolic disturbances appear to underlie the induction of insulin resistance commonly observed with high fructose feeding in both humans and animal models. Fructose induced insulin resistant states are commonly characterized by a profound metabolic dyslipidemia, which appears to result from hepatic and intestinal overproduction of atherogenic lipoprotein particles. Taking into consideration that a typical western diet not only contains high levels of fructose but is also rich in both fat and cholesterol, synergistic interactions among these nutrients can readily occur leading to a greater degree of insulin resistance and dyslipidemia. In conclusion, emerging evidence from recent epidemiological and biochemical studies clearly suggests that the high dietary intake of fructose has rapidly become an important causative factor in the development of the metabolic syndrome. There is an urgent need for increased public awareness of the risks associated with high fructose consumption and greater efforts should be made to curb the supplementation of packaged foods with high fructose additives.
Figure 2 Hepatic fructose metabolism: A highly lipogenic pathway. Fructose is readily absorbed from the diet and rapidly metabolized principally in the liver. Fructose can provide carbon atoms for both the glycerol and the acyl portions of triglyceride. Fructose is thus a highly efficient inducer of de novo lipogenesis. High concentrations of fructose can serve as a relatively unregulated source of acetyl CoA. In contrast to glucose, dietary fructose does NOT stimulate insulin or leptin (which are both important regulators of energy intake and body adiposity). Stimulated triglyceride synthesis is likely to lead to hepatic accumulation of triglyceride, which has been shown to reduce hepatic insulin sensitivity, as well as increased formation of VLDL particles due to higher substrate availability, increased apoB stability, and higher MTP, the critical factor in VLDL assembly.
List of Abbreviations
ACC: acetyl-CoA carboxylase
apoB: apolipoprotein B
ERK: extracellular signal related kinase
FAS: fatty acid synthase
GSK-3: glycogen synthase kinase-3
HDL: high density lipoprotein
HFCS: high fructose corn syrup
IR: insulin receptor
IRS: insulin receptor substrate
LA: alpha-lipoic acid
LDL: low density lipoprotein
LXR: liver X receptor
MAPK: mitogen activated protein kinase
MTP: microsomal triglyceride transfer protein
NEFA: non-esterified fatty acids
PA-1: plasminogen activator inhibitor-1
PI3-kinase: phosphatidylinositol 3 kinase
PKB: protein kinase B
PKC: protein kinase C
PPAR: peroxisome proliferator activated receptor
PTP-1B: protein tyrosine phosphatase-1B
SCD: stearoyl-CoA desaturase
SREBP: sterol regulatory element binding protein
TG: triglyceride
VLDL: very low density lipoprotein
Acknowledgements
This work was supported by operating grants from Heart and Stroke Foundation of Ontario and the Canadian Institutes of Health Research to KA.
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| 15723702 | PMC552336 | CC BY | 2021-01-04 16:37:46 | no | Nutr Metab (Lond). 2005 Feb 21; 2:5 | utf-8 | Nutr Metab (Lond) | 2,005 | 10.1186/1743-7075-2-5 | oa_comm |
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BMC Cell BiolBMC Cell Biology1471-2121BioMed Central London 1471-2121-6-71571590610.1186/1471-2121-6-7Research ArticleContribution of gap junctional communication between tumor cells and astroglia to the invasion of the brain parenchyma by human glioblastomas Oliveira Roxane [email protected] Christo [email protected] Jean Sébastien [email protected] Boüard Sophie [email protected] Stéphane [email protected] Laurent [email protected] Marcienne [email protected] Marc [email protected] INSERM/UPVM 421, Plasticité cellulaire et thérapeutique, Faculté de Médecine, 94010 Créteil cedex France2 Service de neurochirurgie, CHU Henri Mondor, 94010 Créteil cedex France3 INSERM U 114, NeuroBiologie, Collège de France, Place Marcellin Berthelot, 75005 Paris cedex France2005 16 2 2005 6 7 7 10 9 2004 16 2 2005 Copyright © 2005 Oliveira et al; licensee BioMed Central Ltd.2005Oliveira et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Gliomas are "intraparenchymally metastatic" tumors, invading the brain in a non-destructive way that suggests cooperation between glioma cells and their environment. Recent studies using an engineered rodent C6 tumor cell line have pointed to mechanisms of invasion that involved gap junctional communication (GJC), with connexin 43 as a substrate. We explored whether this concept may have clinical relevance by analyzing the participation of GJC in human glioblastoma invasion.
Results
Three complementary in vitro assays were used: (i) seeding on collagen IV, to analyze homocellular interactions between tumor cells (ii) co-cultures with astrocytes, to study glioblastoma/astrocytes relationships and (iii) implantation into organotypic brain slice cultures, that mimic the three-dimensional parenchymal environment. Carbenoxolone, a potent blocker of GJC, inhibited cell migration in the two latter models. It paradoxically increased it in the first one. These results showed that homocellular interaction between tumor cells supports intercellular adhesion, whereas heterocellular glioblastoma/astrocytes interactions through functional GJC conversely support tumor cell migration. As demonstrated for the rodent cell line, connexin 43 may be responsible for this heterocellular functional coupling. Its levels of expression, high in astrocytes, correlated positively with invasiveness in biopsied tumors.
Conclusions
our results underscore the potential clinical relevance of the concept put forward by other authors based on experiments with a rodent cell line, that glioblastoma cells use astrocytes as a substrate for their migration by subverting communication through connexin 43-dependent gap junctions.
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Background
Glioblastoma, the most aggressive primary brain tumor, is invariably associated with profuse and long-distance invasion of the brain parenchyma. Accordingly, this tumor has been defined as "intraparenchymally metastatic" [1]. Intra-parenchymal dissemination is largely responsible for systematic recurrence despite treatments [2], and poor prognosis with a median survival time not exceeding one year [3]. Preferential routes of long-distance dissemination have been identified, among which the basal lamina of blood vessels and fiber tracts [1,4]. Numerous studies have revealed mechanisms of invasion in those conditions, specifying the molecular bases of glioma cells interaction with specific ligands of the brain extracellular matrix, their proteolytic modification by glioma cells, and the corresponding cellular receptors [2,5]. Less is known about the mechanisms that underlie the migration of glioma cells among neural cells, in the brain parenchyma, despite its profuseness in the area that surrounds the tumor mass, and its major role in recurrence.
We have undertaken to identify the mechanisms of this particular type of glioma invasion, on the basis of the hypothesis that cells in the brain parenchyma may play a supportive role in this process. One key for the elaboration of this working hypothesis has been the demonstration that this intra-parenchymal invasion occurs in a non-destructive way (see refs and discussion in Bernstein, 1996), as this suggests that invading human glioblastoma cells may establish relationships of cooperation with their environment and in particular with resident brain cells. We have focused our attention on gap junctions because of two complementary reasons. First, evidence from peripheral tumors such as melanoma has implicated gap junctions in tumor cell migration [6]. Second, gap-junctional communication via connexin 43 (Cx43) between astrocytes and glioma cells has been demonstrated [7], and linked to a phenotypic transformation of astrocyte which may render the brain parenchyma permissive to glioma invasion. In a recent study, Lin et al. have strongly supported this hypothesis by establishing that non-migratory rodent C6 tumor cells display a migratory behavior following transplantation into the rat brain, when engineered to synthesize Cx43 [8]. Although Cx43 was shown to exert additional cell adhesion effects, the authors demonstrated that promotion of migration was not associated with this role but with the establishment of functional gap junctional communication (GJC). Astrocytes -to which glioblastoma cells are phenotypically most closely related- are widely inter-connected through gap junctions, formed predominantly by the protein Cx43 [9] and glioma cells also express Cx43 [10-13].
Building up upon the concepts defined by Zhang et al. (1999) and Lin et al. (2002), we have explored their clinical relevance by looking at the ability of human glioblastoma cells to establish functional gap junctions with astrocytes, and at the role that such functional interaction may play in their migratory behavior. To observe and quantify the migration of human glioblastoma cells in a three-dimensional brain environment ex vivo, our analysis has taken advantage of ISIS, the "intra-slice implantation system" that we have recently developed [14], in addition to in vitro glioma cell cultures and glioma/astroglial co-cultures. The ISIS set-up allows the implantation not only of human tumor cells but also of fragments of biopsied glioblastoma into rodent brain slices maintained in culture [14,15]. It allows for long term analysis of glioma cell migration in a three-dimensional organotypic environment that mimics that of the brain parenchyma and application of pharmacological agents to disrupt GJC.
Results
Heterocellular coupling and migration of human glioblastoma cell lines
Heterocellular coupling was assessed in glioma-astrocytes co-cultures, using the dual-label technique, in which glioma cells were pre-labeled donors cells and astrocytes potential recipient cells labeled through gap-junctional diffusion of the dye. Heterocellular coupling was more extensive for GL15 cells (heterocellular coupling astrocytes/glioma ratio: 4.05 ± 0.6 recipient astrocytes per one GL15 donor cell) than for 8-MG cells (0.6 ± 0.2; p < 0.001) (Fig. 1A).
Figure 1 Heterocellular coupling of glioma cell lines, and migration assay in brain slice cultures. A, heterocellular coupling between astrocytes and human glioma cell lines (using the preloading method, see also fig. 6A). In contrast to 8-MG cells, GL15 cells established extensive GJC with normal astrocytes. Histograms represent mean heterocellular coupling indices (number of recipient astrocytes per double labeled tumor cell) of a minimum of three independent experiments (range 4 – 6; n= 120 to 225 donor cells per group; ± SEM; ***, p < 0.001). B, 8-MG cells and GL15 cells migration in brain slice cultures. Histograms of the average migration indices showing that GL15 were more invasive than 8-MG cells (mean values ± SEM. ***, p < 0.001).
Analysis of tumor cells invasion was assessed in brain slice cultures, in which both homocellular coupling (between glioma cells) and heterocellular coupling with astrocytes can occur. GL15 cells displayed a significantly higher invasive potential than 8-MG cells [(S - S0) / S0 = 7.82 ± 0.68 vs. 2.67 ± 0.67, p < 0.001] (Fig. 1B).
Addition of CBX induced a significant decrease in the heterocellular coupling index of GL15 cells with astrocytes (down to 1.89 ± 0.74; p < 0.001) in co-cultures (Fig. 2B) whereas coupling was similar in control cultures either untreated, or treated with the functionally inactive analogue of CBX, GZA. Inhibition of GJC by CBX following implantation of GL15 into brain slices, resulted in a significant decrease in GL15 cells invasion into the brain parenchyma. The surface ratio (S-S0) / S0 of migration was reduced by inhibition of the gap junction function (5.59 ± 0.49 for CBX-treated vs. 7.82 ± 0.70 in untreated controls; p < 0.01) (Fig. 2C), as well as the number of GL15 cells that left the tumor mass (105 ± 6.3 cells/mm of perimeter vs. 132 ± 7.2; p < 0.01) (Fig. 2D). There was no difference between results obtained in the GZA and control groups. Those results were confirmed when migration of glioma cells out of spheroids was analyzed over an astrocyte monolayer culture in a 42 hours-long time lapse experiment (Fig. 2E).
Figure 2 Effects of inhibition of heterocellular coupling between GL15 cells and astrocytes. A, Photomicrographs in co-cultures (preloading method) showing heterocellular coupling without (upper panel) or with addition of the inhibitor CBX. GL15 donor cells pre-labeled with a non-diffusing membrane-bound dye (DiI, red) and loaded with a fluorescent gap junction-permeable dye (calcein, green) were seeded on a monolayer of unlabeled astrocytes. Functional heterocellular GJC is visualized as the transfer of calcein from DiI-labeled donor cells (small arrows) to surrounding recipients cells. Donors cells appear yellow because of the merge of red and green labeling. CBX reduced the number of calcein-containing (green) recipient astrocytes, (×200). B, Histograms representing mean values of a minimum of three independent runs as in A (range 4 – 6; n = 160 to 225 donor cells per group ± SEM). (***, p < 0.001). C and D, histograms of the migration indices of GL15 in brain slices without or with CBX, illustrating decreased migration in the treated group (mean values ± SEM. **, p < 0.01). E, Cumulative migration indices plotted against time in co-cultures where GL15 cell spheroids were plated upon an astrocyte monolayer, without or with CBX; (S - S0) / S0 values were measured every 12 h, for 48 h. Integrated areas under the two curves in arbitrary units were compared, p < 0.0001.
In contrast, CBX treatment had no significant effect on 8-MG migration, either in glioma-astrocytes co-cultures or following implantation into brain slices (data not shown).
Inhibition of GJC reduces invasion ability of cells either from human glioma xenografts maintained in nude mice or from fresh human glioma biopsies
The composite cellular content of glioma fragments and the technical difficulty in maintaining cells alive after seeding them in culture as cell suspensions, precluded analysis of the functional coupling of tumors, either maintained in nude mice or freshly biopsied. We, therefore, relied upon the effects of CBX to assess whether, similarly to results obtained in established glioma cell lines, inhibition of GJC may affect the migration of tumor cells out of a fragment and into the neural parenchyma of a brain slice.
Invasive capacities of the seven tumors maintained in nude mice were quite heterogeneous. Three were minimally invasive according to our criteria [total number of cells that detached from the tumor mass was less than 15 cells and the distance of migration less than 50 μm (T3, T6 and T8)]. These three tumors were precisely the ones in the series of 7 tumors to not exhibit any response to CBX treatment (Table 1). Among tumors characterized as "invasive" according to our criteria, CBX treatment significantly inhibited invasion in three (T2, T4 and T7), and had no statistically significant effect in one (T1). Inhibition concerned both the surface ratio of migration (35 to 50% decrease) and the number of migratory cells (25% to 40% decrease) (Table 1). Treatment with the inactive analogue GZA had no effect on migration as compared to controls.
Table 1 Cx43 expression and invasive capacity of gliomas maintained in nude mice
Controls CBX
Tumor Tumor§ Cx43 (S-S0)/S0 Cells/mm perimeter (S-S0)/S0 Cells/mm perimeter
T1 TG-4-GEN +++ 1.15 ± 0.23 77 ± 9.4 1.05 ± 0.11 68 ± 5.6
T2 TG-1-HAM +++ 0.56 ± 0.05 81 ± 9.4 0.29 ± 0.08 *** 50 ± 7.5 *
T3 TG-7-ROM + 0a - 0a -
T4 TG-14-RAV +++ 1.11 ± 0.08 73 ± 6.7 0.71 ± 0.04 ** 54 ± 4.7 *
T6 TG-5-RAI +/- 0a - 0a -
T7 TG-14-CHA +++ 0.45 ± 0.08 48 ± 4.2 0.22 ± 0.01 *** 35 ± 4.4 *
T8 TG-20-THO +/- 0a - 0a -
a, cut off : a tumor which exhibited less than 15 migrating cells distance of migration, if any, less than 50 μm, was considered non invasive.
§ Xenograft tumor nomenclature as originally described by Leuraud et al (2003).
n = 7–10 per group and condition. ***, p < 0.001. **, p < 0.01, *, p < 0.05
In order to test whether the results obtained with experimental gliomas could be extrapolated to native tumors, the migration potentials of biopsies from 4 human high grade gliomas (HG) were studied in brain slice culture, with or without addition of CBX or GZA. Overall, CBX treatment reduced migration indices of these four tumors by 30% (range 20 to 50%; p < 0.05 unpaired t test and Mann & Whitney – Table 2); this was the most pronounced for HG-23 (p < 0.02, Fig. 3A).
Table 2 Cx43 expression and invasive capacities of human biopsed gliomass
Controls CBX
Tumor Cx43-IR (S-S0)/S0 (S-S0)/S0
HG-18 + 4.89 ± 0.85 (n = 6) 3.63 ± 0.86 (n = 6)
HG-19 + 8.12 ± 0.83 (n = 9) 6.69 ± 0.88 (n = 7)
HG-22 + 3.64 ± 0.66 (n = 9) 2.92 ± 0.21 (n = 8)
HG-23 +++ 5.48 ± 0.85 (n = 6) 2.92 ± 0.47 (n = 6)
IR: immunoreactivity
Figure 3 Inhibition of gap junctional communication in biopsied human gliomas. A. Vimentin immunostaining of the biopsied human glioblastoma HG-23 7 days after implantation into a cultured brain slice without (left panel) or with (right panel) CBX. Note the inhibition of cell migration when GJC is blocked. B, Connexin 43 immunocytochemistry (DAB, counterstained with hematoxylin) in two glioblastomas showing either moderate (HG-22) or abundant (HG-23) expression of the protein (×200). At the cellular level (inserts, ×1000), staining is dot-like or linear, predominantly at the membrane.
Cx43 expression in glioma cells
In order to investigate whether Cx43 was instrumental in the establishment of functional coupling between glioma cells and astrocytes, Cx43 expression was analyzed in glioma cells from GBM cell lines and fresh human biopsies used in previous experiments. We chose to focus on Cx43 because it is the major Cx in astrocytes and is expressed by glioma cells [10-13].
In agreement with previous studies [16], three isoforms of Cx43 could be separated on SDS-PAGE and visualized by immunoblotting as three distinct bands in control astroglial primary cultures: Cx43-NP, a non-phosphorylated isoform, Cx43-P1 and Cx43-P2, the two phosphorylated isoforms of the protein (Fig. 4). The Cx43-P1 isoform was prominent and Cx43-P2 and Cx43-NP isoforms were equally present (Fig. 4). As for invasive potential, Cx43 expression pattern was different in the two GBM cell lines with the GL15 cells exhibiting a three-fold higher concentration of the Cx43-P1 as compared to 8-MG cells (p < 0.001; Fig; 4A).
Figure 4 Connexin 43 expression in glioma cells. A, Western blot analysis of Cx43 expression in cell lines GL15 (lanes 1 and 2) and 8-MG (lanes 4 and 5), and in primary mouse astrocytes (lane 3). Three isoforms of Cx43 were detected in normal astrocytes, that corresponded to two phosphorylated (Cx43-P1 and Cx43-P2) and one non-phosphorylated (Cx43-NP) isoforms. GL15 cells exhibited higher level of Cx43 protein than 8 MG cells. B, Western blot analysis of Cx43 expression in seven human gliomas maintained in nude mice (T1 to T8). All tumors expressed one phosphorylated isoform. In addition, the non-phosphorylated isoform was detected in 4 tumor samples (T1, T2, T4 and T7). Tubulin was used as internal control The gels shown, are representative of a minimum of three independent experiments.
Histological analysis of Cx43 immunoreactivity in brain slices implanted with GL15 cells, showed that the protein was overexpressed in the tumor margin where glioma cells and astrocytes processes were intimately apposed and intermingled (Fig. 5). Confocal microscopy of brain slices allowed us to identify regions of enhanced immunostaining for Cx43 at the interface between GL15 cells and astrocytes, suggesting the existence of heterocellular gap junctions (Fig. 5). Interestingly, these astrocytes displayed a bipolar morphology that extended in arrays a long process that paralleled the outward migrating cells. This was not observed when 8-MG glioma cells were implanted.
Figure 5 Cx43 immunoreactivity in brain slice culture injected with GL15 glioma cells. A, 3D projection of 15 consecutive confocal sections (0.5 μm) (×630), showing cooperation between migrating GL15 glioma cells (vimentin imunostaining, blue) with host astrocytes of the brain slice (GFAP immunostaining, red). Cx43 protein (green), is overexpressed by both tumor cells and astrocytes at the tumor margin where their processes are entwined. B, a single confocal section and two orthogonal projections (right and lower margins) corresponding to the overlayed perdendicular line. Enlarged view, of area boxed in B shows punctuate Cx43 immunoreactivity corresponding to gap junction plaques, at the interface of glioma cells and astrocytes strongly suggesting formation of heterocellular gap junctions. Note astrocyte morphologic changes which extend bipolar processes parallel to the long axes of the tumor cells
A general expression of the P1 isoform was observed in the seven tumors maintained in nude mice, and a variable expression of the Cx43-NP isoform. The four invasive tumors (T1, T2, T4 and T7) expressed abundantly both Cx43 isoforms Cx43-NP and Cx43-P1, whereas the three others (T3, T6 and T8) which were not invasive and did not exhibit any response to CBX treatment, expressed lower levels of Cx43-P1 isoform and did not present detectable levels of Cx43-NP. (Fig. 4B). No expression of Cx43-P2 was recorded.
The amount of tissue available for experimental analysis did not allow us to carry out western blotting on freshly biopsied tumor fragments. Thus Cx43 expression in the human tumor biopsies was evaluated by immunohistochemistry on paraffin embedded tissue section.
Cx43 immunostaining was seen in all 4 specimens. Staining was patchy, and both the intensity of staining and the number of immunostained cells in patches differed from one sample to another. In the typical case, a positive microscopic field contained several strongly stained cells, and several unequivocally labeled cells of lower intensity (Fig. 3B). Homogenous fields of particularly strong immunolabeling intensity was observed in the HG-23 specimen.
Homocellular coupling and migration of human glioblastoma cell lines
We have next evaluated homocellular coupling between glioma cells by the scrape loading method, in which homocellular coupling is demonstrated by the diffusion of the dye from mechanically lesioned cells to their neighbors in a monolayer culture. Assay revealed extensive coupling of GL15 (homocellular coupling index: 63.75 ± 4.4 %), comparable to that displayed by astrocytes in parallel control experiments (60 ± 5.2%), whereas 8-MG cells were only weakly coupled (12.7 ± 1.87 %; p < 0.001 vs. GL15), a result in keeping with their low level of Cx43 expression. Homocellular coupling of all cell types was strongly affected by addition of the gap junctional blocker CBX: down to 7.4 ± 1.3 % for astrocytes (p < 0.001), to 1.97 ± 0.47 for GL15 (p < 0.001) and to 2.2 ± 0.47 for 8-MG cells (p < 0.001) (Fig. 6), whereas GZA had no effect.
Figure 6 Homocellular coupling in human glioma cell lines using the scrape loading method. A, Dye coupling in absence (upper row) or presence (lower row) of the gap junctional blocker CBX. After scraping, injured cells were loaded with Lucifer Yellow (LY) and Rhodamin Dextran, and GJC was assessed by the number of neighboring cells that secondarily acquired green fluorescence. Left panel, confluent monolayer of GL15 cells in phase contrast. Right panel, centrifugal (left to right) diffusion of the dye tracer LY (green) from initially loaded injured cells (yellow, because of the merge of red and green) in the same monolayer. (×100). B, histogram representing the homocellular coupling indices (mean values ± SEM) from 4 to 6 independent cultures for each condition and cell type (astrocytes, GL15 or 8-MG). Homocellular coupling index = % labeled cells out of a total of ~400–800 analyzed cells per cell culture. Addition of CBX resulted in a dramatic decrease of the homocoupling in all cases ***, p < 0.001.
To ensure that CBX-induced decreased migration of GL15 cells was mediated by the effect of the compound on gap junction communication, rather than more generally on the phenotype of tumor cells, analysis of tumor cell migration was performed in experimental conditions in which only homocellular coupling could exist, i.e. after seeding of glioma spheroids on a collagen IV matrix. Surprisingly, when homocellular coupling between tumor cells was inhibited by CBX, both tumor cell lines displayed a significant increase in migration out of the spheroids [(S-S0) / S0 = 55 ± 4.1 and 25.4 ± 5.25 for GL15 and 8 MG, respectively, p < 0.001 vs. untreated in both cases] (Fig 7).
Figure 7 Migration assay of glioma cell lines on collagen IV. A, vimentin-immunostained GL15 and 8-MG spheroids, after 4 days of culture on collagen IV without (upper row) and with (lower row) addition of CBX, (x50). B, (S - S0) / S0 ratios (mean values ± SEM) obtained from 10 to 30 spheroids per group and condition. Addition of CBX enhanced spheroid disintegration and centrifugal dispersion of migrating cells. ***, p < 0.001.
Induction of glioma cell migration in vivo by overexpression of Cx43
In order to validate further the hypothesis of a role of GJC formed by Cx43 in tumor cell migration, we have used a technique adapted from that used by Lin et al. (2002) [8]. C6 tumor cells have been genetically engineered by transfection, using a plasmid containing a fusion gene encoding GFP attached to the carboxyl terminus of Cx43. The gap junctions formed by this chimeric protein were dye-permeable [17]. C6 cells are known to proliferate without migrating out of the tumor mass, following implantation into the rat brain, and do not express a significant amount of Cx43. By transplanting mixed population of native and GFP-Cx43-transfected cells, we explored the capacity for the latter to display specifically a migratory behavior. Indeed, GFP-Cx43 expressing C6 cells were preferentially located at the periphery of the tumor mass and some were even observed scattered in the surrounding brain parenchyma, strongly suggesting a migratory behavior (Fig. 8). This was not observed with C6-GFP cells which were, as classically described, only present in the tumor mass.
Figure 8 In vivo parenchymal invasion of C6 glioma cells overexpressing the fusion protein GFP-Cx43. A, vimentin-immunolabeled C6 glioma cells (red) 8 days after striatal injection in the nude mice brain. C6 cells overexpressing the Cx43-GFP protein in green (arrow heads), are preferentially located at the tumor margin and display an invasive phenotype (×100). B, Microscopic field of the area boxed in A, at a higher magnificence (×200). C, D, confocal section (1 μm) of the border of another tumor showing invasive C6-Cx43-GFP cells at the tumor periphery migrating out. C6 cells which did not express Cx43-GFP protein, only labeled in red for vimentin remain in the tumor mass. E, is the overlay of the two channels. Migrating C6-Cx43-GFP cells appear yellow because of the merge of red and green. Dashed lines indicate the extremity of the tumor mass. Scale bar, 25 μm. tm, tumor mass.
Discussion
The main result of this study is the demonstration that functional GJC between tumor cells and host astrocytes is instrumental in the invasion of the brain parenchyma by human glioblastoma cells. Establishment of functional coupling between those two cell populations may be mediated by the gap junction protein Cx43 as ability of glioblastoma cells of various origins to migrate out of the tumor bulk into the brain parenchyma, appeared related to the level of Cx43 expression. In support of this hypothesis, overexpression of a fusion Cx43-GFP gene construct triggered a migratory behavior in otherwise non-infiltrative C6 glioma cells implanted into the brain. The similarities between those results and mechanisms of neural precursor cell migration during embryonic development suggest that glioma cells may subvert and take advantage of a physiological mechanism of cell migration into the brain. In contrast, homocellular interaction between glioma cells through gap junctions hampers their dissemination out of the tumor mass, pointing to an additional role for gap junctions, in cell-to-cell adhesion.
Over the past decade, studies on GJC in gliomas have mainly addressed its role in uncontrolled cell proliferation [18-20], by analogy with epithelial cancers in which a low level of connexin expression is a sign of profoundly perturbed tissue homeostasis and malignant evolution [21]. The notion that, in gliomas, GJC may also favor migration draws scientific argumentation from embryology and from observations that other neurectodermal tumors such as melanoma need to establish GJC with host cells in order to migrate out [6]. This concept has been strongly supported recently by the demonstration that a C6 rodent glioma cell line that had been genetically engineered to express Cx43 displayed a migratory behavior following transplantation into the rat brain [8]. This phenomenon was the more striking that the value of the rodent C6 cell line as an experimental model for glioblastoma has always been questioned because it did not reproduce the infiltrative phenotype which is a major characteristic of the human brain tumor. The induction of C6 migration was related to the establishment of functional GJC between glioma cells and host astrocytes through homotypic Cx43-Cx43 gap junctions.
Several lines of evidence drawn from our results converge to indicate that this concept is clinically relevant, as GJC with astrocytes also plays a role in the migration of brain tumor cells for human glioblastomas. Experimental results obtained here with native human glioblastoma cells of various origins, can indeed be interpreted as a demonstration of a similar role for GJC. First, pharmacological blockade of GJC by the specific inhibitor CBX [22] had negative effects on tumor cell migration, whereas its inactive analogue GZA [23] did not. Second, the gap junction protein Cx43, which is overexpressed by reactive astrocytes in the margin of glioblastomas [10], was also expressed by all studied human tumors, including biopsy specimens, providing a molecular basis for the potential formation of homotypic heterocellular gap junctions. Last, close membrane appositions at regions of reinforcement of Cx43 immunostaining, suggesting intercellular contacts between astrocytes and glioma cells migrating out of a tumor spheroid or the body mass, were observed in co-cultures of glioma cells on an astroglial monolayer, as well as following implantation of tumors into brain slices maintained in organotypic cultures.
The pharmacological approach used here had both advantages and drawbacks, which should be indicated as they may limit the conclusions to be drawn. The main advantage of that technique is the fact that it allowed us to study GJC inhibition in fresh tumor samples, in which biological properties and diversity of glioblastoma cells are well preserved, in contrast to the clonal selection and genetic variation that are commonly observed with cells lines. Conversely, we probably introduced some biological variability in the experimental system. This may be relevant to the fact that GJC inhibition with CBX did not abolish but rather reduced the rate of human glioma cell migration in our study, whereas Lin et al. (2002) obtained an "all-or-nothing" effect following genetic engineering of the C6 line. The partial effect on migration observed here may be due to the partial pharmacological inhibition consistent with the results of other studies [24,25] that demonstrated that CBX only reduced GJC by 50%. Alternatively, glioblastoma cell migration into the brain parenchyma does not relate exclusively to cell-to-cell interaction with astrocytes. Interactions between tumor cell and matrix components (reviewed by Giese et al., 2003) [5] are also major actors in glioma cell migration, and they are most likely not effected by inhibition of GJC, at least not directly. The use of a pharmacological compound also leads to questions about specificity. Various compounds used to block GJC have additional non-specific and toxic effects, among which halotane, octanol or acidifying agents. CBX has been shown to be nontoxic and has no effect on cell proliferation, total protein synthesis or cell viability [23,26,27]. This is ascribed, by some authors, to its mode of action, through direct binding on connexon particles [22]. We have checked the specificity of the GJC effects via different experimental ways, including a systematic negative control with glycyrrhyzic acid (GZA), the CBX precursor which only differs from it by its inability to disrupt GJC. CBX and its inactive analog GZA are known to both interact with mineralocorticoïd and glucocorticoïd receptors (see discussion in [28]), but these receptors do not mediate CBX-decreased GJC [23]. We also tested CBX effect on cell apoptosis, cell proliferation or cell viability in our conditions. No significant effect was observed. Although it has been reported that Cx43 may induce cell growth [18,29], we did not observe any effect of CBX on cell proliferation. It is interesting to mention that CBX-decreased GJC is mainly due to alteration of gap junction channel permeability, whereas Cx overexpression is sufficient to regulate cell growth [20,30]. As an interesting positive control of the specificity of the effects recorded in co-cultures and following implantation of tumor cells into brain slices, we have observed an opposite effect of CBX on cell migration when only homocellular interaction between glioblastoma cells was concerned (on collagen IV). Altogether, these results support the conclusion that non-specific pharmacological effects, if any, did not alter results of our study in a consequential manner.
Levels of Cx43 expression were positively related to glioma invasiveness. Analysis of Cx43 expression by western blot allowed detection of two distinct isoforms of the Cx43 protein, Cx43-P and Cx43-NP. Both isoforms were detected in the four highly invasive tumors (T1, T2, T4, T7), whereas the three minimally invasive tumors (T3, T6, T8) displayed no detectable levels of Cx43-P isoform. The presence of both Cx43 isoforms strongly correlated with the invasive capacities of the tumor. However, whether the migratory capacity may depend on the overall levels of expression, and/or upon the concomitant presence of both Cx43 isoforms is, at this stage, a matter of speculation. Published data on the association of either isoform with the functional state of gap junctions are conflicting. Phosphorylation of Cx43 either on serine or tyrosine residues has been reported to disrupt GJC (for review see [31,32]). This could in part explain why T3, T6 and T8 tumors, which only displayed the phosphorylated isoform -the inactive isoform of Cx43-, were non-invasive tumors. However, the reverse association between Cx43 dephosphorylation and GJC inactivation has been suggested in astrocytes [33]. Further biochemical analysis of Cx43 isoforms function in these cells may reveal whether the pattern of Cx43 isoform expression is involved in the invasive process and how this pattern can be altered by specific phosphorylation. On the other hand, it cannot be excluded that the abundance of both -P and -NP isoforms in migrating tumors simply relates to a dynamic turnover of connexons between the cytoplasm (Cx43-NP) and the cell membrane of a migrating cell, where Cx43-P may participate in short-lived contacts with host astrocytes [21]. Recent data have shown that CBX itself did not have any effect on the phosphorylation pattern of Cx43 [34].
In the biopsy specimens, levels of Cx43 expression were different from one tumor to another, and from one zone to another within a specimen, in agreement with results reported by other authors [10-13]. Interestingly, the highest expression of Cx43 was seen in HG-23, the tumor in which migration was the most substantially inhibited by CBX. In addition, transfection of C6 glioma cells with a Cx43 protein fused to a green fluorescent protein allowed us to trace C6-Cx43 cells after intrastriatal stereotactic implantation. Consistent with results of Lin et al (2002) and [35], only C6-Cx43 cells switched to an invasive phenotype providing a direct evidence of the positive correlation between glioma cells Cx43 expression and invasive potential. We hypothesize that Cx43 may favor glioma cells migration by facilitating glioma cells to form gap junction interactions with host astrocytes that will allow them, through currently unknown molecular mechanisms, to drag out from the tumor mass.
A peculiar morphological finding in this study was the phenotypic alteration of astroglial processes around tumors. Changes in the astroglial phenotype induced by glioma cells through GJC have been observed by other authors [7]. The ISIS protocol used here allowed us to gain a comprehensive three-dimensional view of the astroglial meshwork, and to identify the radial orientation of astrocytic processes toward the body of the tumors. This organization is reminiscent of the scaffolding of radial glial cells observed in the developing neocortex in vivo [36]and in vitro [37]. Radial glial cells extending long processes from the ventricular zone to the cortical plate [36]communicate with migrating neurons through Cx43 gap junctions [38-40]. Although tentatively, we may therefore speculate that astroglial Cx43 provide similarly support and guidance to the migrating tumor cells. In the same way as immature neuronal cells do [38,39,41], glioma cells may trigger the phenotypic change of surrounding astrocytes into radial glia-like cells, in order to make them suitable as a substrate for migration.
The three different migration assays allowed us to explore the importance of intercellular interactions between migrating cells and cellular environment occurring during invasion. We have shown that inhibition of homocellular GJC in GL15 spheroids seeded on collagen IV (i.e. in the absence of astrocytes), boosted glioma cell motility, resulting in a higher centrifugal dispersion of cells. This result is in keeping with that obtained by Lin et al (2002) in an experiment in which Cx43 was shown to mediate glioma cell adherence and aggregation. However, accelerated motility of GL15 glioma cells when CBX blocked homocellular glioma/glioma GJC was not observed when glioma cells additionally formed heterocellular GJC with astrocytes, i.e. on astrocyte monolayer or in brain slices. Quite the contrary, heterocellular glioma-astrocytes GJC seemed to be necessary for the invasive process to occur normally, as CBX significantly blocked it in those conditions.
Conclusions
The purpose of our study was to determine whether the concept that functional gap junctional communication with host astrocytes facilitates invasion of malignant glioma cells put forward by other authors on the basis of experiments using a rodent glioma model (Lin et al, 2002), indeed apply to human glioblastoma and may, as a consequence, bear clinical relevance. This has indeed been clearly confirmed by the inhibitory effect of the specific GJC blocker, carbenoxolone, on the migration of human glioblastoma cells. Results obtained ex vivo on different supports of migration point to a functional difference between homocellular (glioma-glioma) and heterocellular (glioma-astrocytes) GJC. This may eventually be of prime therapeutic interest, by revealing mechanisms by which glioma cells disengage themselves from their neighbors in the tumor bulk, and establish new contacts with host astrocytes in order to migrate away [2,7].
Methods
The role of GJC in human glioma invasion was investigated in vitro using three different types of preparations, namely glioblastoma cell lines, xenografts in nude mice, and freshly biopsied tumors. Because of selection for genotypes, the original cell heterogeneity of gliomas is restricted in xenografts and absent from cell lines. These models, therefore, do not fully reproduce the cell content of in vivo gliomas but, reciprocally, they present the advantage of being homogenous, easy to handle, and to allow analysis of large numbers of cells. ISIS uniquely permits to study, in addition, the invasiveness of freshly biopsied tumors with preserved cell repertoire. In those conditions, however, investigation is limited by the amount of available tissue.
Human glioma cells
The two human glioblastoma (GB) cell lines used in the study, GL15 [42] and 8-MG [43] were grown as cell culture in glioma-cell medium: 50% minimum essential medium (MEM) and 50% Dubelco Modified Eagle's Medium (DMEM) complemented with 10% fetal calf serum (ATGC Biotechnological, France), glutamine 2 mM, D-glucose 3.3 mM, Penicillin 100 UI/mL, Streptomycin 100 μg/mL (all from Eurobio, France) at 37°C in a 5% CO2 humidified incubator. On confluence, cells were trypsinized, centrifuged at 300 G for 10 minutes and resuspended in 1 mL of glioma-cell medium. 5 × 105 cells were seeded in 1.5% agar-coated flasks (25 cm3, Falcon, France) for 7–10 days to obtain GL15 and 8-MG spheroids. Spheroids with a diameter ranging from 50–100 μm were selected for migration assays.
Seven grade III-IV human glioma biopsies xenografted and maintained subcutaneously in Swiss Nu/Nu mice (Charles River, France), kindly provided to us by Dr M-F. Poupon and P. Leuraud [44], were used in this study. For the sake of simplicity these tumors are referred to in the text as T1, T2, T3, T4, T6, T7 and T8 (see table 1). Mice were sacrificed when the tumor bulk reached a diameter larger than 1 cm. The tumors were retrieved and immediately transferred in the glioma-cell medium. Areas of necrosis and hemorrhage were identified under a microscope and discarded. The remaining tissue was cut into three fragments that were used as follows: -one was immediately implanted into a healthy nude mouse, in order to maintain the model; -a second one was immediately frozen for Western Blot analyses; -the third one was cut into smaller pieces for implantation into rodent brain slices maintained in organotypic cultures (see below).
Finally, four fresh human biopsy specimens were obtained from stereotactic or resection biopsies in the operating room. All samples were retrieved by the neurosurgeon from areas of enhanced contrast on magnetic resonance imaging, which correspond to a hypercellular zone on histological examination of the tumor. After retrieval, samples were immediately transferred in glioma-cell medium and prepared for implantation into brain slices. Pathological analysis of the same tumors was carried out in parallel on adjacent specimens; it systematically included immunohistochemistry for the connexin protein Cx43 in addition to usual stains. According to the WHO 2000 classification, these tumors were glioblastomas in three cases, an anaplastic grade III astrocytoma in one. For the sake of simplicity the generic term of glioblastoma will be used in the rest of the text.
Functional assessment of gap junctional communication
Functional assessment of gap junctional communication (GJC) was performed on glioblastoma cell lines. Homocellular GJC was determined by the scrape-loading/dye transfer technique [45,46]. Briefly, either GL15, 8-MG or astrocytic cell cultures were incubated for 10 minutes in HEPES buffer solution (140 mM NaCl, 5.5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2 10 mM glucose and 10 mM HEPES pH 7.3). Cells were then washed in a Ca2+-free HEPES solution to prevent uncoupling. After scraping with the razor blade cells were incubated in the same solution containing 1 mg/ml of Lucifer Yellow (LY) (Sigma, France) and 1 mg/mL of Rhodamin Dextran 10,000 MV (Molecular Probes) for 1 minute. Dye transfer through GJC was assessed 10 minutes after scraping, using an Axioplan-2 microscope and appropriate filters (Carl Zeiss, Inc; Oberkochen, Germany). Fluorescent and bright light images digitized with a CoolSNAP camera (Ropper Scientific; Tuscon, AZ) from every microscopic field were merged, and the homocellular coupling index was calculated as the ratio of the number of LY-labeled cells to the total number of cells.
Heterocellular GJC was evaluated in glioma/astroglial cells co-cultures by a method adapted from Goldberg et al. (1995) [27]. Tumor cells were loaded for 20 min with a dual-label dye solution containing isotonic PBS-glucose (0.3M), 5 μM Calcein-AM and 10 μM DiI (both from Molecular Probes, France). They were then rinsed three times with isotonic PBS-glucose 0.3M, trypsinized and centrifuged. Five hundred Calcein/DiI-labeled cells (donor cells) were seeded on an unlabeled confluent astrocytic monolayer (recipient cells), and incubated at 37°C in a 5% CO2 humidified incubator for 20 h. Heterocellular coupling was calculated as the ratio of recipient cells (calcein-labeled astrocytes) to donor cells (Calcein/DiI-labeled glioblastoma cells). It is important to underline that this technique is different from the scrape loading method and uses another dye tracer to assess the gap junctional coupling. Hence, their results cannot be readily compared in quantitative terms.
Effects of inhibition of GJC were evaluated by treating the cultures with carbenoxolone (CBX); controls received its inactive analog, glycyrrhyzic acid (GZA) [23] (both from Sigma, France). CBX is a widely used specific inhibitor of GJC likely exerting its action by changing the conformation of gap-junction channels [22], while GZA has no effect on GJC. CBX and GZA treatments were carried out at a concentration of 30 μM. Cytotoxicity of these molecules on the two glioblastoma cell lines and on astrocytes had been tested in preliminary experiments. There was no significant increase in the lactate dehydrogenase medium/cell ratio for up to 7 days at the concentrations used (Cytotoxicity Detection Kit, Roche Molecular Biochemicals, France).
Cell migration assays
Migration of glioblastoma cell lines was analyzed in three different experimental in vitro set ups: on collagen IV-coated cell culture dishes, after seeding on an astrocytic monolayer culture, and following implantation into organotypic brain slices (ISIS, Intra-Slice Implantation System). Migration out of tumor fragments issued from xenografted gliomas or from freshly biopsied specimens were only studied using the ISIS method.
Migration of GL15 and 8 MG cells out of spheroids deposited onto collagen-coated (0.5 μg/mL; Sigma, France) cell culture dishes was analyzed over 4 days.
For astrocytic co-cultures, primary astrocyte cultures were obtained from cerebral hemispheres of neonatal C57/bl6 mice (Charles River, France) as previously described [47]. Cells were grown in astrocyte medium: MEM (GIBCO, France) complemented with 10% FCS (ATGC Biotechnological, France), glutamine 2 mM, D-glucose 33 mM, Penicillin 100 UI/mL, Streptomycin 100 μg/mL and amino acids 0.5X (all from Eurobio, France). Medium was changed every 3 days. On confluence, GL15 and 8-MG spheroids were seeded on top of the astroglial monolayer, and cultures were continued for 4 days.
The ISIS migration assay was carried out as described previously [14], using brain slices obtained from postnatal day 6 C57/bl6 mice. Briefly, after decapitation the two hemispheres were separated and 400 μm-thick coronal slices were cut using a McIllwain tissue chopper (Mickle Laboratory, England). Slices were transferred into brain slices medium: MEM (GIBCO, France), 1 g/l D-glucose, 10% heat-inactivated fetal calf serum, 0.1 g/l transferrin, 16 μg/l putrescin, 40 μg/l N-selenium, 30 μg/l tri-iodothyronin, 5 mg/l insulin, and 60 μg/l progesterone (all from Sigma, France). Slices were separated and transferred onto Millicell-CM membranes (Millipore, France). The Millicell-CM membranes were kept in six-well plates, above 1 ml of brain slice medium. Slices were incubated at 37°C in a humid atmosphere with 5% CO2. The medium was changed three times a week. 24 hours after brain slice preparation, GL15 and 8-MG spheroids or small fragments of xenografted tumors and freshly biopsied specimens were deposited onto the slice surface and gently pushed with the tip of a needle, until the tumor tissue was well enveloped by the parenchyma of the slice. Cultures were maintained for 5 or 7 days for cell lines and tumor fragments, respectively.
In all experimental conditions, effects of the blockade of GJC were assessed using CBX (30 μM for collagen-coated and astrocytic co-cultures; 60 μM for brain slice cultures, a stronger dose because of the thinness of the slice), with two negative controls: untreated and GZA supplemented to the medium instead of CBX. These compounds were added to the culture medium 24 h after glioma spheroid or tumor implantation.
In vivo experiments
For a control experiment, we transfected the C6 glioma cell line with a plasmid vector encoding either a green fluorescent protein (GFP) fused to the carboxyl terminus of Cx43 [17] kindly provided by Dr P. Martin, or else only GFP. C6-Cx43-GFP cells and C6-GFP control cells were implanted into the striatum of nude mice (5. 104 cells in 1 μL) (n = 5). Mice were sacrificed at day 8 and perfused with paraformaldehyde (4%) 8 days after implantation. Brains were sectioned serially on cryostat at 30 μm thickness as previously described [48].
Immunoblotting and immunohistochemistry
Immunoblot analysis for Cx43 was performed on cells and tumor samples that were suspended in lysis buffer containing Tris base 50 mM, pH 7.4, NaCl 150 mM, Triton X-100 0.5%, EDTA 1 mM, protease inhibitor cocktail 0.5% (Sigma, St. Quentin, France) and triturated to homogeneity. Homogenates were centrifuged at 13,000 G for 10 or 30 minutes (for cell lines and tumor samples, respectively), and supernatants were aliquoted and stored at -80°C. Total protein content was determined by the Lowry method with bovine serum albumin as a standard. Total protein (30 μg) was boiled for 5 min after addition of 10% glycerol/5% mercaptoethanol and separated on SDS-PAGE 12.5% gels, transferred on PVDF membrane (Bio-RAD, France) under classical conditions [49] and immunoblotted overnight at 4°C, using a polyclonal anti-Cx43 antibody (1:500, Zymed, France). Bound primary antibody was detected with a horseradish peroxydase-coupled anti-rabbit antibody (1:5,000; Amersham Pharmacia Biotech, France). In preliminary positive control experiments, Cx43 was revealed as three bands of about 41, 43 and 45 kDa, representing three different isoforms in heart sample. The two higher molecular mass bands have been shown to correspond to phosphorylated forms of the protein, and the 41 kDa migrating species to the unphosphorylated form of the protein [16].
Immunohistochemistry was carried out on cell and slice cultures and brain slices from in vivo experiments following fixation using 4% paraformaldehyde in PBS at 4°C (30 min for mono- and co-cultures, 4 h for ISIS). For fluorescent immunostaining, cultures were rinsed several times in PBS, then incubated for 1 hour in PBS containing 10% normal horse or goat serum and 0.6% Triton X-100 (Sigma). They were incubated overnight at 4°C with monoclonal antibodies against vimentin (1/400, Neo Markers, France), or polyclonal antibodies against glial fibrillary acidic protein-GFAP (1/100, Dako) or Green Fluorescent Protein-GFP (1/200,. Molecular Probes). After 3 washes in PBS, they were then incubated with a PBS solution containing anti-rabbit or anti-mouse immunoglobulin conjugated either to fluorescein isothiocyanate-FITC, AMCA (1/400; Vector, France), or to cyamidine-Cy3 (1/1000; Jackson Immuno Research, France). For human biopsies, immunocytochemical detection of Cx43 was carried out on formalin-fixed, paraffin-embedded 5 μm sections. Antigen-retrieval consisted in heating slides immersed in citrate buffer (pH 6) in a microwave oven (3 × 5 min at 750 W). To block non-specific binding sites slides were incubated in PBS containing 10% BSA. A primary polyclonal antibody (Zymed) directed against the intracellular portion of the Cx43 molecule was applied (1:200) to sections overnight. Bound primary antibodies were detected by a Vector Elite ABC kit (Vector Laboratories, Burlingame, California) following the manufacturer's instructions and using DAB as the chromogen. Slides were lightly counterstained in Harris's hematoxylin. Negative controls had the primary antibodies omitted. A specimen of normal human cortex from epilepsy surgery showed diffuse staining of astrocytic cells accentuated at perivascular and superficial locations, corresponding to astrocytic endfeet and the glia limitans, respectively.
Quantification and statistical analysis
Fluorescent microscopy on a Zeiss Axioplan2 microscope and image analysis using the KS.400 (3.0 version) software were performed to quantify invasion of glioma cells, according to techniques that have been detailed elsewhere [14]. Two invasion parameters were assessed, (i) the maximum area of invasion, given as the ratio (S-S0) / S0, where S is the maximum area containing migrating glioma cells and S0 is the initial tumoral mass area, this ratio compensating for the heterogeneous sizes of implanted spheroids and tumor fragments, and (ii) the number of invading cells (cells/mm of S0 perimeter). For collagen-coated migration assay and astrocytic co-cultures, only the maximum area of invasion was assessed. The statistical significance was evaluated using Mann & Whitney test and Student unpaired t test.
Authors'contribution
ROconceived and designed the study, performed acquisition, analysis and interpretation of data and drafted the manuscript. CCconceived and designed the study, performed acquisition, analysis and interpretation of data and drafted the manuscript. JSGparticipated in the design of the study and carried out xenografts in nude mice. SdB participated in the design of the study. SP provided human biopsies tissue and diagnosis. LV participated in the design of the study. MT have been involved in revising the article critically for important intellectual content. MPconceived, designed and coordinated the study, drafted the manuscript and have given final approval of the version to be published. All authors read and approved the final manuscript.
Acknowledgements
This work was supported by INSERM. The authors are grateful to Drs. M.F. Poupon and P. Leraud for providing the glioma xenografts, to Dr. P. Dreyfus and S. Marty for help with the microscopic images and Dr P. Martin for providing the Cx43-GFP plasmid
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| 15715906 | PMC553963 | CC BY | 2021-01-04 16:39:11 | no | BMC Cell Biol. 2005 Feb 16; 6:7 | utf-8 | BMC Cell Biol | 2,005 | 10.1186/1471-2121-6-7 | oa_comm |
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BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-6-361572534710.1186/1471-2105-6-36SoftwareGANN: Genetic algorithm neural networks for the detection of conserved combinations of features in DNA Beiko Robert G [email protected] Robert L [email protected] Institute for Molecular Bioscience, The University of Queensland, Brisbane 4072, Australia2 Department of Biology, University of Ottawa, Ottawa, ON, K1N 6N5, Canada3 Genome Atlantic, Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, B3H 1X5, Canada2005 22 2 2005 6 36 36 5 8 2004 22 2 2005 Copyright © 2005 Beiko and Charlebois; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The multitude of motif detection algorithms developed to date have largely focused on the detection of patterns in primary sequence. Since sequence-dependent DNA structure and flexibility may also play a role in protein-DNA interactions, the simultaneous exploration of sequence- and structure-based hypotheses about the composition of binding sites and the ordering of features in a regulatory region should be considered as well. The consideration of structural features requires the development of new detection tools that can deal with data types other than primary sequence.
Results
GANN (available at ) is a machine learning tool for the detection of conserved features in DNA. The software suite contains programs to extract different regions of genomic DNA from flat files and convert these sequences to indices that reflect sequence and structural composition or the presence of specific protein binding sites. The machine learning component allows the classification of different types of sequences based on subsamples of these indices, and can identify the best combinations of indices and machine learning architecture for sequence discrimination. Another key feature of GANN is the replicated splitting of data into training and test sets, and the implementation of negative controls. In validation experiments, GANN successfully merged important sequence and structural features to yield good predictive models for synthetic and real regulatory regions.
Conclusion
GANN is a flexible tool that can search through large sets of sequence and structural feature combinations to identify those that best characterize a set of sequences.
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Background
The minimal requirement for transcriptional activation is recruitment of an RNA polymerase complex to a promoter sequence of DNA upstream of an open reading frame (ORF). Most genes are also potentially under the control of DNA-binding regulatory proteins or transcription factors that can activate or silence transcription. In bacteria, activator and repressor proteins bind to operator sequences that are typically found near the promoter, and promoter specificity is typically conferred through the sigma subunit of RNA polymerase, which binds the promoter directly [1]. Eukaryotic transcription factors interact with DNA within the promoter, and are responsible for recruitment of the RNA polymerase complex [2]. Regulatory proteins also bind to conserved sites near the promoter region, as well as to enhancers that can be far (> 10 000 nucleotides) upstream or downstream of the promoter. In all domains of life, transcription factors that bind near the promoter are typically involved in either stabilizing or disrupting the initiation of transcription, while distal enhancer sequences are needed to destabilize the nucleosomes that usually prevent the initiation of transcription in eukaryotes [3].
The identity and spacing of these protein binding sites are key contributors to the responsiveness of a gene to changing cellular conditions, and sets of genes or operons that are expressed under similar conditions often have similar sets of regulatory elements in their 5' upstream regions. Recent programs such as ClusterBuster [4] and Promoter2.0 [5] recognize the need to detect combinations of binding sites in order to characterize promoters or whole regulatory regions. An additional challenge is the possibility of multiple cluster types within a class of genes that respond to the same regulatory stimulus. Artificial neural networks (ANNs) are suited to the task of discovering complex interactions within a set of features, and identifying multiple alternative solutions that yield the same type of response. This type of problem is not linearly separable [6], and would apply to classification problems such as the regulatory cascade of genes that is induced by lipopolysaccharide in mammals, where the entire set of genes is upregulated in response to a stimulus, but time or subresponse specificity is conferred by one of a set of regulatory modules [7,8].
Another issue in the detection and modelling of regulatory regions is the assumption of additivity in DNA-protein interactions when position-specific scoring matrices (PSSMs) are used to model binding sites. In fact, binding affinity has been shown in some cases to depend on interactions between sites [9-11], which suggests that more-sophisticated modelling schemes may be necessary to build accurate models of binding site affinity. PSSMs are a useful and versatile tool and may be adequate for binding site modelling in many cases (see for instance [12]), but the additional flexibility of ANNs may be useful in representing non-additive relationships among components of a binding site.
The genetic algorithm (GA) is a powerful tool for combinatorial problems of model optimization and feature selection when the 'model space' is complex and has many local optima. Genetic algorithms carry out a number of simultaneous searches in model space, with one or more recombination operators to periodically combine the results of two or more searches, permitting large scale 'jumps' out of locally optimal regions. GAs have been applied to several tasks in computational biology, including sequence alignment (SAGA: [13]) and phylogenetic inference (MetaPIGA: [14]). ModuleSearcher [15] is a recent application of genetic algorithms to the problem of cis-regulatory module detection, with the stochastic GA approach shown to yield similar outcomes to an exact search method in substantially less time. ClusterScan [16] is another recent approach that uses genetic algorithms to detect optimal combinations of binding sites from the TRANSFAC database [17].
Another important question in modelling regulatory regions involves the representation of binding sites. While position-specific scoring matrices (PSSMs) are a popular and effective way of representing conserved sites [18], other strategies such as consensus sequences, sequence composition and structural features [19] can be considered as well. Structural features should be of particular interest, since DNA deformability appears to play a role in at least some regulatory interactions such as the binding of Escherichia coli integration host factor (IHF) to its target sequence [20], the correct orientation of both halves of bacterial promoter sequences [21] and the dynamics of histone/DNA interactions [22]. DNA structural properties have been derived from crystal and nuclear magnetic resonance experiments and from theoretical simulations, and different oligonucleotides have different propensities toward unwinding, wrapping around other molecules, and deformation in response to ligand binding (reviewed in [23]). While there is still considerable controversy in the structural field about issues such as A-tract curvature [24], and there have been questions about the role of experimental conditions in determining results [25-27], carefully selected parameters can permit the testing of specific structural hypotheses pertaining to regulatory protein-DNA interactions.
We have developed GANN, a software suite that uses machine learning methods to identify combinations of the features listed above that best distinguish between a positive set (containing, for instance, a set of regulatory regions from co-expressed genes) and a negative set. GANN implements all of the binding site representations described above, allowing examination of models of different complexity as warranted by the type of binding site modelled and the amount of training data that is available.
Implementation
GANN contains a set of programs for sequence extraction, retrieval and grouping of requested patterns, neural network analysis of these patterns and collection of results. Each program in the suite can accept either the output of the previous program, or an appropriate set of data generated from an external method. The components of GANN and the flow of data through the system are shown in Figure 1. 'Indices' and the core machine learning component are implemented in C++, while the other programs that read, interpret and combine text files are written in Perl.
Sequence extraction
The first program (GetSeq) reads in either a GenBank file of annotated genome sequence, or raw sequence and a list of open reading frames (ORFs) of interest generated with the NeuroGadgets Inc. Bioinformatics Web Service [28]. GetSeq identifies and extracts upstream intergenic regions of a specified length and labels them as the positive set, and can also extract negative set sequences from intergenic regions that are not immediately upstream of ORFs, or directly from the protein-coding regions.
Generation of sequence and structure indices
The Indices program takes as input a set of positive and negative set sequences (such as those generated by GetSeq), and can compute various properties of the sequences. The input sequences can be subdivided into overlapping windows of any size prior to the calculation of index values. The following indices can be calculated:
- Oligonucleotide frequencies are computed by counting the number of instances of a given k-mer within a window, then dividing by the length of that window. The program can determine the frequency of all k-mers of a specific length, or can assess any user-specified set of k-mers, which may include IUPAC notation to represent degenerate nucleotides.
- User-specified PSSMs can be counted for each sequence window. The user provides a set of scores for each type of nucleotide at each position within a PSSM, and a threshold score. The program will then count and record the number of sequence instances within each window that yield a PSSM score greater than the specified threshold.
- Structure and flexibility rules are implemented via a text file, by assigning floating-point values to each k-mer of a given length. The average score for a given sequence window is then computed by adding the scores for each overlapping k-mer within the sequence, and dividing by the total number of k-mers considered. Any numeric encoding of a complete set of k-mers can be specified: features sampled from publications such as [29-31] are available at the GANN website (see below).
After the extraction of indices, the Combine program merges the different index files into a single large file that is used as the input for the machine-learning software. Combine also allows the computation of Z-scores, thus representing each index value in terms of the number of standard deviations from the mean, and can identify peak values for a given index across a set of windows. Combine randomly subdivides the positive and negative index sets into training and test sets, and can also generate a negative control by randomly reassigning some positive and negative set members to the opposite category, yielding a disruption of patterns that were previously consistent within a single set. This type of control sets a 'baseline' for classification accuracy that can be compared to real experimental results.
Pattern classification
The core of GANN is the neural network classification system. The indices generated from 'Indices' and 'Combine' are presented as input to an artificial neural network, which is trained with either backpropagation or a genetic algorithm to maximize the discrimination between the positive and negative sets. Since the number of indices associated with each sequence is potentially very large, the Outer Genetic Algorithm (OGA) presents random subsets from the pool of indices to a series of neural networks. The unit of selection for the OGA is a 'Chromosome' that contains a predetermined number of indices sampled from the larger pool, and a set of parameters that define the architecture and connectivity of the ANN. The constitution of a population of OGA Chromosomes is determined randomly in the first generation, with random sampling of indices from the pool and ANN parameters sampled randomly from within a set of ranges specified by the user. Each OGA Chromosome is used to construct an ANN, which is then trained to yield optimal predictive accuracy on the training set defined by the Combine program above. At the end of training, performance on the test set is evaluated, and the fitness of the OGA Chromosome is equal to its predictive accuracy on the test set samples. The predictive accuracy is defined as follows:
Where TP and TN are the number of correctly classified positive and negative test set examples, and ||Pos|| and ||Neg|| the size of the positive and negative test sets, respectively. This formula assigns equal weight to the positive and negative sets regardless of their size, so the ANN cannot achieve an artificially high score by predicting every case as a member of the larger (training or test) set.
The OGA Chromosomes with highest fitness are then permitted to 'recombine', yielding new subsets of indices that are trained in the same manner, while less successful indices are gradually lost from the population. The classification potential of indices can be evaluated by examining the scores of neural networks that include these indices in their input set, and through a 'population genetics' approach that traces the frequency of indices through several rounds of OGA recombination and selection.
Training of the neural networks is performed using either backpropagation of errors [32] or with an 'inner' genetic algorithm (IGA). An IGA Chromosome consists of a set of floating-point values, each representing a connection weight within the ANN that is being trained. The fitness of each IGA Chromosome within a population is equal to the predictive accuracy of the specified ANN as defined in the equation above, but on the training set. IGA Chromosomes with relatively high fitness are then subjected to stochastic recombination and mutation of parameters to yield a new population of Chromosomes that are used in the next round of training. While gradient-descent training methods for ANNs (such as backpropagation) can easily get trapped in local optima of the solution space, the recombination option of genetic algorithms permits a search to 'jump' through the solution space and escape local optima. The optimisation of network architecture and connection weights is similar to the 'structure evolution' method of [33], which has been applied to problems of biological pattern detection [34,35], but our method differs in the partitioning of the connection weight and architecture components of the optimisation.
There are many variables within GANN whose values can be specified by the user. While most default settings will be adequate in most situations, parameters such as the number of feature combinations generated by the OGA and the number of features in each combination should be chosen carefully. The 'DefineVars' program provides a set of menus that allow the user to set these parameters and write them to a configuration file that is input to GANN.
In addition to reporting the scores of trained artificial neural networks, GANN will save information about the topology, connection weights and constituent indices of each neural network instance that achieves a generalization score above a specified minimum threshold. If GANN is invoked with any of these saved neural networks as input in addition to a table of indices, then it will use the input neural network to classify the new table. This process can yield functional predictions for sequences whose true classification is unknown.
Results and discussion
Two sets of detection experiments, both based on DNA sequences extracted from the Escherichia coli K12 genome, are presented to illustrate the performance of GANN. Both of these experiments included a set of 250 nucleotide sequences, each 100 nucleotides (nt) in length, which were extracted from between convergently transcribed genes in the E. coli genome using the GetSeq program. These sequences were chosen because they are intergenic like upstream regulatory regions and not subject to the evolutionary constraints of protein-coding sequences, but are not expected to contain functional transcriptional regulatory features since they are exclusively 'downstream' of one gene in each direction. In the first experiment, we created an artificial positive set by inserting conserved sequences into a subset of the 250 sequences, with the remainder constituting the negative set. The entire set of 250 sequences was used as the negative set in the second experiment, while the positive set consisted of 212 upstream regulatory regions containing experimentally validated binding sites for the σ70 protein of E. coli.
Several run parameters were consistent across both experiments. Each OGA Chromosome contained a total of 8 indices, to allow the simultaneous representation of several sequence and structure properties. The population size (= number of OGA Chromosomes) was determined by multiplying the total number of indices by 10, then dividing by the number of indices (8) per OGA Chromosome. This formula ensured that indices would be represented 10 times each on average in the initial randomly generated population, and 99.9% of all indices should occur at least 3 times in the population according to the Poisson distribution. Thirty rounds of OGA Chromosome evaluation and selection were performed in each run. While GANN can evolve ANN architecture and learning parameters as well as combinations of indices, we chose reasonable ANN parameters (available at the GANN website) and fixed them for the entire run.
The performance of different sets of indices was expressed in terms of the predictive accuracy (= score on the test set of sequences) described above. Differences in predictive accuracy are expected across replicates, because random partitioning of sequences into training and test sets is likely to yield variation in the frequency of some features that do not define the whole set. However, average predictive accuracy can be estimated by taking the mean across replicates. Indices that are retained in every replicate of an experimental run are more likely to reflect true characteristics of the sequences under consideration, though the redundancy of many indices (different window sizes, different percentiles for PSSM scores, and correlated frequencies of some k-mers) may yield multiple alternative solutions that are equally good. A final indicator of index performance is the composition of OGA Chromosomes that yield high predictive accuracy: if a set of features is important for characterization of a set of sequences, then each of those features should be represented by at least one index in the best OGA Chromosomes.
Experiment 1 – Synthetic positive set
In the first experiment, the positive set was constructed by adding conserved features to 76 of the 250 sequences (~30%) described above. Each member of the positive set was modified by adding a nucleotide decamer with high conformational mobility (CM). Five thousand unique decamers were generated randomly, and each of these was assigned a CM score based on the dinucleotide table in [30]. Decamers from this set that scored in the top 5% of all CM values were selected at random to be added to members of the positive set. One of two types of conserved binding site was also added to each member of the positive set. The set of experimentally validated binding sites for cAMP receptor protein (CRP) and leucine-responsive regulatory protein (Lrp) were extracted from RegulonDB [36], and each positive set sequence gained a binding site randomly selected from one list or the other.
The construction of synthetic conserved regions is summarized in Figure 2. 'Type A' positive set sequences consist of a high CM decamer beginning anywhere between positions 10 and 20, and a randomly chosen CRP binding site of length 19 that starts between positions 65 and 70. The order of patterns is inverted in the 'Type B' sequences, with the 12 nt Lrp binding site beginning between positions 25 and 30, and the high CM decamer starting anywhere between positions 75 and 80. A total of 34 Type A and 42 Type B sequences were generated.
Once the positive and negative set sequences were obtained and assembled, the Indices program was used to extract several different types of information from them, with varying window sizes depending on the features being examined. The overlap between adjacent windows was chosen to be 50% of the window length, so if a window of size 10 covered sites 1–10 in a sequence, the next window would cover sites 6–15.
- PSSMs for Lrp and CRP binding sites were constructed from the set of binding sites in RegulonDB. Frequency matrices for each site were constructed by dividing the number of occurrences of each residue at each site by the total number of sites (72 Lrp, 128 CRP). These frequencies were then divided by the 'background' frequency of each corresponding nucleotide in the set of intergenic sequences. Since the background frequency of each nucleotide was within the range 0.250 ± 0.005, background frequencies of 0.25 were assigned to each nucleotide. The PSSM was then obtained by taking the natural logarithm of each value in the corrected frequency matrix. Threshold values for PSSM predictions were determined by scoring 50 000 random sequences of the appropriate length against the PSSM, and identifying the scores that corresponded to the 99th, 95th, 90th, and 80th percentiles. The number of sequence matches above each PSSM threshold was computed for windows of size 20 and 40.
- The conformational mobility of sequence windows of size 10 and 20 was computed according to the dinucleotide values in [30].
- Counts of all k-mers of size 1 (mononucleotides) and size 2 (dinucleotides) were computed for windows of size 10 and 20.
The computations described above yielded a total of 450 indices: 360 describing k-mer counts, 64 describing the counts of PSSM 'hits' at different thresholds, and 36 describing the conformational mobility of different windows of sequence. These indices were then combined in four different ways to yield separate tests of different subsets. The k-mer frequencies were included in every set as a 'background' measure of predictive power with no explicit hypothesis. Set 1.1 included only the k-mer frequencies, while set 1.2 added the indices of conformational mobility, set 1.3 included the PSSM scores, and set 1.4 included all three types of index. We initially performed runs where index values were not standardized, but found that indices with values that were not close to zero, particularly the CM indices which ranged between 40 and 70, did not perform well and were consistently eliminated from the population of OGA Chromosomes. In response to this, we standardized all indices for the experiments described below and in Experiment 2.
Set 1.4, with the full set of 450 indices, was used to define the number of OGA Chromosomes. The formula at the beginning of this section yielded a recommendation of 562.5 OGA Chromosomes per generation, which was rounded up to 600 and applied to all four sets. 'Combine' was used to randomly subdivide the positive and negative set sequences into training and test sets with a ratio of 2:1. This random reassignment was repeated five times, and five corresponding negative control sets were generated as described in Generation of Sequence and Structure Indices above.
The mean of the best generalization scores achieved in each replicate over 30 rounds of OGA evaluation and selection is shown in Figure 3. The 4 groups of negative control runs corresponding to the four data sets all yielded a mean best score between 0.78 and 0.79, and the range of scores in each case did not overlap with the range of the corresponding five experimental replicates. However, the average generalization score of experimental set 1.1 was only 5–6 % higher than the corresponding negative control runs. Set 1.2, which included CM as well as k-mer counts, yielded a mean generalization score of 0.880, a substantial improvement over set 1.1 with no overlap in the range of maximum scores between the two sets. Set 1.3, which considered k-mer counts and PSSM scores at several thresholds, yielded a mean best generalization score of 0.883, which was substantially better than set 1.1 and indistinguishable from set 1.2. Finally, set 1.4 yielded a small improvement over sets 1.2 and 1.3 in generalization score, with a mean of 0.900. These results suggest that the inclusion of PSSMs and flexibility indices yielded a substantial increase in predictive accuracy over the background of k-mer counts, with the combination of the two possibly producing a further slight increase.
Figure 4 shows the change in the mean, maximum and minimum generalization scores for the 600 OGA Chromosomes in each of 30 training rounds for set 1.4. The mean over all five replicated runs is shown for both the experimental and negative control runs. There is an upward trend with all six values, which shows that improvements in the mean performance are due to both the creation of new, advantageous combinations of indices by the OGA as evidenced by the increase in the maximum score, and through the elimination of bad indices, shown with the increase in the minimum score. The difference in mean generalization score between the experimental and negative control runs is very low (< 0.025) in the first OGA generation, but increases rapidly to 0.11 – 0.12 within the first ten generations of optimisation. This trend is consistent with the idea that many indices in the experimental runs are not good at distinguishing between the positive and negative sequence sets, and their replacement with more copies of good indices yields better predictive accuracy. However, poor indices are expected to persist to some degree through the population, since they can 'hitchhike' with good indices through many rounds of OGA training and may even increase in frequency if they are associated with an otherwise good combination of indices. The low (< 0.7) predictive accuracy of some experimental OGA Chromosomes in the last round of training may be due to recombination events that merge sets of hitchhiking indices.
If sets of indices that yield the best predictive accuracy are preferentially selected for recombination by the OGA, then good indices should increase in frequency with successive rounds of testing and recombination. Only five indices were present in the final population of all five replicated experimental runs of set 1.4: one measuring the count of sites scoring in the 99th percentile of the CRP matrix between positions 60 and 99, another measuring the same quantity for Lrp sites between positions 20 and 59, an index describing the conformational mobility between sites 80 and 90, and two indices representing the count of GC dinucleotides and C mononucleotides between positions 29 and 39. The first three indices are easy to understand, as they correspond directly to features (binding sites and a high CM region) that were deliberately inserted into the positive sequence set. However, the third index is less clear until the Lrp sites from RegulonDB are examined in detail: these sites are G+C poor in general, and of the 72 × 11 = 792 dinucleotides contained in the full set of Lrp binding sites, only 20 of these are GC steps. If all 16 possible dinucleotides were present with equal frequencies, then the pair would be present 49 or 50 times, so GC is strongly underrepresented in this data set. Thus, it appears that the GC content in this region is included in many OGA Chromosomes because it is another indicator of the presence or absence of the Lrp binding site in this region.
Experiment 2 – σ70 positive set
The second experiment tested the ability of GANN to distinguish between the 250 unmodified intergenic sequences described earlier, and a set of sequences containing binding sites that are recognized by the 'housekeeping' σ70 subunit of RNA polymerase in E. coli. The 212 promoter-containing sequences in the positive set were extracted from a larger set defined in [37]. Where multiple promoter sequences were identified in a single upstream region, one of these sequences was chosen at random for inclusion in the positive set. All sequences in this experiment were 100 nt in length, and the positive set sequences were aligned at the transcription start sites of the relevant promoters. The -35 box (consensus 'TTGACA') was typically contained between positions 40 and 49 in the sequence, while the Pribnow box (consensus 'TATAAT') was located approximately between positions 65 and 70 in the positive sequences.
Indices were constructed in a similar manner as in Experiment 1. Separate PSSMs for the -10 and -35 boxes recognized by σ70 were constructed from 250 promoter sequences in the data set, again with background frequencies of 25% for each nucleotide. As with CRP and Lrp above, the 99th, 95th, 90th and 80th percentile scores were generated for the -10 and -35 boxes from a set of random sequences, though only 1000 random sequences were generated for each case. Since the two halves of the σ70 consensus sequence are only six bases in length, indices based on PSSM matches for a window size of 10 nt as well as 20 nt and 40 nt were calculated. Indices of conformational mobility and k-mer frequency were calculated as in Experiment 1 above. A total of 618 standardized indices were generated in this experiment: 360 describing k-mer counts, 222 describing the counts of matches to the -10 and -35 PSSMs at different thresholds, and 36 describing the conformational mobility of different windows of sequence. Five sets of experiments were performed, with five experimental and five negative control runs in each. All sets (2.1 to 2.5) included the k-mer count indices, and these were the only indices considered in set 2.1. Sets 2.2 and 2.3 also included the -35 and -10 PSSMs respectively, while set 2.4 included both. All 618 indices were considered by set 2.5.
Runs were performed as in Experiment 1 above, with the exception of the OGA Chromosome population size. With 618 indices in total, the formula described at the beginning of this section yielded a recommendation of 772.5 OGA Chromosomes per generation, which was rounded up to 800 and applied to all five sets.
The mean of the best generalization scores achieved in each replicate over 30 rounds of OGA evaluation and selection is shown in Figure 5. Remarkably, there was no substantial difference between any of the experimental sets, which yielded mean predictive accuracies between 0.828 for set 2.2 and 0.843 for set 2.5. This range was smaller than that of the predictive accuracies of the five negative control treatments, which ranged from 0.701 for set 2.2 to 0.737 for set 2.4. No clear trend exists for either the experimental or negative control sets, suggesting that the CM and PSSM indices did not yield any improvement in predictive accuracy over the k-mer counts alone, and that the inclusion of the σ70 PSSMs did not yield a more-precise model of the promoter sequence.
While the predictive accuracy did not change across multiple sets of experiments, the type of indices that were selected by the OGA varied from set to set. The most successful index overall described the frequency of the dinucleotide 'TA' between positions 65 and 74, which corresponded to the position of the Pribnow box in the positive set sequences. This index was the only one present in all five replicates of sets 2.1, 2.3 and 2.4 after 30 rounds of OGA training, and was one of only three such indices for set 2.2. The other two indices that were retained by all five replicates of set 2.2 described the frequency of TA between positions 60 and 69, and the frequency of CA between positions 55 and 64. No single index was retained in all five replicate runs of set 2.5. In all sets that included PSSM representations, the appropriate PSSM component(s) were always retained in four out of five replicated runs. While PSSMs with a very high score threshold (99th percentile) were favoured in Experiment 1, the PSSMs most commonly retained in the σ70 experiments favoured lower percentile thresholds, with a roughly even distribution of indices representing the 95th, 90th and 80th percentiles. This effect may be due to greater degeneracy of σ70 sites relative to the CRP and Lrp sites modelled earlier. Finally, four out of five replicate experimental runs of set 2.5 retained a CM index that covered positions 30–50 in the sequence. While no single index was retained in all five replicate runs of set 2.5, the OGA Chromosomes with the highest generalization scores all contained at least one instance of a PSSM for each of the two halves of the σ70 consensus and a flexibility feature. Thus, more-selective indices such as those based on PSSMs were included in the majority of OGA Chromosomes when they were present in the experimental set, even if the gain in predictive accuracy was marginal.
Conclusion
We have explored several features of GANN, most notably the ability to build classification rules for positive set members that form natural subsets, and the capacity to search through large sets of DNA sequence and structural indices to find combinations that yield optimal predictive accuracy. Our generalization accuracy of ~84% on the σ70 promoter set is similar to the sensitivity of 86% and specificity of 85% reported by [38], though these results may not be directly comparable due to differences in the size of the data set and the definition of 'negative' examples. While our use of PSSMs in these experiments implies acceptance of the statistical mechanical theory of binding sites [39], GANN could also be used to build models that take into account interactions between individual residues within a binding site. The model thus constructed could then be compared against a traditional PSSM to see if better predictive accuracy is obtained on a test set of sequences. In focusing on the generation and testing of combinations of indices, we have not examined the performance of GANN when ANN architectural parameters are optimised alongside index combinations. One approach that avoids dealing with too many interactions at once can be to first use GANN to screen a large set of indices and generate a smaller list of indices with predictive power, and to perform a subsequent run where this smaller set of indices are examined in combination with variable ANN parameters.
In Experiment 1, we found that indices with mean values that are not close to zero should be standardized. A disadvantage of this approach is that standardization of a column of values is entirely sample dependent, since different finite samples from the same population of values will typically have different means and standard deviations, which may limit the accuracy of an ANN trained on one sample that is used to classify other subsamples from the same population. If standardization is to be applied, then there should be sufficient values in each index to yield a stable estimate of the population mean, as indicated with a low standard error. This is of particular concern since biological sequence samples often do not represent a random sample of all possible sequences, leading to biased estimates of sample mean and standard deviation.
The two primary goals of GANN are to allow multiple alternative representations of DNA features, and to permit the discovery of important combinations of these features through the hybrid genetic algorithm / neural network approach. A long-term goal is to use GANN to identify important combinations of motifs predicted from programs such as PatSer [40], which would permit the application of GANN to the task of identifying complex regulatory features in multicellular eukaryotes. The software packages are released under the GNU GPL and have been successfully tested and run on Win32 systems and on several flavours of UNIX. Complete documentation for GANN and the key files used in the experiments described in this manuscript are available at the project Web site.
Availability and requirements
- Project name: GANN
- Project home page:
- Operating systems: Win32, UNIX
- Programming language: C++, Perl
- Other requirements: none
- License: GNU GPL
- Any restrictions to use by non-academics: none
Abbreviations
CM – Conformational mobility
GANN – Genetic Algorithm Neural Networks
OGA – Outer Genetic Algorithm
PSSM – Position-specific scoring matrix
Authors' contributions
RGB was responsible for software design and implementation, and manuscript writing. RLC contributed to the design, and to the implementation of the genetic algorithm classes in C++.
Figures and Tables
Figure 1 Programs and data flow for GANN.
Figure 2 Construction of the synthetic positive set of sequences in Experiment 1. The range of starting positions and length of each added feature are indicated (CM = conformational mobility, CRP = cAMP receptor protein, Lrp = leucine-responsive regulatory protein).
Figure 3 Maximum predictive accuracy of OGA Chromosomes on the generalization set in Experiment 1. Mean ± standard deviations are shown for the five replicates of the experimental (E) and negative control (N) runs for the four data sets (1.1 to 1.4) used.
Figure 4 Change in the mean (squares), maximum (triangles) and minimum (circles) generalization scores for the 600 OGA Chromosomes in each of 30 training rounds for set 1.4. Plotted values represent the mean of each value across all 5 replicates of either the experimental (filled shapes, solid line) or negative control runs (empty shapes, dashed line).
Figure 5 Maximum predictive accuracy of OGA Chromosomes on the generalization set in Experiment 2. Mean ± standard deviations are shown for the five replicates of the experimental (E) and negative control (N) runs for the five data sets (2.1 to 2.5) used.
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| 15725347 | PMC553964 | CC BY | 2021-01-04 16:02:51 | no | BMC Bioinformatics. 2005 Feb 22; 6:36 | utf-8 | BMC Bioinformatics | 2,005 | 10.1186/1471-2105-6-36 | oa_comm |
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BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-6-201571792710.1186/1471-2164-6-20Research ArticleThe domain architecture of large guanine nucleotide exchange factors for the small GTP-binding protein Arf Mouratou Barbara [email protected] Valerie [email protected] Alexandra [email protected] Jean [email protected] David J [email protected] Niko [email protected]ürgens Gerd [email protected]çon Paul [email protected] Jacqueline [email protected] Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, avenue de la Terrasse, 91198 Gif sur Yvette cedex, France2 Centre de Génétique Moléculaire, CNRS, Gif-sur-Yvette, France3 Department of Cell Biology, University of Alberta, Edmonton, Canada4 Center of Plant Molecular Biology, Universitaet Tuebingen, Tuebingen, Germany5 Plant Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, USA2005 17 2 2005 6 20 20 4 11 2004 17 2 2005 Copyright © 2005 Mouratou et al; licensee BioMed Central Ltd.2005Mouratou et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Small G proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors (GEFs) that stimulate the exchange of the tightly bound GDP nucleotide by GTP. The catalytic domain responsible for nucleotide exchange is in general associated with non-catalytic domains that define the spatio-temporal conditions of activation. In the case of small G proteins of the Arf subfamily, which are major regulators of membrane trafficking, GEFs form a heterogeneous family whose only common characteristic is the well-characterized Sec7 catalytic domain. In contrast, the function of non-catalytic domains and how they regulate/cooperate with the catalytic domain is essentially unknown.
Results
Based on Sec7-containing sequences from fully-annotated eukaryotic genomes, including our annotation of these sequences from Paramecium, we have investigated the domain architecture of large ArfGEFs of the BIG and GBF subfamilies, which are involved in Golgi traffic. Multiple sequence alignments combined with the analysis of predicted secondary structures, non-structured regions and splicing patterns, identifies five novel non-catalytic structural domains which are common to both subfamilies, revealing that they share a conserved modular organization. We also report a novel ArfGEF subfamily with a domain organization so far unique to alveolates, which we name TBS (TBC-Sec7).
Conclusion
Our analysis unifies the BIG and GBF subfamilies into a higher order subfamily, which, together with their being the only subfamilies common to all eukaryotes, suggests that they descend from a common ancestor from which species-specific ArfGEFs have subsequently evolved. Our identification of a conserved modular architecture provides a background for future functional investigation of non-catalytic domains.
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Background
Guanine Nucleotide Exchange Factors (GEFs) are obligatory components of signaling cascades regulated by small GTP-binding proteins (called small G proteins hereafter). Their biochemical activity is to stimulate the dissociation of the tightly bound GDP nucleotide from the small G protein in response to cellular signals. Thereby, they favor the binding of the more abundant cellular GTP, organizing the active conformation of the small G protein which can recruit its effectors (reviewed in [1]). Each small G protein family features its own ensemble of GEFs characterized by a conserved catalytic domain responsible for nucleotide exchange, which is generally combined with non-catalytic domains that define the spatio-temporal conditions of activation. In the case of small G proteins of the Arf family, which are major regulators in membrane trafficking (reviewed in [2]), the exchange domain is a conserved module of ~200 amino acids called the Sec7 domain [3]. Its biochemical (reviewed in [4]) and structural [5,6] mechanisms have been investigated in detail. Remarkably, the Sec7 domain is the only domain that is conserved in all ArfGEFs (reviewed in [7,8]) and it is to some extent interchangeable between species [9]. In contrast, little is known about the functions of the other domains, which are likely to determine intracellular localization of ArfGEFs and their responsiveness to specific signals.
As for most small G proteins, Arf family members are outnumbered by ArfGEFs in many species. In humans for instance, 5 Arf proteins have been identified, and there are at least 13 proteins carrying a Sec7 domain, of which most have been characterized as bona fide ArfGEFs (reviewed in [7,8]). Thus an individual Arf protein may be activated by more than one GEF, emphasizing that essential aspects in building up the Arf responses may be encoded by the modular architecture of their GEFs. Sequence similarity in the non-catalytic regions forms the basis for the classification of ArfGEFs into subfamilies. 8 subfamilies are currently identified in eukaryotes with sizes ranging from small (~40–80 kD including CYH, EFA6 and FBS), to medium (~100–150 kD, including BRAG/LONER, SYT1, SYT2) and large (~170–200 kD) ArfGEFs (reviewed in [7,8]). Large ArfGEFs comprise two subfamilies which we will refer to as the BIG and GBF subfamilies after the name of their human representatives. The GBF subfamily includes human GBF1 [10], Arabidopsis GNOM [11] and Saccharomyces Gea1 and Gea2 [12], the BIG subfamily human BIG1 and BIG2 [13,14] and yeast Sec7p [15]. An additional subfamily called RalF is found in Rickettsie and Legionella bacteria, likely acting on an host Arf pathway [16]. Analysis of the CYH and EFA6 subfamilies, present only in multicellular animals, and that of the large ArfGEFs, found in all eukaryotes, have yielded most of the functional data currently available. CYH and EFA6 are active on Arf6 at the plasma membrane where they may function in the crosstalk of membrane traffic, cytoskeleton dynamics and signalling in endosomal pathways (reviewed in [17]). Most members of the BIG and GBF subfamilies characterized so far function in vesicular trafficking at the Golgi [12,14,18], except for BIG2, which also localizes on recycling endosomes [19], and GNOM which acts in the endosomal recycling pathway [11].
The domain architecture of non-catalytic regions of ArfGEFs, hence their contribution to specific aspects of the build-up of the Arf response, is essentially not established except for those ArfGEFs with domains found in other classes of cellular regulators. The known domains include membrane-interacting PH domains in the CYH (reviewed in [20]), EFA6 [21] and possibly BRAG/LONER[22] subfamilies, and a putative F-box in the FBS subfamily [23], a protein-protein interaction domain that has been involved in the recruitment of substrates to the SCF ubiquitination machinery. Coiled-coil structures have also been predicted in the N-terminus of the CYH subfamily and in the C-terminus of the EFA6 subfamily. In CYH, they are involved in dimerization [3], recruitment of partners [24] and Golgi targeting [25], and in actin remodeling functions in the case of EFA6 [21]. On the other hand, although the functions of BIG and GBF subfamilies have been the subject of many investigations, their architecture is barely described, making it difficult to associate biochemical activities with their molecular structure.
Here we investigate the domain architecture in the BIG and GBF subfamilies, including all sequences from fully annotated eukaryotic genomes and our novel annotation of Sec7-containing proteins from the Paramecium tetraurelia alveolate. Sequence comparisons combined with secondary structures and splicing patterns analysis identifies five novel domains that are conserved between BIG and GBF subfamilies, thus unifying them as a higher order subfamily with a probable common ancestor. Our analysis of Sec7-domain containing sequences from Paramecium also introduces a novel subfamily of ArfGEFs unique to alveolates, which we call TBS (TBC-Sec7).
Results and discussion
A conserved domain architecture in BIG and GBF subfamilies
The BIG and GBF subfamilies are the only ArfGEFs subfamilies common to all eukaryotes [8] and the sole ArfGEFs present in plants [26] (Figure 1). They are therefore possible representatives of ancestral ArfGEF functions and may provide a model to understand the nature and implementation of activities associated with the exchange function carried by the conserved Sec7 domain. However, domain 'hunting' in BIG and GBF subfamilies was complicated by the facts that the Sec7 domain is their only domain that could be identified from known domain repertoires, and that their poorly characterized non-catalytic regions were not found outside these ArfGEF subfamilies. Alternatively, we based our search of candidate structural domains in BIGs and GBFs on the bioinformatics analysis of their own sequences, taking advantage of the growing number of sequences from fully annotated genomes from mammals, insects, plants, nematode, and fungi, to which we included our annotation of Sec7-containing proteins from the newly sequenced genome of Paramecium.
Figure 1 Venn diagram of the nine Sec7-containing subfamilies sorted according to the species where each subfamily has been found. The TBS subfamily was identified in this study. The BIG and GBF subfamilies are merged in a higher order subfamily (GBG), and are the only subfamily common to all eukaryotes.
Multiple alignments of 42 sequences (listed in Table 1) revealed that the BIG and GBF subfamilies share an unexpected conserved architecture (schematized in Figure 2). Two homology domains are located in N-terminus of the Sec7 domain – the DCB (~150 aa) and HUS (Homology Upstream of Sec7, ~170 aa) domains – and three in its C-terminus -the HDS1 (Homology Downstream of Sec7, ~130 aa), HDS2 (~160 aa) and HDS3 (~120 aa) domains (Figure 3,4,5,6,7). In Arabidopsis GNOM, the DCB domain is included in an N-terminal region of ~250 residues involved in dimerization and possibly binding to cyclophilin5 and called the Dimerization/Cyclophilin Binding region [27], after which the new domain was named (Figure 3). All domains are predicted to have a high content of α-helices that co-align in the multiple sequence alignments, reinforcing the prediction of sequence similarities and suggesting that these domains form folded structural units that may share common functional features. Except for the N-terminal DCB domain which is also found in the yeast protein Ysl2p [28], all of them are unique to these two ArfGEFs subfamilies within the detection limits of the BLAST search. The HUS domain features a remarkably conserved N(Y/F)DC(D/N) motif, which we call the HUS box, which is predicted to locate in a loop where it may be available for functional interactions (Figure 4). The N- and C-terminal ends of BIGs and GBFs are more variable, including an unusual enrichment in Asp/Glu or Pro residues in some members. A specific feature of BIG members is that their C-terminus is in general less variable than that of GBFs, and is predicted with a significant amount of secondary structures. In contrast to the predicted structural domains, the intervening regions are highly variable in length and do not yield aligned sequences. Analysis of their amino-acid composition reveals a paucity of hydrophobic residues which is predicted to associate with an essentially unfolded conformation, suggesting that they act as linkers to tether the functional domains together.
Table 1 BIG and GBF protein sequences used in this study.
Species Protein name a Accession Number Size in amino acids
Metazoa Ag Q7PWN5 EAA14874 1522
Q7PXQ7 EAA00837 1285
Ce Q9XWG5 NP_493386 1628
Q9XTF0 NP_499522 1820
Dm Q9VJW1 AAF53331 1653
Q9V696 AAF58532 1983
Hs BIG1 Q9Y6D6 1849
BIG2 Q9Y6D5 1785
GBF1 Q92538 1859
Rn BIG1 XP_232614 1987
BIG2 Q7TSU1 1791
GBF1 XP_347197 1883
Fungi Ca EAL04295 EAL04295 1839
EAL02873 EAL02873 1015
Nc Q7SAX4 EAA33549 1940
Q7SAL8 EAA33457 1626
Sc SEC7 P11075 2009
GEA1 P47102 1408
GEA2 P39993 1459
Sp SC71 Q9UT02 1811
SC72 Q9P7V5 1822
Q9P7R8 NP_596613 1462
Viridiplantae At At1g01960 Q9LPC5 1750
At3g43300 NP_189916 1728
At3g60860 Q9LZX8 1793
At4g35380 O65490 1711
At4g38200 NP_195533 1698
GNOM Q42510, At1g13980 1451
GNL1 Q9FLY5, At5g39500 1443
GNL2 NP_197462, At5g19160 1375
Os 9631.m01366 Q8S565 1789
9630.m00920 Q9XGN9 1687
9634.m04029 - 1704
9635.m03752 - 1680
9631.m04495 - 1456
9630.m02122 - 1396
9632.m00175 Q7XT11 1407
Alveolata Pt GGG1 CR533425 1615
GGG2 CR533424 1628
GGG3 CR533423 1598
GGG4 CR533422 1599
GGG5 CR533421 1435
a Unnamed sequences are designated by their NCBI accession number, AGI (Arabidopsis Genome Initiative) locus numbers for At and TIGR model temporary IDs for Os. BIG and GBF subfamily members are in normal and bold characters respectively, except for Pt members which have not been assigned to either subfamily (see also Figure 8). Species abbreviations are: Ag, Anopheles gambiae; Ce, Caenorhabditis. elegans; Dm, Drosophila melanogaster; Hs, Homo sapiens; Rn, Rattus norvegicus; Ca, Candida albicans; Nc, Neurospora crassa; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; At, Arabidopsis thaliana; Os, Oryza sativa; Pt, Parameciumtetraurelia).
Figure 2 The common domain architecture of the BIG and GBF subfamilies. From N- to C-terminus : DCB , HUS, Sec7, HDS1, HDS2, HDS3. Linker regions of variable length and sequence are shown in grey, with alternate splicing sites in human GBF1, BIG1 and BIG2 in black, white and grey diamond shapes respectively. Interactions reported in the litterature are indicated in boxes of width corresponding to the mapped regions, except for myosin IXb interaction which was studied only with full-length BIG1. Arrows indicate predicted Protein kinase A-anchoring motifs. 1 [45]; 2 [27]; 3 [46]; 4 [47]; 5 [48]; 6 [49]; 7 [50]; 8 [51].
Figure 3 The conserved domains of the BIG/GBF subfamily: DCB domain. Multiple sequence alignement of the conserved domains from BIG and GBF representative sequences showing secondary structure predictions that co-align in all sequences. Colour coding is red for invariant residues, yellow for a sequence similarity score threshold of 0.15 using the BLOSUM62 matrix. The gap in helix 4 is due to an insert in the drosophila Q9V696 sequence, and may be resulting from a sequence annotation error.
Figure 4 The conserved domains of the BIG/GBF subfamily: HUS domain. See Figure 3 legend for alignment details. The highly conserved HUS motif is boxed in blue. The gap in helix 5 domain is due to an insert in the Arabidopsis 3g43300 sequence, and may be resulting from a sequence annotation error.
Figure 5 The conserved domains of the BIG/GBF subfamily: HDS1 domain. See Figure 3 legend for alignment details.
Figure 6 The conserved domains of the BIG/GBF subfamily: HDS2 domain. See Figure 3 legend for alignment details.
Figure 7 The conserved domains of the BIG/GBF subfamily: HDS3 domain. See Figure 3 legend for alignment details.
To further investigate the predicted organization of BIGs and GBFs in 6 conserved helical domains connected by variable linkers, splicing patterns of human BIGs and GBFs were analyzed in the large number of cDNAs and ESTs in the databases that correspond to GBF/BIG transcripts. This revealed the use of alternate splice donor and acceptor sites predicted to yield proteins with insertions and deletions ranging from 1 to 38 residues, and a number of splice variants arising from exon skipping (Table 2). Strikingly, all observed sequence variations occur in regions identified as linkers between conserved domains (Figure 2). Together with our domain analysis, this suggests that splicing at non-canonical exon/intron boundaries is only tolerated in regions of the protein where the impact upon folding of domains with essential function would be minimal.
Table 2 Alternate splice variants of human GBF1, BIG1 and BIG2 a,b
Change in protein Apparent cause of variation in transcript
GBF1 Extra Q at 337, 55 residues upstream of HUS domain Insertion of 3 nucleotides (nt) resulting from use of alternate 3' acceptor site within intron during splicing of exons 10 and 11
New Ser and loss of 14 residues at 613, between HUS and Sec7 domains Loss of 36 nt resulting from use of alternate 5' donor site within exon 15 during splicing with exon 16
Loss of VSQD at 1494, 38 residues upstream of HDS3 Loss of 12 nt resulting from use of alternate 5' donor site within exon 33 during splicing with exon 34
Frame-shift at 1625 causing loss of last 19 residues of HDS3 Intron retention between exons 36 and 37 leading to frame shift and premature termination
Loss of 38 residues starting at 1784, near C-terminus Loss of 114 nt resulting from use of novel cryptic splice donor and acceptor sites within exon 40.
BIG1 Frame-shift at 1340, 32 residues upstream of HDS3 Loss of 59 nt resulting from use of alternate 5' donor site within exon 28 during splicing with exon 29
Loss of VSEKPL at 1557, 68 residues downstream of HDS3 Loss of 18 nt resulting from use of alternate 5' donor site within exon 33 during splicing to exon 34
New T and loss of 33 residues at 1607, 118 residues downstream of HDS3 Loss of 96 nt resulting from use of alternate 3' acceptor site within exon 35 during splicing with exon 34
BIG2 Frame-shift at 1542, 106 residues downstream of HDS3 Loss of exon 35 resulting from splicing of 5'donor site of exon 34 with 3' acceptor site of exon 36
a All changes were expressed relative to the reference sequence stored under accession number NM_004193 (hGBF1), NM_006421 (hBIG1) and NM_006420.1 (hBIG2).
b All variants are supported by one or more cDNA/ESTs as detailed in the Aceview for each gene that can be obtained at [38].
Evolution of BIGs and GFBs from a common ancestor
Combined, our analysis reveals that the BIG and GBF subfamilies share the same overall domain organization, and are likely to descend from a common ancestor gene that duplicated first to form the BIG and GBF groups, and again within these groups to yield species-specific BIG and GBF members. These two subfamilies can therefore be unified as a higher order ArfGEF subfamily (called below GBG for GBF/BIG GEFs), from which unrooted phylogenetic trees can be built (Figure 8). Unlike previous phylogenetic analysis which compared ArfGEFs based on their Sec7 domains after diverging non-catalytic regions have been trimmed [8], our trees were established from the simultaneous alignment of all 6 conserved domains (DCB, HUS, Sec7, HDS1, HDS2 and HDS3), excluding variable linkers. The same tree topology was obtained with both neighbor-joining and maximum likelihood methods, and was retained using any one of the new conserved domains alone (data not shown). Bootstrap analysis strongly supported this topology for most branches. Only a few small branches located at the base of the groups were found in less than 60% of the trials in one of the two methods, but this never occurred with both methods simultaneously.
Figure 8 Unrooted neighbour-joining phylogenetic tree of the BIG/GBF subfamily. Colour coding for the main groups is green for plants, marine blue for fungi, orange and red for animals, cyan for protists. Branches found in less than 60% bootstrap trials by either the neighbor-joining or the maximum likelihood method are in dotted lines. Species abbreviations are as in Table 1.
The tree topology strongly suggests that in most organisms, GBG members sort in separate branches, corresponding to their classification in BIGs and GBFs. Remarkably, our annotation of Sec7-containing proteins in the genome of Paramecium reveals the first departure from this distribution, as all GBGs in this species are located in a single branch, which is closer to the BIGs. This unexpected tree topology may indicate that alveolates diverged from animals/fungi and plants before the duplication of an ancestral GBG into the BIG and GBF, and that GBGs in that organism are representative of this ancestral gene. Alternatively, duplication may have been followed by loss of GBF genes. Current knowledge of the phylogenetic branching of alveolates relative to the plants and animal/fungi branches does not permit resolution between those two possibilities.
GBGs in plants: refining the functional evolution of BIGs and GBFs
In fungi and mammals, BIGs and GBFs are represented by only one or two members, whose functions in vesicular trafficking at the Golgi within each group appear largely overlapping [12,14]. In contrast, plants encode a large number of GBGs in both the BIG and GBF branches but lack other ArfGEFs (Figures 1, 8). In Arabidopsis, none of the GBGs map to duplicated chromosomes where identical functions may be encoded [26,29]. In addition, comparative analysis with the rice genome nearing completion identifies at least five branches each represented by one rice and one or two Arabidopsis homologs (Figure 8). This correspondence between two highly divergent plant species indicates that GBGs diversified early during plant evolution, probably reflecting functional specialization along with the establishment of plant multicellularity. While GNOM has a plant-specific function in recycling plasma-membrane proteins needed for cell-cell communication and cell polarity establishment [11], possibly closer to the function of EFA6 or CYH subfamilies in metazoans, other plant GBGs are expected to fulfill the presumed ancient function of regulating Golgi trafficking exemplified by mammalian and yeast GBGs. Comparison of orthologous pairs in plants further reveals that they have different sensitivities to Brefeldin A (a widely used fungal inhibitor of Golgi traffic) as predicted from the sequences of the binding site of the drug carried by the Sec7 domain [6]. This observation clearly illustrates that differences in outcome following BrefeldinA treatment may not reflect differences in underlying molecular mechanisms, but instead simply reflect neutral sequence differences at the Sec7 domains between species. In particular, not all BIGs may be BFA-sensitive or GBFs BFA-resistant, unlike suggested by their original nomenclature.
A novel ArfGEF subfamily in alveolates
A remarkable evolutionary feature of ArfGEFs is that while GBGs seem to be ubiquitous to all eukaryotes, fungi and animals kingdoms evolved their own ArfGEFs subfamilies unrelated to those of the other kingdoms. We thus addressed the question of whether Paramecium, which has a large number of GBGs (at least five, of which four are present as pairs as the result of recent duplications) but appears to lack the specialization into the BIG and GBF subgroups, has the same ArfGEF distribution as plants or features a second ArfGEF subfamily. We thus searched the newly sequenced genome from Paramecium tetraurelia and the available alveolate genomes from Cryptosporidium parvum and Tetrahymena thermophila for additional Sec7-containing proteins. This identified a novel putative ArfGEF subfamily characterized by the association of the Sec7 domain with a TBC (Tre/Bub2/Cdc16) domain (Figure 9), which was found only in the protists kingdom. The TBC domain is predicted to carry a GAP (GTPase activating protein) activity towards small G proteins of the Rab family [30], suggesting a potential crosstalk between Rab and Arf pathways. Such a relationship between these two small G proteins families, which are major regulators of membrane traffic, would not be unprecedented, as for example the SYT1 ArfGEF gene was identified in yeast by its genetic interactions with Rab proteins in the exocytic pathway [31]. Interestingly, alveolates have specialized exocytic pathways based on a membrane organelle lying beneath the plasma membrane, the trichocyst, where this unique ArfGEF family may potentially function.
Figure 9 TBS: a novel ArfGEF subfamily in alveolates. Top: Domain structure of the TBS subfamily. Below: Sequences of the TBC domain from Paramecium TBS aligned with TBC domains from known RabGAPs. Secondary structures are from the crystal structure of yeast GYP1 [30].
Conclusion
A conserved scenario for the activation of Arf proteins by their GEFs?
The identification of a conserved modular architecture in all GBG subfamily members suggests that the mechanistic basis for their activation of Arf is likely to follow a similar scenario. Candidate functions for the conserved domains include oligomerization, the collection of input signals, membrane localization, regulation of the exchange activity, scaffolding of Arf proteins to their downstream effectors, not excluding signaling to partners outside the Arf pathways. Dimerization has been reported in the BIG subgroup for BIG1, which forms heterodimers with the highly homologous BIG2 ArfGEF [14], and in the GBF subgroup for GNOM, which forms homodimers [27]. The conservation of the DCB domain in GBGs, which is responsible for the dimerization of GNOM, suggests that such a dimerization function may be general to this domain in GBGs. Another unresolved issue is the conservation of the cellular partners effecting the functions associated with the conserved domains. Our identification of an almost invariant motif in the HUS domain argues in favor of this domain interacting with a conserved partner. However, the ancient divergence into the BIG and GBF groups and their subsequent divergence into species-specific members suggest that specialized requirements are likely to have evolved in most organisms, possibly yielding less conserved partners outside the Sec7 and HUS domains. Finally, whereas in plants all ArfGEFs are predicted to function according to the scheme defined by the conserved domains, other species have additional ArfGEF subfamilies with a modular architecture unrelated to that of the GBG subfamily. It is not known to what extent the GBG's scenario for Arf activation will also apply to non-GBGs ArfGEFs, acting alone or in association with protein partners. In the case of the GBGs, our definition of the structural homology domains as reported here should now provide a robust background for future investigations of their interactions and functions.
Methods
Protein sequence databases were searched with amino acid sequences from human BIG1, human GBF1 and Arabidopsis GNOM using the BLAST algorithm [32]. Paramecium tetraurelia genes were identified with the BLAST algorithm using genome sequence data from Genoscope [33] and manually annotated using Artemis [34]. Tetrahymena sequences were retrieved from the Tetrahymena thermophila genome sequencing project server [35]. Arabidopsis sequences were retrieved from the Arabidopsis Genome Initiative database [36], rice sequences from the TIGR Rice annotation project [37]. Splice variants for hGBF1, hBIG1 and hBIG2 were identified from information provided under Aceview in the December (03) release for their respective listings at the NCBI [38]. Multiple sequence alignments were performed using ClustalW [39] with default alignment parameters or T-coffee [40,41]. Reliability of the alignments was evaluated according to the T-coffee score, and ranged from average to good for all predicted domains. Average sequence identities were respectively 24 % (DCB domain), 26 % (HUS domain), 44% (Sec7 domain), 26% (HDS1 domain), 28% (HDS2 domain) and 21% (HDS3 domain). Aligned sequences were displayed with ESPript [42] using a similarity global score of 0.15 calculated using the BLOSUM62 matrix. Unrooted phylogenetic trees were generated using the neighbor-joining algorithm of ClustalW excluding gapped regions, and with a maximum likelihood method using the PHYML package [43]. Phylogenetic trees for individual domains was performed on the subset of sequences used in Figure 3. The reliability of the trees was assessed by a bootstrap analysis (1000 replicates). Trees were drawn with TreeView version 1.6.6. Secondary structure predictions on aligned sequences were carried out with the PHD program along with the ClustalW multiple alignment [39]. Non-structured linkers poor in hydrophobic residues were predicted with the PONDR algorithm [44].
Abbreviations
GEF: Guanine nucleotide exchange factor. CYH: cytohesins/ARNO; EFA: Exchange Factor for Arf6; FBS: F-Box/Sec7; TBS: TBC/Sec7; GBF: Golgi-associated BFA-resistant guanine nucleotide exchange Factor; BIG: BFA-Inhibited Guanine nucleotide exchange factor; GBG: GBF/BIG Gefs; SYT1: Suppressor of ypt. DCB: Dimerization/Cyclophilin Binding; HUS: Homology Upstream of Sec7; HDS: Homology Downstream of Sec7; TBC: Tre/Bub2/Cdc16; SCF: Skp1/Cull1/F box.
Authors' contributions
B.M. and V.B. carried out sequence and phylogenetic analysis. A.J. participated in the domain analysis. J.Co. annotated Paramecium sequences. D.S. and P.M. performed splicing pattern analysis. N.K. and G.J. analyzed the distribution of large ArfGEFs in plants. J.Ch. conceived and coordinated the study and wrote the manuscript.
Acknowledgements
This work was supported by a Human Frontiers in Science Program grant to G.J., P.M. and J.Ch. We thank Genoscope for access to Paramecium whole genome shotgun primary data and Linda Sperling (CNRS, Gif-sur-Yvette) for help with annotations.
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| 15717927 | PMC553965 | CC BY | 2021-01-04 16:39:33 | no | BMC Genomics. 2005 Feb 17; 6:20 | utf-8 | BMC Genomics | 2,005 | 10.1186/1471-2164-6-20 | oa_comm |
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BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-5-171571792610.1186/1471-2407-5-17Research ArticleExpression analysis of the mouse S100A7/psoriasin gene in skin inflammation and mammary tumorigenesis Webb Meghan [email protected] Ethan D [email protected] Michael [email protected] Salem [email protected] Gefei [email protected]'ar Abdullah [email protected] Linda J [email protected] Yulian [email protected] Alberto [email protected] Yvonne [email protected] Robert [email protected] Leigh C [email protected] Peter H [email protected] Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Canada2 Dept of Physiology, University of Manitoba, Winnipeg, Canada3 Dept of Pathology, University of Manitoba, Winnipeg, Canada4 Dept of Biochemistry & Medical Genetics, University of Manitoba, Winnipeg, Canada5 Dept of Biology, University of Winnipeg, Winnipeg, Canada2005 17 2 2005 5 17 17 27 10 2004 17 2 2005 Copyright © 2005 Webb et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The human psoriasin (S100A7) gene has been implicated in inflammation and tumor progression. Implementation of a mouse model would facilitate further investigation of its function, however little is known of the murine psoriasin gene. In this study we have cloned the cDNA and characterized the expression of the potential murine ortholog of human S100A7/psoriasin in skin inflammation and mammary tumorigenesis.
Methods
On the basis of chromosomal location, phylogenetic analysis, amino acid sequence similarity, conservation of a putative Jab1-binding motif, and similarities of the patterns of mouse S100A7/psoriasin gene expression (measured by RT-PCR and in-situ hybridization) with those of human S100A7/psoriasin, we propose that mouse S100A7/psoriasin is the murine ortholog of human psoriasin/S100A7.
Results
Although mouse S100A7/psoriasin is poorly conserved relative to other S100 family members, its pattern of expression parallels that of the human psoriasin gene. In murine skin S100A7/psoriasin was significantly upregulated in relation to inflammation. In murine mammary gland expression is also upregulated in mammary tumors, where it is localized to areas of squamous differentiation. This mirrors the context of expression in human tumor types where both squamous and glandular differentiation occur, including cervical and lung carcinomas. Additionally, mouse S100A7/psoriasin possesses a putative Jab1 binding motif that mediates many downstream functions of the human S100A7 gene.
Conclusion
These observations and results support the hypothesis that the mouse S100A7 gene is structurally and functionally similar to human S100A7 and may offer a relevant model system for studying its normal biological function and putative role in tumor progression.
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Background
Human S100A7 or psoriasin was first identified as an over-expressed secreted protein in psoriatic skin[1]. More recently its expression in both pre-invasive (ductal carcinoma in situ, DCIS) and invasive human breast cancer was demonstrated [2]. Although highly expressed in DCIS and generally down-regulated in invasive breast cancer, the expression of psoriasin/S100A7 in both in-situ and invasive breast cancer is correlated with markers of poor prognosis [3,4] and in invasive carcinoma also with poor clinical outcome [5]. Support for psoriasin/S100A7 having a functional role in this aggressive phenotype is shown by the observation of increased growth and tumorigenesis when breast cancer cells over-expressing psoriasin/S100A7 are grown as xenografts in nude mice [6]. This activity may in part be mediated by the ability of psoriasin/S100A7 to interact with c-Jun activation domain-binding protein 1 (Jab1) [6] and enhance pro-survival pathways [7] and protect against anoikis in human breast and head and neck squamous cancer cells [8]. However, further investigation of the physiological and pathophysiological function of psoriasin/S100A7 can only be effectively undertaken using genetic manipulation of an animal model. To undertake such studies we need to characterize and clone the mouse psoriasin gene. Interestingly, during the preparation of this manuscript Marenholz etal., [9] have argued that the possible ancestral homolog of the human S100A7 paralogs [10] has been identified in the mouse on the basis of genome sequence analysis and gene orientation, but has not been characterized fully as yet. The following study provides evidence of the cloning of a cDNA of the putative mouse ortholog of human psoriasin/S100A7, investigates its expression under conditions of mouse mammary tumorigenesis and skin inflammation, and confirms that the factors involved in its regulation are comparable to those involved in regulation of the human counterpart.
Methods
Tissues
Murine studies were conducted in accordance with the principles and procedures recommended and approved by the University of Manitoba Animal Care Review Board. Mammary tumors in CD1 mice were generated chemically using 7, 12-dimethylbenz anthracine (DMBA) as previously described [11]. Freshly dissected mouse tissues were either stored frozen at -70°C, or processed to generate formalin fixed paraffin-embedded tissue blocks.
Acute dermatitis in C57/B6 mice was induced by the topical application of 20% croton oil (dissolved in dimethyl sulfoxide) to a 1 cm length mid-tail portion [12]. The tail skins were stimulated continuously with croton oil every 4 hours for 24 hours. Mice were divided into five time-groups (each containing 3 mice) that were designated 0, 4, 8, 16, and 24 hours. At each time-point, the mice were sacrificed. The tail-skins were harvested and paired specimens of normal and inflamed skin tissue were fixed in 3.7% formaldehyde in 0.1 M phosphate buffered saline for 16–18 hours, followed by paraffin embedding.
Sections from the above blocks were used for preparation of haematoxylin and eosin stained sections for light microscopic examination, in situ hybridization (ISH), and immunohistochemistry (IHC).
Human tumor tissues were obtained from the Department of Pathology, University of Manitoba. All cases were coded and therefore anonymous and prior approval was obtained from the University of Manitoba Research Ethics Board and the Pathology Access to Tissue Committee. Different cervical tumor pathologies (total n = 39 cases) including in-situ adenocarcinoma (n = 10) and squamous cell carcinoma (n = 9), as well as invasive adenocarcinoma (n = 10) and squamous carcinoma (n = 10) were selected for one cohort study. In addition, different invasive lung tumor types (total n = 78 cases) including mesothelioma (n = 10), small cell carcinoma (n = 15), adenocarcinoma (n = 28), and squamous carcinoma (n = 25) were selected to form another study cohort. Squamous differentiation within both murine and human tumors was determined by standard morphological criteria including cytoplasmic keratinization and cellular stratification relative to keratin pearls.
In-situ hybridization
Paraffin embedded 5 μm tissue sections were analyzed by in-situ hybridization according to a previously described protocol [13]. The plasmid pCR4-TOPO-mPsor-ORF, consisted of pCR4-TOPO plasmid (Invitrogen Canada Inc, Burlington, ON) containing a 344 base pair insert of the mouse psoriasin cDNA, also known as mouse S100A15 (from nucleotide 94 to 437 as numbered in AY465109, and from 1 to 344 as numbered in AY582964). One microgram of linearized template DNA was used to generate 35S-UTP-labeled sense and antisense cRNA probes using the Riboprobe System (Promega, Madison, WI) according to the manufacturer's instructions. Sense probes were used as controls. In-situ hybridization and washing conditions were as previously described[2]. Sections were developed using Kodak NTB-2 photographic emulsion and counter-stained with Lee's stain after 2–6 weeks.
Levels of mouse S100A7/psoriasin RNA expression were assessed by microscopic examination at low power magnification and with reference to the negative sense control. This was done by scoring the estimated average signal intensity (on a scale of 0 to 3), where 0 is no expression and 3 is a high proportion of strong focal expression.
Immunohistochemistry
Immunohistochemistry (IHC) was performed on serial 5 μm sections from a representative, formalin fixed paraffin embedded tissue block from each tumor. For human tumor blocks, psoriasin/S100A7 IHC was performed essentially as described [5] and human psoriasin/S100A7 was detected using a previsouly characterized rabbit polyclonal antibody [3,5]. For murine tumors estrogen receptor-alpha (ERα) was detected using an affinity purified rabbit polyclonal antibody, MC-20, raised against a C-terminal peptide of mouse ERα (#sc-542, Santa Cruz Biotechnology Inc, CA). Antibodies were applied using an automated tissue immunostainer (Discovery module, Ventana Medical System), 3, 3-diaminobenzidine IHC kit and bulk reagents were supplied by the manufacturer. Briefly, the Discovery staining protocol was set to "Standard Cell Conditioning", followed by 60 minutes incubation at 42°C with primary antibody and 30 minutes incubation at 42°C with secondary antibody (goat anti-rabbit-IgG-HRP, Jackson Immuno Research Labs Inc). Primary antibody concentrations initially applied to the Ventana instrument were 1:200 for ERα and 1:200 for the secondary antibody translating into final dilutions of 1:600 after 1:3 dilution with buffer dispensed onto the slide with the primary antibody. Slides were counterstained with hematoxylin.
Levels of psoriasin/S100A7 and ERα expression were scored semi-quantitatively in tissue sections, under the light microscope. Scores were obtained by estimating average signal intensity (scale of 0 to 3) and the proportion of epithelial cells showing a positive signal (0–100%). The intensity and proportion scores were then multiplied to give an overall IHC-score.
RNA extraction and reverse transcription
Total mouse RNA was extracted using Trizol™ reagent (Invitrogen) according to the manufacturer's instructions, and the integrity of the RNA was confirmed by denaturing gel electrophoresis as previously described [14]. RNAs from the various frozen tissues were reverse transcribed. One μg of total RNA was reverse transcribed in a final volume of 30 μl composed of 50 mM Tris-HC1 (pH 8.3), 75 mM KC1, 3 mM MgC12, 0.5 μM random hexamers (Invitrogen) 0.5 mM dNTPs, 0.01 mM DTT in the presence of 300 units of MMLV-RT (Invitrogen), and 4 units RNase inhibitor at 37°C for 1 hour, followed by 5 minutes at 95°C and kept at -20°C until used.
PCR conditions
The primer pairs used were as follows;
Mouse S100A7/psoriasin C-terminus
5'-ATG CCA GAC ACA CCA GTG GAG-3' (sense; nucleotides 111–131 in GenBank acc. AY465109) and 5'-GGT AGT CCT TCA CCA GCT TGC-3' (antisense; nucleotides 358–378).
Mouse S100A7/psoriasin open reading frame
5'-TGA AGG GTC CAT CAG TCA-3' (sense; nucleotides 94–111 in GenBank acc. AY465109) and 5'-CTA GTA GAG GCT GTG CT-3' (antisense; nucleotides 421–437).
Mouse β-actin
Primers were designed according to the mRNA sequence (GenBank acc. NM_007393): 5'-TCT ACG AGG GCT ATG CTC TCC-3' (sense; nucleotides 574–594) and 5'-GGA TGC CAC AGG ATT CCA TAC-3' (antisense; nucleotides 883–903). According to the chromosome 5 genomic contig sequence (GenBank acc. NT_039324), these primers span an 87-bp intron with the antisense primer binding across the intron-exon boundary.
PCR reactions were performed essentially as previously described [3]. To amplify cDNA corresponding to mouse S100A7/psoriasin, an initial 2 minutes at 94°C was followed by 36 cycles (30 seconds at 94°C, 30 seconds at 56°C, 30 seconds at 72°C). Twenty six cycles were used to amplify β-actin cDNA (30 seconds at 94°C, 30 seconds at 58°C, 30 seconds at 72°C). PCR products were separated on 1.5% agarose gels containing ethidium bromide (0.1 μg/ml) as previously described [13]. Identity of the 344 bp product corresponding to mouse S100A7/psoriasin and the 330 bp product corresponding to β-actin were confirmed by subcloning and sequencing as described previously [13].
Semi-quantitative PCR analyses were performed using three independent PCRs for each sample for both mouse S100A7/psoriasin and β-actin. Signals visualized with UV irradiation on a GelDoc2000/ChemiDoc System (Bio-Rad), were quantified by densitometry using the Quantity One software (version 4.2; Bio-Rad). Mouse S100A7/psoriasin expression was standardized to β-actin expression assessed from the same cDNA in separate PCR reactions and run in parallel on separate gels. The standardized mean of each triplicate PCR was then expressed relative to the levels in a "moderately expressing" sample selected for each batch of cDNAs to be analyzed.
Phylogenetic analysis
Amino acid sequences of S100 family genes were aligned using Clustal X [15]. This alignment was used to construct a phylogenetic tree based on a Poisson corrected neighbour-joining distance method [16] available in the computer software package MEGA v3.0 [17]. The reliability of the phylogeny's interior branches was tested by a bootstrap test with 1000 replications [18]. The human sequences used, were GenBank Accession numbers: AAH05019 (S100A14), NP_789793 (5100A15), NP_006262 (S100A1), NP_005969 (S100A2), NP_002951 (S100A3), AAH00838 (S100A4), NP_002953 (S100A5), AAH09017 (S100A6), AAH34687 (S100A7), XP_060509 (S100A7L-2), AAH05928 (S100A8), AAH47681 (S100A9), AAH15973 (S100A10), AAH14354 (S100A11), NP_005612 (S100A12), NP_002952 (S100A13). The mouse sequences used, were GenBank Accession numbers: NP_035439 (S100A1), NP_035440 (S100A3), NP_035441 (S100A4), NP_035442 (S100A5), AAH03832 (S100A6), NP_955454 (S100A7/15), NP_038678 (S100A8), AAH27635 (S100A9), AAH25044 (S100A10), AAH21916 (S100A11), NP_033139 (S100A13), AAH25607 (S100A14).
Results
Identification and cloning of mouse S100A7/psoriasin cDNA
A BLASTP search of mouse sequence databases was performed using the human psoriasin (S100A7) amino acid sequence and identified a mouse sequence highly similar to human S100A7, which had been previously predicted by automated computational analysis (GenBank acc. XM_143311, later replaced by AY465109). Using this predicted sequence in a BLASTN search, a mouse skin expressed sequence tag (EST) (GenBank acc. AA792680) containing a highly similar sequence was identified. A pair of mouse "psoriasin"-specific PCR primers was designed that would only amplify a sequence of mouse psoriasin contained within the EST. According to the recently posted genomic sequence (GenBank acc. AY465110) these primers span a 1992-bp intron, thus the potential amplification of contaminating genomic DNA is minimized (Figure 1). An additional set of mouse psoriasin primers ("psoriasin-ORF") was later designed to amplify the entire predicted open reading frame (Figure 1). These primers also span the 1992-bp intron. Both sets of putative mouse psoriasin primers were used for RT-PCR and semi-quantitatively measure the potential expression of the putative mouse psoriasin in mouse skin, mammary gland and mouse mammary tumors. These tissues were chosen because the human psoriasin/S100A7 was described as differentially expressed in skin and breast. The results, using primers that amplified the entire predicted ORF of the putative mouse psoriasin, are shown in Figure 2, with β-actin as a control in parallel. A 344 bp PCR product was amplified from cDNA derived from normal mouse skin and mammary gland using mouse psoriasin primers (see above for description). Based on our PCR results, mouse psoriasin mRNA is expressed in skin as well as in mammary gland where it is more highly expressed in mammary tumors than in normal mammary gland (Figure 2a). The psoriasin RT-PCR product and the 330 bp product generated using the mouse β-actin primers were both cloned and sequenced to confirm their identities. The nucleotide sequence of the putative mouse psoriasin RT-PCR product (GenBank acc. AY582964) was 100% identical to the coding sequence of the putative mRNA in the GenBank sequence database (GenBank acc. XM_143311). Start/stop codons, PCR primer binding sites, and intron/exon boundaries are indicated in Figure 1. However, during the course of this study the predicted mRNA sequence of GenBank acc. XM_143311, named as 'similar to the S100 calcium-binding protein A7 (psoriasin)' was removed and apparently replaced by another entry GenBank acc. XM_356221 for a predicted mRNA that was named as 'similar to the S100 calcium-binding proteins A15'. GenBank acc. AY465109 then appeared as a cDNA cloned from mouse skin and was named as 'Mus musculus S100 calcium-binding protein A15 mRNA'. The amino acid sequences of the various Genbank entries believed to describe the mouse psoriasin are aligned in Figure 3 to illustrate that the amino acid sequences of the ORF from each entry are identical. From here on, we will refer to this gene as mouse S100A7/psoriasin. In addition, according to the recently posted genomic sequence (GenBank acc. AY465110 and NT_078386), the mouse S100A7/psoriasin gene maps within the murine S100 cluster on chromosome 3 (LOC381493, NCBI Map Viewer Link), in a region similar to the human S100 cluster on chromosome 1q21 [10,19]. Our sequence database searches and RT-PCR experiments demonstrate a putative mouse ortholog of the human psoriasin gene exists and like human psoriasin, it is expressed in skin and mammary tissue.
Phylogenetic analysis
The putative mouse psoriasin gene (originally "Similar to S100A7") has been renamed "mouse S100A15" in Genbank (AY465110) [20]. This name implies higher homology to human S100A15 than to human psoriasin/S100A7. However, it has been shown that human S100A7 and S100A15 diverged sometime during primate evolution [10]. A phylogenetic tree was constructed to examine the evolutionary relationships between the mouse and human S100 genes (Figure 4). The results are consistent with the divergence of human S100A7 and S100A15 occurring by duplication after the human/mouse split in evolution, and thus the mouse gene is likely ancestral to both human paralogs.
Presence of putative Jab1 binding motif in mouse S100A7/psoriasin
A notable structural feature of the human psoriasin/S100A7 gene is a consensus Jab1-binding domain sequence that we have speculated may confer the ability of human psoriasin/S100A7 to interact with Jab1 and so play an important role in the mechanism by which human psoriasin/S100A7 medicates its action [6]. Jab1 was originally identified as a factor influencing c-Jun transcription of AP-1 regulated genes [21]. It is now known that Jab1 is a component of a multimeric protein complex (the CSN/COP9 signalosome) and that Jab1 interacts with many components of cell signalling pathways [22]. This may be in the context of either phosphorylation or proteasomal activities as Jab1 is also the only known deneddylating protein active in control of the SCF-cullin ubiquitin ligases [23].
A putative Jab1-binding motif is also present in the murine S100A7/psoriasin sequence but is absent from the human S100A15 sequence (Figure 5). Of the three amino acid residues known to be conserved in the Jab-1 binding motif of Jab-1 binding proteins (see highlighted residues in Figure 5), one is different in the mouse S100A7/psoriasin. However, the change from aspartic acid in the human S100A7 to a glutamic acid in mouse S100A7/psoriasin is a conservative change and the residue remains acidic. In contrast, the equivalent residue in human S100A15 is threonine, a neutral residue containing an hydroxyl group in its aliphatic side chain, and also having the potential to be post-translationally modified by phosphorylation. These observations support further our hypothesis that the so-called mouse S100A15/psoriasin gene encodes a product that is functionally more similar to human psoriasin/S100A7 than human S100A15. Since the human and mouse Jab1 proteins are 99% identical (Genbank accession number, mouse Jab1 AAC33900; human Jab1 NP_00364) we speculate that conservation of the Jab1-binding domain in the mouse protein results in conservation of the interaction as well.
Expression of mouse S100A7/psoriasin during mouse mammary tumorigenesis
Our initial results shown in Figure 2 suggest that the expression of mouse S100A7/psoriasin may be upregulated during mammary tumorigenesis. This was of interest since we and others have previously found that human psoriasin/S100A7 expression is highly up-regulated in human breast cancer compared to normal breast tissue [2,24]. To confirm and extend this observation, further analysis of murine mammary tumors was conducted by both semi-quantitative RT-PCR and by in situ hybridization.
cDNAs were generated from matched normal mammary gland and mammary gland tumors from DMBA-treated CD1 wild-type mice (n = 6, same as in Figure 2A). Semi-quantitative RT-PCR analysis showed significantly increased expression of mouse S100A7/psoriasin mRNA in mammary gland tumors relative to matched normal mammary gland (p < 0.05) by Wilcoxon signed-rank test, one-sided (Figure 2B).
To confirm this differential expression and to determine which cell type within the mammary tumors was expressing mouse S100A7/psoriasin, in situ hybridization was performed on sections from paraffin-embedded tissue blocks corresponding to the fresh frozen samples from which RNA was extracted to generate cDNAs used for RT-PCR analysis. A strong but focal signal was observed in all six mammary gland tumors while the signal was weak or undetectable in all of the matched normal mammary gland tissues (Table 1, Figure 6). Assessment of the sections at high magnification and correlation with serial sections stained only by H&E, revealed that the S100A7/psoriasin expression was usually confined to a subset of epithelial tumor cells that showed morphological features of squamous differentiation (Figure 6). Within these regions of squamous differentiation expression was usually decreased with proximity to the squamous surface. Semi-quantitative RT-PCR and in situ hybridization results showed a significant positive correlation (Spearman's rank correlation coefficient r = 0.63, p < 0.02) (Table 1).
Although up-regulation of mouse S100A7/psoriasin expression occurred in DMBA induced tumors compared to adjacent normal mammary gland tissue, expression levels showed a wide-scatter amongst the mammary tumors (Figure 2). This was of interest since ERα has been reported to have variable expression in chemical carcinogen induced tumors [25-27] and we have previously shown an inverse association of human psoriasin/S100A7 expression with ERα in breast cancer [3,5]. We therefore compared the expression of mouse S100A7/psoriasin as determined by in situ hydridization with ERα expression assessed by IHC in adjacent sections from the same tumors. An inverse association between mouse S100A7/psoriasin and ERα expression was clearly evident but categorical contingency analysis did not reach statistical significance (Fisher's exact test, p = 0.073), likely due to small numbers. However detailed comparison within tumors showed that in tumors that expressed both genes, mouse S100A7/psoriasin expression was restricted to areas within heterogeneously positive ERα tumors that lacked ERα expression. Interestingly, mouse mammary tumors that develop due to targeting of the neu oncogene to the mouse mammary gland (MMTV-neu) did not express mouse S100A7/psoriasin RNA nor ERα (data not shown).
Expression of mouse S100A7/psoriasin in a model of skin inflammation
Human psoriasin/S100A7 was originally cloned from psoriatic skin, an inflammatory skin disease [1]. Mouse S100A7/psoriasin was however first identified in an expression library constructed from normal mouse skin (Genbank Acc AA792680 and AY465109) and our data have confirmed mouse S100A7/psoriasin RNA expression in normal mouse skin (see Figure 2A). To determine if mouse S100A7/psoriasin expression is upregulated in inflammation, ISH was performed on sections from normal mouse tissues (see Figure 2A) as well as tissue from a model of mouse skin inflammation. As seen in mammary tumor tissues, a strong but focal signal was observed in some samples and assessment of the sections at high magnification revealed that the mouse S100A7/psoriasin expression was confined to a subset of epithelial cells surrounding hair shafts (Figure 7A) consistent with the localization of human psoriasin/S100A7 in normal human skin [3]. No signal was observed using a mouse S100A7/psoriasin sense probe. In addition we have speculated that mouse S100A7/psoriasin expression would mimic its human counterpart and be upregulated under conditions of skin inflammation. A significant increase in mouse S100A7/psoriasin RNA expression is seen at 24 hrs after croton oil application correlating with the development of inflammation characterized by leucocytic inflammation, (Figure 7B, Table 2).
Expression of human psoriasin/S100A7 in relation to differentiation in human tumors
The pattern of mouse S100A7/psoriasin expression observed in mouse tumors and skin suggested an association of expression with the process of squamous differentiation. Studies of human psoriasin /S100A7 support a similar association in skin and bladder cancer [28,29]. To determine further the relation between psoriasin and different pathways of epithelial differentiation we therefore examined human psoriasin expression relative to different human tumor types in the cervix and lung, since overt squamous differentiation is rare in human breast cancer. In tumors in both cervix and lung, psoriasin expression was significantly correlated with squamous differentiation (p < 0.0001). Expression was observed in over 90% of squamous carcinomas in both organs, but was a rare occurrence (<10%) in adenocarcinomas and absent in other tumor types in the lung (Figure 8). As we have previously observed in both breast and skin, psoriasin expression in the cervix was also most highly expressed in squamous carcinoma-in-situ with lower levels in invasive squamous carcinoma (p = 0.0013).
Discussion
We have used structural and expression analysis to show that a gene currently classified as mouse S100A15 (GenBank acc. AY465110) which maps within the murine S100 cluster on chromosome 3 (see acc. NT_078386, NCBI Map View Link and LOC381493) [10] should be reclassified as mouse S100A17/psoriasin, as suggested by Marenholz et al. [9]. This gene is not highly conserved relative to other members of the S100 family (~40% amino acid sequence similarity of mouse S100A7 with either human S100A7 and/or human S100A15, compared to >60% amino acid sequence similarity of most other known mouse S100A proteins to their human counterparts, unpublished data) [10]. However, phylogenetic analysis shows that while mouse retained an ancestral S100A7/psoriasin gene, the human ortholog underwent duplication and functional differentiation making the assignment of orthology/paralogy only on the basis of phyogenetic information uncertain.
In the human, the closely related psoriasin/S100A7 and S100A15 appear to be functionally distinct. Although both were isolated as overexpressed genes in human psoriatic skin [1,20], we have previously identified S100A7 to be differentially expressed in neoplastic mammary gland [30]. Neither gene is substantially detectable in database analysis of the available normal human mammary gland libraries, and only psoriasin/S100A7 is present at high copy numbers (up to 2,300 tags per 200,000) in several mammary gland neoplasia libraries, whereas S100A15 is absent or rarely detectable (<10 tags per 200,000) [30]. Additionally, psoriasin/S100A7 is expressed in several SAGE database libraries representing human skin neoplasia, while S100A15 is not detected (data not shown). Thus, despite some similarities in their occurrence and expression in association with psoriasis [20] these closely related human genes show dissimilar expression patterns in mammary tumors suggesting distinct functional roles. In contrast, our studies show that the pattern of gene expression of mouse S100A7/psoriasin during mammary tumorigenesis and skin inflammation occurs in a similar pattern to that seen with human psoriasin/S100A7 [2,3]. Furthermore, while mouse S100A7/psoriasin shows approximately equivalent amino acid sequence similarity to both human S100A7 and human S100A15 proteins, both mouse S100A7/psoriasin and human psoriasin/S100A7 contain a putative Jab1-binding motif that is functionally significant in the human at least [6,7] but this motif is not found in human S100A15. We conclude on the basis of chromosomal location, phylogenetic analysis, amino acid sequence similarity, conservation of a putative Jab1-binding motif, and similarities in patterns of expression, that mouse S100A7/ psoriasin is the murine ortholog of human psoriasin/S100A7.
Expression of human psoriasin/S100A7 was originally attributed to skin pathologies where abnormal squamous differentiation occurs [1]. In breast tumors where psoriasin/S100A7 is also expressed, overt squamous differentiation is rare. However it has been detected and suggested as a marker of squamous differentiation in a subtype of bladder cancer [29]. Extending the latter finding our data here shows that in cervix and lung, two tissues where squamous and glandular differentiation are both common differentiation pathways for carcinomas, psoriasin/S100A7 is commonly expressed and almost exclusively associated with squamous tumor subtypes. The parallel finding that mouse S100A7/psoriasin was distinctively associated with areas of squamous differentiation within murine breast adenocarcinomas suggests that similar factors are involved in the regulation of both psoriasin/S100A7 and murine S100A7/psoriasin genes, and is in keeping with a similar role for the murine gene.
A role for human psoriasin/S100A7 in inflammation has been suggested, since psoriasin/S100A7 can be secreted and was shown to be chemotactic for neutrophils and CD4+ T-cells in vitro [31]. Our current data from a model of mouse skin inflammation is also consistent with such a role, since a significant upregulation in mouse S100A7/psoriasin expression occurs simultaneously with the acute phase of skin inflammation after application of croton oil to the mouse tail skin. The role for psoriasin/S100A7 in tumorigenesis may be related to the activity of pro-survival pathways [7] and acquisition of apoptosis resistance [8]. Our results here also provide indirect support for this hypothesis, as mouse S100A7/psoriasin RNA was expressed in keratinocytes at the margin of squamous differentiation, where resistance to apoptotic stimuli may be an important component of the sequence of differentiation and to allow time for production of large amounts of keratin before finally undergoing desquamation, denucleation and cell death [32].
Finally, psoriasin has been implicated in human breast cancer progression. Specifically, psoriasin/S100A7 has been associated with the pre-invasive DCIS phenotype [2], augmentation of several characteristics of malignancy in vitro and in vivo [4,6] and with poor outcome in invasive estrogen receptor-negative tumors [3,5]. Our current results also support the involvement of S100A7/psoriasin in murine mammary tumorigenesis. Additionally, differential expression of mouse S100A7/psoriasin was observed between DMBA and MMTV-neu induced mammary tumors, and a strong trend towards a negative correlation between mouse S100A7/psoriasin and estrogen receptor alpha expression emerged in the DMBA induced mammary tumors. Such data are consistent with the pattern of expression of human psoriasin/S100A7 in human breast tumors, and are consistent with the view that mouse S100A7/psoriasin subserves similar roles to human psoriasin/S100A7 in mammary tumorigenesis and breast cancer progression. It is nevertheless possible that human psoriasin/S100A7 and mouse S100A7/psoriasin have several functions, depending on cellular context. This is reflected by differences already observed in localization of expression in different cell types within a tissue or subcellular localization, since psoriasin has been found in the nucleus, in the cytoplasm, at the cell periphery/plasma membrane and it can also be secreted [33,34].
Conclusion
S100 proteins have been known to be differentially expressed during tumorigenesis as well as other disease states for some time. However, the functional roles they may play in disease processes are poorly understood [33,34]. In breast cancer, psoriasin/S100A7 is associated with important biological and clinical aspects of the disease [30] and the identification of its potential ortholog in the mouse is an important step to facilitate understanding of its function and mechanism of action. The results presented in this study strongly support the hypothesis that mouse S100A7 is the murine ortholog of human psoriasin/S100A7, and provide a rationale for the manipulation of mouse S100A7/psoriasin in mice to gain important insights into the function of human psoriasin/S100A7.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
MW carried out the database searches, cloning, sequencing, ISH, RT-PCR of mouse S100A7/psoriasin, phylogenetic analysis and co-ordinating frozen tissue and tissue block collection from other authors. She was involved in data analysis and writing of the manuscript and critically revising it.
EE helped MW in database searches and motif analyses. He was involved in writing and revising it critically.
ML carried out the mouse skin inflammation experiments and prepared the tissue blocks. He was involved in writing and revising it critically.
SA, GQ and AA were involved in carrying out the tissue analysis and data acquisition and analysis in Figure 8.
LS-C supervised and carried out some of the ISH.
YN carried out the IHC.
AC supervised and helped carry out the phylogenetic analysis. He was involved in writing the manuscript and revising it critically.
YM and RS provided the mouse mammary tumor tissues, other mouse tissues and respective blocks for analysis. They were both involved in writing the manuscript and revising it critically.
LCM is the Principal Investigator of the project and was responsible for the overall supervision and co-ordination of the project. She is the corresponding author and contributed to the writing, critical revision together with draft preparation, final preparation for manuscript submission, preparation and submission of all revisions.
PHW is the co-P.I. of the project. He supervised and was involved in the interpretation of all histopathology, ISH and IHC. He contributed to all data analysis, to the writing of the manuscript and its critical revision.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This work was supported by grants from the Canadian Institutes for Health Research (CIHR), the Canadian Breast Cancer Research Alliance (CBCRA) and the Canadian Foundation for Innovation (CFI). P. H. W. is a CIHR/MRC Scientist. Y.M. is an MMSF career awardee. E.E. is a recipient of a Terry Fox research studentship from the National Cancer Institute of Canada.
Figures and Tables
Figure 1 Cloned mouse S100A7/psoriasin sequence, and gene structure A The mouse S100A7 gene sequence (GenBank acc. AY465110) describes a 3829 bp gene with 3 exons and 2 introns. B The sequence of our mPsor-ORF-TOPO clone isolated by RT-PCR of RNA from a DMBA mouse mammary tumor is shown compared with the mouse S100A15 cDNA sequence (GenBank acc. AY465109) isolated from mouse skin. Predicted introns are inserted into the cDNA sequences (black), the predicted open reading frame (ORF) and translated products (amino acid one letter code in italics below the nucleotide sequence) are shown with start and stop codons (green). Vector sequences of the mPsor-ORF-TOPO are indicated in light grey. Sense (>) and antisense (<) primer annealing sites are indicated for both primer sets: mouse psoriasin (blue) and mouse psoriasin-ORF (red).
Figure 2 A Top panel Expression of mouse S100A7/psoriasin in normal skin RNA from normal mouse skin from 6 different animals (1–6) was subjected to RT-PCR analysis using mouse psoriasin-ORF primers and β-actin primers as described in Materials and Methods. PCR products of 344 bp for mouse S100A7/psoriasin and 330 bp for β-actin are shown. Bottom panel Expression of mouse S100A7/psoriasin in normal mouse mammary gland) and DMB A induced mammary tumors RNA from freshly frozen matched normal mammary gland (Normal MG) and DMBA induced mammary tumors (MG tumor) from 6 different CD1 mice (1–6) was extracted and subjected to RT-PCR analysis using mouse psoriasin-ORF primers and β-actin primers as described in Materials and Methods. PCR products of 344 bp for mouse S100A7/psoriasin and 330 bp for β-actin are shown. B Semi-quantitative analysis of the results shown in bottom panel of A Data points represent the means of three independent PCR reactions. The value of mouse S100A7 normalized to β-actin for each matched normal MG and MG tumor sample are joined by red lines. Blue lines represent the median value of mouse S100A7/psoriasin expression for each tissue type. Increased expression in MG tumors is significant (p < 0.05, one sided Wilcoxon signed rank test).
Figure 3 Alignment of the translation products of the predicted ORFs from the various GenBank entries related to mouse S100A7/psoriasin.
Figure 4 Phylogenetic analysis of human and mouse S100 proteins A neighbour-joining tree of human (Hs) and mouse (Mm) S100 proteins (1 through 15) was constructed using the Poisson correction model and the bootstrap percentage from 1000 replicates is indicated at each node. Results are consistent with the human S100A7 duplications (blue sub-tree) occurring after the divergence of mouse and human in evolution (red node).
Figure 5 Conservation of the Jab1 binding motif in human psoriasin/S100A7 and mouse S100A7/psoriasin Human psoriasin/S100A7 contains a Jab1 binding domain identified previously in p27 and LFA-1. Mouse S100A7/psoriasin also contains the Jab1 binding motif but human S100A15 does not. Note that human S100A15 has a significant change, from a charged (D) to a neutral (T) amino acid, in the first of the essential amino acids (color coded) required for Jab1 binding. The change seen in this residue in the mouse S100A7/psoriasin protein is a conservative change and the residue (E) remains negatively charged. Amino acid color-coding: non-polar (yellow), neutral polar (green), negatively charged (red). Hs = homo sapiens, Mm = Mus musculus. Adapted from [6].
Figure 6 A Strong focal expression of mouse S100A7/psoriasin RNA in mammary gland tumors compared to little expression in matched normal mammary gland tissue using in situ hybridization Sections from formalin fixed paraffin-embedded mammary tissue samples matched to the frozen tissue from which RNA was previously isolated for RT-PCR analysis were used for in situ hybridization analysis of mouse S100A7/psoriasin expression: A, C, D, F show hybridization with mouse S100A7/psoriasin 35S-labeled antisense cRNA; B, E show hybridization with mouse S100A7/psoriasin 35S-labeled sense cRNA. A = high magnification (400X) section of normal mammary gland, hybridized with mouse S100A7/psoriasin 35S-labeled antisense cRNA. B = high magnification (400X) adjacent section to A hybridized with mouse S100A7/psoriasin 35S-labeled sense cRNA. C = low magnification (100X) of section from the matched DMBA mammary tumor from same animal shown in A hybridized with mouse S100A7/psoriasin 35S-labeled antisense cRNA. D = higher magnification (400X) of C. E = high magnification (400X) of adjacent section from the same mammary tumor shown in D, hybridized with mouse S100A7/psoriasin 35S-labeled sense cRNA. F = low magnification (100X) of a single tissue section (tissue from a different mouse to that shown in A-E)) hybridized with mouse S100A7/psoriasin 35S-labeled antisense cRNA, where strong focal expression of mouse S100A7/psoriasin RNA is observed in the mammary gland tumor (black arrow) with little or no expression in the surrounding normal mammary gland tissue. B Localization of mouse S100A7/psoriasin expression to areas of squamous differentiation in DMBA-induced mammary gland tumors Sections from formalin fixed paraffin-embedded DMBA-induced mammary tumor samples matched to the frozen tissue from which RNA was previously isolated for RT-PCR analysis were used for in situ hybridization analysis of mouse S100A7/psoriasin expression. A = low magnification (100X) of tumor section hybridized with mouse S100A7/psoriasin 35S-labeled antisense cRNA. B = higher magnification of A (200X), C = high magnification of B. D = adjacent section to that seen in A, B, C, and at same magnification as B but hybridized with mouse S100A7/psoriasin 35S-labeled sense cRNA. E and F are haematoxylin and eosin stains of adjacent sections to that shown in A, B, C, and D. E is the same magnification as in B, and F is the same magnification as that shown in C. Squamous differentiation in the haematoxylin and eosin stained adjacent sections is visualized as a characteristic "pearling" pattern where epithelial tumor cells (purple) differentiate into keratinocytes (pink) before denucleating and sloughing off into the inner lumen (white). Thus cells on the outer margin of the pearled area are undergoing squamous differentiation, and those correspond to cells expressing mouse S100A7/psoriasin RNA, in A, B, and C.
Figure 7 A Localization of mouse S100A7/psoriasin expression to areas of squamous differentiation in skin Sections from formalin fixed paraffin-embedded normal mouse skin samples matched to the frozen tissue from which RNA was previously isolated for RT-PCR analysis were used for in situ hybridization analysis of mouse S100A7/psoriasin expression. A, B, C increasing magnifications (original is 200X) of sections hybridized with mouse S100A7/psoriasin 35S-labeled antisense cRNA, showing strong focal expression of mouse S100A7/psoriasin RNA in a particular subset of cells surrounding the hair shafts, which are associated with squamous differentiation. D, adjacent section hybridized with mouse S100A7/psoriasin 35S-labeled sense cRNA. B Upregulation of mouse S100A7/psoriasin expression in a model of skin inflammation Sections from formalin fixed paraffin-embedded mouse skin tissue at 0 mins and 24 hours after croton application to the tail skin as described in Materials and Methods. A and D, haematoxylin and eosin stained sections, from 0 and 24 hr treated skin, respectively. B and E adjacent sections from 0 and 24 hr croton oil treated skin, respectively, hybridized with mouse S100A7/psoriasin 35S-labeled sense cRNA. C and F, adjacent sections from 0 and 24 hr croton oil treated skin, respectively, hybridized with mouse S100A7/psoriasin 35S-labeled antisense cRNA.
Figure 8 Expression of psoriasin in human lung and cervical tumors determined by immunohistochemistry Upper panel shows the scatter plot chart of level of expression (assessed by IHC score) in different lung tumor types (SqC = squamous cell carcinoma, AdC = adenocarcinoma, SmC = small cell carcinoma, Mes = mesothelioma). Bars represent the median scores. P value determined by t-test. Lower panel shows the scatter plot chart of level of expression (assessed by IHC score) in different cervical lesions corresponding to early stages of cervical tumor progression (metaplasia, CIN = cervical intra-epithelial squamous neoplasia, stages 1, 2, and 3, SqC = squamous cell carcinoma invasive, AdC = cervical adenocarcinoma, in-situ and invasive). P values determined by t-test.
Table 1 Correlation of mouse S100A7/psoriasin expression using RT-PCR and in situ hydridization in normal mammary gland and DMBA-induced mammary tumors Mouse S100A7/psoriasin mRNA levels were determined in extracts from frozen tissue samples of matched normal mammary gland and DMBA-induced mammary tumors (n = 6) by semi-quantitative RT-PCR as described in Materials and Methods. Sections from the corresponding adjacent tissue samples which had been formalin fixed and paraffin-embedded, where used for in situ hybridization (ISH) with 35S-labelled antisense mouse S100A7/psoriasin probes. Specific hybridization was scored on a scale of 0 to 3 as described in Materials and Methods. Results of the two analyses were correlated using Spearmans test (r = 0.63, p < 0.02).
Relative mouse S100A7/psoriasin expression
Sample RT-PCR ISH
Normal MG 1 0.37 0
2 0.16 0
3 0.14 0
4 0.21 <0.5
5 0.73 <0.5
6 0.42 0
MG Tumor 1 1.50 2
2 1.63 3
3 1.12 2
4 0.22 1
5 1.22 1
6 0.13 1
Table 2 Inflammatory response to topical application of croton oil to the skin.
0 hours median [range] 24 hours median [range]
Experiment #1 9 [3-21] 15 [4-32]***
Experiment #2 10 [0-19] 20 [10-52]***
Experiment #3 13 [0-24] 25 [9-48]***
The number of nuclei per 50 μm2 area of dermis was used as a parameter to evaluate inflammation.
Note – in all three experiments, the medians were found to be significantly different from each other (P < 0.0001) using the Mann-Whitney U test.
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| 15717926 | PMC553966 | CC BY | 2021-01-04 16:03:07 | no | BMC Cancer. 2005 Feb 17; 5:17 | utf-8 | BMC Cancer | 2,005 | 10.1186/1471-2407-5-17 | oa_comm |
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BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-5-191573331610.1186/1471-2458-5-19Research ArticleComparison of patterns of use, beliefs, and attitudes related to waterpipe between beginning and established smokers Asfar Taghrid [email protected] Kenneth D [email protected] Thomas [email protected] Wasim [email protected] Syrian Center for Tobacco Studies, Aleppo, Syria2 Department of Health & Sport Sciences, and Center for Community Health, University of Memphis, Memphis, USA3 Department of Psychology, Virginia Commonwealth University, Richmond, USA4 Institute of Epidemiology and Social Medicine, University of Muenster, Muenster, Germany2005 25 2 2005 5 19 19 9 9 2004 25 2 2005 Copyright © 2005 Asfar et al; licensee BioMed Central Ltd.2005Asfar et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
To compare patterns of use, beliefs, and attitudes related to waterpipe smoking between university students (beginning smokers) and café customers (established smokers) in Aleppo Syria, in order to explore the evolution of this smoking method.
Methods
Two cross-sectional surveys were conducted among representative samples of university students (total 587, 48.4% men, mean age 22 years), and waterpipe users among cafe' customers (total 268, 60% men, mean age 30 years) in Aleppo, Syria. We used interviewer-administered questionnaire inquiring about pattern of waterpipe smoking (initiation, frequency), situational characteristics of use (partner, place, sharing), beliefs related to waterpipe smoking (harmful/addictive properties of waterpipe), attitudes related to waterpipe smoking (confidence in quitting, will to quit, motivation for quitting, past year quit attempt), and cigarette smoking.
Results
Daily and regular patterns of smoking become more prevalent with increased duration of smoking, but intermittent smoking remains the predominant pattern of waterpipe use. Women seem to be drawn later to the habit, which seem to escape the usual taboo against women's cigarette smoking. Patterns and context of waterpipe use tend to change with progress of the practice affecting frequency, setting, and sharing of waterpipe. Unlike beginners, established waterpipe smokers seem more smoking-method oriented, more hooked on the habit, less willing to quit, and less likely to foresee challenges to quitting.
Conclusion
Use patterns and attitudes related to waterpipe smoking evolve to accommodate the change in dependence and life circumstances of the smoker. Most of use features, beliefs, attitudes, as well as time-course seem unique to this smoking method requiring novel approach to intervention.
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Background
Waterpipe, is a generic name for tobacco use methods that share a common feature; passage of smoke through the water before inhalation. It is known under different names and shapes in different cultures and countries (e.g. shisha, narghile, hookah, hubble bubble) [1]. Although waterpipe smoking is centuries old, its use was declining until recent years when it witnessed a boom in popularity among Arab communities around the world [1,2]. Recent estimates show that this smoking habit has reached a quarter of some groups in the EMR, affecting youths and women among others [3-6].
Studies on the health effects of this smoking method are scanty, and often suffer from poor control of other confounding factors (e.g. cigarette smoking). We are just beginning to learn in a more methodological way the full spectrum of harmful effects of this smoking method and about shared and distinguishing features from cigarette smoking. For example, in regards to CO, one of the main cardiovascular risks of cigarette smoking, waterpipes are at least as efficient as cigarettes in delivering this toxic gas to smokers' lung [1]. On the other hand, tobacco smoke generated by this method seems to contain larger amounts of heavy metals such as arsenic, cobalt, chromium, and lead [7]. In addition, this smoking method is efficient in delivering nicotine to smokers, who tend to show signs of tobacco use dependence [8,9].
Because of the increasing trend of this smoking method, together with its health damaging and addictive potentials the need to develop ways to intervene with smokers and prevent initiation seems timely. Intervention efforts to deal with this emerging public health problem however, are hindered by the inadequacy of data characterizing various aspects related to patterns of waterpipe use and dependence [10].
We recently conducted a survey looking at prevalence, beliefs, and attitudes related to this smoking method among university students in Aleppo, Syria. Waterpipe was practiced by a quarter of male and 5% of female students and was characterized by intermittent and social use [3]. Also, smokers among university students seem to be at the initial stages of their waterpipe practice [3]. Informed by these results, a second survey was carried out among older, more established, smokers in cafés in Aleppo. In this report we compare the two groups in terms of pattern of use, beliefs, and attitudes related to waterpipe smoking in order to gain insights on the natural history of this smoking method and inform intervention development.
Methods
The methods of these surveys conducted in 2003 are described in details elsewhere [3,9]. Briefly, these surveys were conducted among representative samples of university students (total 587, 48.4% men, mean age 22 years, age range 18–30 years) of whom 86 (15%) were waterpipe smokers, and waterpipe users in coffee shops in Aleppo (total 268, 60% men, mean age 30 years, age range 18–68 years). The students survey was carried out at Aleppo University's dormitories (total 19), where four women's and four men's dormitories were selected randomly, and within each dormitory, a sampling frame was used to recruit about 75 participants. In the cafe' survey, 17 cafes were selected out of total 112 in Aleppo, and within each cafe' participants were recruited by random selection of pre-numbered waterpipes. Unlike the café survey, the university survey included both waterpipe smokers and non-smokers, and thus only waterpipe smokers (15%) among students were included in the current analysis (total 86, 82.5% men, mean age 22 years) (Table 1).
Table 1 Basic indicators of the café survey population in comparison to University students
University students (n= 86) n (%) Café customers (n = 268) n (%)
Gender
Male 71 (82.6) 161 (60,1)
Female 15 (17.4) 107 (39.9)
Residence
City 43 (50.0) 264 (98,5)
Country 43 (50.0) 4 (1.5)
Religion
Muslim 81 (94.2) 201 (75.3)
Christian 5 (5.8) 66 (24.7)
Marital status
Married 1 (1.2) 128 (47.8)
Single, divorced, or widowed 85 (98.8) 140 (52.2)
Frequency of narghile use
Daily 5 (5.8) 64 (24.0)
Less than daily 81 (94.2) 203 (75.7)
Mean ± SD Mean ± SD
Age 22.3 ± 2.3 30.1 ± 10.2
Economic status (DI) 2.0 ± 0.7 1.2 ± 0.6
Number of years of education 14.7 ± 1.3 12.5 ± 3.6
The questionnaires used in these surveys were developed from standard instruments modified to suit waterpipe smoking [11-13]. The protocol and informed consent document were approved by the Institutional Review Boards at the Syrian Society Against Cancer and The University of Memphis.
Generally, the two groups were compared on the following dimensions; 1- socio-demographics including age, gender, number of years of formal education, economic status assessed by the density index (DI = number of household members/number of rooms in the house), 2- smoking characteristics including frequency of use (daily, occasional), age of initiation of use, age of initiation of daily use, cigarette smoking status (past month cigarette smoking, ex-daily smoking for current non-smokers), 3- situational characteristics of waterpipe use including waterpipe smoking initiation (alone, with friends, with family), current waterpipe smoking (alone, with friends, with family), usual place of waterpipe smoking (open end question categorized later into home/dorms, cafes, other or no particular place), waterpipe sharing with others, usual sharing companion (as an open end question categorized later into friend, family), whether use is periodic/seasonal, the season of increased use (as an open end question categorized later into holidays, summer/spring, stress/exams, and other), 4- quit attitude (waterpipe, cigarette) including belief of own ability to quit, will to quit, past year quit attempt, quit motivation (open end question categorized later into health, cost, and other), perceived major challenge for quitting (open end question categorized into friends, addiction and habit, boredom and free time, no challenge, other or non specific answer), 5- perceived main health hazard of waterpipe smoking (open end question categorized into cardiovascular effects, respiratory effects, cancer, other bodily effects, none or don't know), harmful and addictive effects of cigarettes compared to waterpipe (categorized into cigarettes are more harmful, both methods are equally harmful, and waterpipe is more harmful, and the same categories were used for the comparison of addictiveness), and finally 6- family's attitude towards waterpipe and/or cigarette smoking categorized into (friendly, normal, not friendly), and smokers' perception of the extent they are hooked on waterpipe (categorized into not at all, somewhat, and very hooked) (tables 1, 2, 3). Participants were asked as well on the average time of each waterpipe smoking bout, and daily waterpipe smokers were asked about their average monthly expenditure on waterpipe smoking.
Table 2 Waterpipe smoking patterns among café customers (established) compared to those of university students (beginners)
Beginners (students, n = 86) n (%) Established (customers, n = 268) n (%)
Frequency of waterpipe smoking
Daily 5 (5.8) 64 (24.0%)*
Occasionally 81 (94.2) 203 (76.0)*
First experience with waterpipe smoking
Alone 2 (2.3) 25 (9.3)*
With friend 69 (80.2) 165 (61.6)*
With family 15 (17.4) 75 (28.0)
Current use of waterpipe
Alone 1 (1.2) 31 (11.6)*
With friends 69 (80.2) 175 (65.3)*
With family 16 (18.6) 62 (23.2)*
Place of usual waterpipe smoking
Home 48 (55.8) 51 (19.2)*
Café/restaurant 24 (27.9) 207 (77.8)*
Other or no particular place 14 (16.3) 8 (3.0)*
Share the same waterpipe 83 (96.5) 117 (43.8)*
Individual most commonly waterpipe is shared with
Friend 78 (94.0) 49 (41.9)*
Family 5 (6.0) 66 (56.4)*
Current cigarette smoking 41 (47.7) 71 (26.5)*
Ex-cigarette smoking 8 (17.8) 24 (12.2)
Seasonal increase in waterpipe smoking frequency 60 (69.8) 131 (48.9)*
Period of increased waterpipe smoking frequency
Holiday 15 (25.0) 25 (19.1)
Summer 31 (51.7) 91 (69.5)*
Stress/exams 12 (20.0) 5 (3.8)*
Other or no specific answer 2 (3.3) 10 (7.7)
Mean ± SD Mean ± SD
Age of initiation of waterpipe smoking (years) 19.6 ± 2.6 24.3 ± 8.3*
Age of initiation of daily waterpipe smoking (years) 21.8 ± 3.6 24.7 ± 8.8
Duration of waterpipe smoking (years) 2.7 ± 1.9 5.9 ± 6.2*
Monthly cost of waterpipe smoking 680 ± 507 2366 ± 1918*
Age of initiation of daily cigarette smoking (years) 18.8 ± 2.4 19.1 ± 4.1
* p < 0.05 using the Chi2 test for dichotomous variables, and Student's t test or the Mann-Whitney test as appropriate for continuous variables (comparison is always between beginners and established smokers).
Table 3 Attitudes and beliefs related to waterpipe quitting among café customers and university students
Beginners (students, n = 86) n (%) Established (customers, n = 268) n (%)
Belief can quit waterpipe any time 77 (89.5) 231 (86.5)
Will to quit waterpipe 35 (40.7) 76 (28.4)*
Last year quit attempt (waterpipe) 23 (65.7) 45 (59.2)
Belief can quit cigarettes anytime 19 (46.3) 36 (50.7)
Will to quit cigarettes 29 (70.7) 45 (63.4)
Last year quit attempt (cigarettes) 26 (89.7) 32 (71.1)
Main motivation for quitting waterpipe**
Health 33 (91.6) 79 (103.8)
Cost 3 (8.7) 3 (6.5)
Other 3 (8.6) 4 (5.3)
Main challenge for quitting waterpipe
Friends 10 (28.6) 6 (7.9)*
Addiction and habit 6 (17.1) 5 (6.6)
Boredom and free time 3 (8.6) 7 (9.2)
No challenge 13 (37.1) 47 (61.8)*
Other or no specific 3 (8.6) 11 (14.5)
Main health hazard of waterpipe
Cardiovascular effects 5 (5.8) 25 (9.4)
Respiratory effects 41 (47.7) 98 (36.7)*
Cancer 25 (29.1) 96 (36.0)
Other bodily effects 11 (12.7) 13 (4.8)*
None, don't know, none specific 4 (4.7) 35 (13.0)*
Belief about the addictive effects of waterpipe compared to cigarettes
Cigarettes are more addictive 77 (89.5) 221 (82.8)
Equally addictive 7 (8.1) 27 (10.1)
Waterpipe is more addictive 2 (2.3) 19 (7.1)
Belief about harmful effects of waterpipe compared to cigarettes
Cigarettes are more harmful 32 (37.6) 124 (46.4)
Equally harmful 11 (12.9) 49 (18.4)
Waterpipe is more harmful 42 (49.4) 94 (35.2)*
* p < 0.05 using the Chi2 test ** Multiple responses were allowed (percentages add to more than 100)
Statistical analysis
Frequency analyses of main study indices were tabulated and differences between the two groups were assessed using the Chi2 test for dichotomous variables. Measures of central tendency were calculated for continuous variables, expressed as mean ± standard deviation (SD), and were compared between the two groups using Student's t test or the Mann-Whitney test as appropriate. Spearman correlation coefficient was calculated for the relation between duration of waterpipe smoking and frequency of use (categorized as daily, weekly, monthly) in the café study. SPSS statistical software (release 11) was used for the analysis with p value <0.05 considered significant.
Results
Overall, the two samples combined show that daily waterpipe smoking was seen among 19.5% of participants (23.3% of smoking men and 12.3% of smoking women), while occasional smoking was seen among 80.2% of participants (76.3% of smoking men and 87.7% of smoking women). More than half of waterpipe users initiated use and currently use waterpipe with a friend (Table 2). About a quarter of waterpipe users (26.5%) smoke cigarettes as well. Duration of use and frequency of use (daily, weekly, monthly) were correlated (Spearman correlation coefficient 0.14, p = 0.02). Mean duration of waterpipe smoking session among café customers was 71.1 ± 35.8 minutes.
Comparison of patterns of initiation and current use of waterpipe between café customers and university students is detailed in Table 2. Mainly, café customers initiate waterpipe use and daily use later and have longer duration of waterpipe smoking compared to students. University students were more likely to start, currently smoke, and share the same waterpipe with their friends compared to their café counterparts (Table 2). Also, a marked difference between café customers and students is that cigarette smoking was more common among students waterpipe smokers than café ones (p < 0.01), still ex-cigarette smoking did not differ between the two groups. Students as well were more likely to have a seasonal pattern of waterpipe use, which was more associated with exams and stress compared to café customers (Table 2).
Quit attitude of café customers shows that the majority (86.5%) believe that they can quit waterpipe any time, but only a minority (28.4%) are interested in quitting, mainly out of health motivation (Table 3). In comparison, interest in quitting was higher among students and friends were more likely to be cited as the main challenge to quitting (Table 3). This was different from cigarette smokers, where only about a half of those (in both groups) believed they can quit anytime and the majority were willing to quit. Waterpipe users among café customers equally acknowledge respiratory disease and cancer as associated with waterpipe smoking.
Attitude of families towards participants' waterpipe smoking for both café customers and students are depicted in figure 1, showing more tolerance for women waterpipe smoking than for men's in general. Figure 2 illustrates participants (both groups) self perception of being hooked on waterpipe according to their frequency of smoking, were these dimensions were strongly associated among café customers but not students (p < 0.001, p = 0.1, respectively).
Figure 1 Family attitude towards participants (café customers, university students) waterpipe smoking showing more tolerant attitude for women's waterpipe smoking compared to men's. This unique observation marks the first incident in this region where a smoking method by women is more tolerated than by men.
Figure 2 Self-perceived dependence among waterpipe smokers according to their frequency of waterpipe use (café customers compared to students). It shows that dependence is related to frequency of use, and that at comparable level of use, established smokers among café customers are more likely to perceive themselves as being hooked on the waterpipe compared to students.
Finally, both mean education years and DI differed significantly between café customers (mean education years12.5 ± 3.6; mean DI 1.2 ± 0.6) and students (mean education years 14.7 ± 1.4, mean DI 2.0 ± 0.7) (p < 0.01 for both).
Discussion
Obviously café customers are different from university students in many attributes that can reflect on waterpipe smoking and attitudes towards it. The longer duration of waterpipe smoking of café customers compared to students suggests that these two groups have been sampled at different stages of their waterpipe smoking practice. As such the two studied groups can provide an opportunity to look at features relevant to initiation and maintenance of waterpipe smoking. Such information can advance our understanding of this unique and increasingly popular smoking method and aid the design of intervention strategies. Generally, it looks that smoking patterns and quit attitude are mostly shaped by the stage of smoking, while beliefs and knowledge related to waterpipe tend to follow a more subtle roots likely reflecting cultural and socio-economic attributes.
The first important characteristic that differentiates waterpipe use patterns in the two studied groups is that daily use was much higher among café customers practiced by about a quarter of waterpipe smokers. This is likely to reflect the time-course of waterpipe smoking, with regular use becomes more noticed as duration of smoking increases. In fact duration of waterpipe smoking was correlated to frequency of smoking (daily, weekly, and monthly) among café customers. Still, unlike cigarette smoking, which is practiced mainly on a daily basis in the two studied populations and in the Syrian society in general [13], intermittent use seems to be the predominant pattern of waterpipe smoking. This observation is supported by data from other countries in the EMR highlighting the unique social dimension of waterpipe use [4,14]. However, with average smoking bout of more than one hour and the potentially high levels of different toxicants in waterpipe smoke (arsenic, cobalt, chromium, lead) [7], even intermittent use can be associated with significant health hazards to smokers.
Age of initiation of waterpipe differed significantly between men and women for both studied groups (analysis not shown). This may explain why the proportion of women among café customers is double that of students, although one should bear in mind the limitation of the samples for such a comparison. We have to be mindful as well that unlike cigarette smoking we are still at the expansion stage of the waterpipe epidemic, where people of different age groups are joining the new hype at relatively the same time period. An indication of this fact is that while age of initiation of waterpipe differed between the two studied groups, age of initiation of daily cigarette smoking was somehow identical. The tolerant attitude towards waterpipe smoking by women can aid its spread among them (Figure 1) [3,15]. Being perceived as closer to the local traditions, waterpipe smoking may escape the societal taboos of cigarette smoking by women in the EMR [15,16].
The social dimension, which is a salient feature of waterpipe smoking, seems also to differ between the two studied groups. So while students mainly initiate and currently practice the habit with their friends, more smokers among café customers are initiating or currently practicing the habit with family members or alone, despite being sampled in a social setting. It appears that the social context of waterpipe smoking changes with the change of both personal and smoking related characteristics (marriage, increased dependence). Our previous analysis of factors related to frequency of waterpipe use, as a marker of dependence, has lead us to suggest that as waterpipe smokers become more dependant the social pattern of smoking is gradually replaced with more individual one [9]. Sharing the same waterpipe and seasonal increase of waterpipe smoking, another important features of waterpipe smoking among students, were less noticed among café customers. It is likely that as waterpipe smokers move from the phase of experimentation to regular use they become more dependant, as such the habit is increasingly practiced in a way to satisfy dependence rather than as a pure social utility. Noticeably, at comparable level of use (daily) café customers perceive themselves to be more hooked on waterpipe than students (Figure 2).
Waterpipe quit attitude and perceived challenges to quitting differed between café customers and university students, with café customers demonstrating lower interest in quitting and lower perception of challenges to quitting (Table 3). This can be related to the fact that while students waterpipe smokers come mainly from cigarette smokers (with higher cigarette smoking levels compared to their peers, 3), waterpipe smokers among café customers seem to be more smoking method-oriented (with lower cigarette smoking levels than that of adults in Syria, 13). The fact that ex-cigarette smoking did not differ between the two groups supports this argument. In addition, café customers were in disagreement with students about which smoking method is more harmful, favoring the waterpipe (less harmful). Finally, waterpipe smokers in cafés seem to be more hooked on this smoking method compared to students, and dependence among café customers was inversely related to their willingness to quit [17]. Thus, the difference of quit attitude between the two groups is likely to different stage and orientation along waterpipe smoking practice. The predominance of intermittent use among both groups on the other hand, may have created false perception of easiness of quitting waterpipe. This is likely to be a false perception as about two thirds of those willing to quit waterpipe in both groups made an unsuccessful quit attempt last year. In fact unlike the long held belief linking dependence and difficulty in quitting to daily/frequent use, new studies among youth cigarette smoking suggests that dependence and difficulty in quitting can develop at low levels of consumption [18,19].
As mentioned above a limitation of this study is that the café sample cannot be considered representative of adult smokers in the community. Also, the study consists of two cross sectional surveys in two populations presumably at two different stages of their waterpipe practice. Still, the two groups are not totally distinct from each other as they overlap in terms of age and smoking characteristics. However, as noted from the discussion we are mindful of these limitations throughout analyses and interpretation of the study's results.
This study shows the predominance of intermittent use of this smoking method as well as its increasing popularity among women who are drawn to the habit relatively later than men. The social context of waterpipe smoking, which is a defining feature of this smoking method, tends to change to accommodate smoker's life and dependence progress. Our data suggest that at early stages of waterpipe use, users tend to come from cigarette smokers or people with liberal attitude towards smoking in general, but at more advanced stages of use smokers tend to be more smoking method-oriented and less keen on quitting. There is a general belief of easiness to quit waterpipe compared to cigarette, but this is likely to be an under-estimation as it is not translated into high success rate of quit attempts. Intervention or prevention strategies to curb this emerging epidemic should take into consideration the unique use features of this smoking method as well as smokers' perceptions and attitudes towards it.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
TA designed the study and wrote the first draft. KDW and TE participated in the study design and co-authored the manuscript. WM participated in the study design and wrote the final draft.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This study is supported by USPHS grant R01 TW05962
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| 15733316 | PMC553967 | CC BY | 2021-01-04 16:28:55 | no | BMC Public Health. 2005 Feb 25; 5:19 | utf-8 | BMC Public Health | 2,005 | 10.1186/1471-2458-5-19 | oa_comm |
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BMC Med EducBMC Medical Education1472-6920BioMed Central London 1472-6920-5-81572369910.1186/1472-6920-5-8Research ArticleDetermining the quality of educational climate across multiple undergraduate teaching sites using the DREEM inventory Varma Rajesh [email protected] Ekta [email protected] Janesh K [email protected] Academic Department of Obstetrics and Gynaecology, Birmingham Women's Hospital, Birmingham, B15 2TG, UK2005 21 2 2005 5 8 8 26 11 2004 21 2 2005 Copyright © 2005 Varma et al; licensee BioMed Central Ltd.2005Varma et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Our obstetrics and gynaecology undergraduate teaching module allocates 40–50 final year medical students to eight teaching hospital sites in the West Midlands region. Based on student feedback and concerns relating to the impact of new curriculum changes, we wished to objectively assess whether the educational environment perceived by students varied at different teaching hospital centres, and whether the environment was at an acceptable standard.
Methods
A Dundee Ready Education Environment (DREEM) Questionnaire, a measure of educational environment, was administered to 206 students immediately following completion of the teaching module.
Results
The overall mean DREEM score was 139/200 (70%). There were no differences in the education climate between the teaching centres.
Conclusion
Further research on the use of DREEM inventory, with follow up surveys, may be useful for educators to ensure and maintain high quality educational environments despite students being placed at different teaching centres.
==== Body
Background
The undergraduate curriculum at our medical school was redesigned in 1998/99 to bring it in line with recommendations suggested by the General Medical Council (GMC) in Tomorrow's Doctors [1]. Obstetrics and Gynaecology is taught as a final year module. Around 20–30 students, of a total year group of around 200 students, are allocated to eight teaching hospital sites in the West Midlands region, and remain their for the length of the module (eight weeks). Throughout the placement, all formal lectures take place at the principal Teaching Hospital (Birmingham Women's Hospital). A comprehensive course handbook and web-based multiple choice formative assessment accompany the module, and detail the teaching, practical and assessment objectives for students and clinicians. We have aimed to ensure there are no significant differences in the way the curriculum is delivered between centres. All 200 students sit the final exam in Obstetrics and Gynaecology straight after completing the 8-week course module.
Based on previous student feedback reporting differences in educational experiences, together with our concerns relating to the impact of new curriculum changes, we wished to objectively assess whether the educational environment perceived by students varied at different teaching hospital centres, and whether the environment was at an acceptable standard. In particular, was there any potential loss of teaching experience when students were placed away from the principal Teaching Hospital. Thus, the null hypothesis we wished to test was that there was no difference in the learning environment between centres. Several questionnaire-based educational tools are available that set out to 'quantify' the educational environment [2-4]. However, we chose to use the Dundee Ready Education Environment Measure (DREEM) inventory, as more studies had evaluated and validated this method [5,6]. The DREEM inventory consists of 50 questions, each scoring 4, giving a total maximum individual DREEM score of 200. The five domains that comprise the DREEM are depicted in Table 1.
Table 1 Domains of DREEM questionnaire
TOPIC Number of questions Maximum DREEM Score
Students' Perception of Learning 12 48
Students' Perception of Teachers 11 44
Students' Academic Self-Perceptions 8 32
Students' Perception of Atmosphere 12 48
Students' Social Self-Perceptions 7 28
Total 50 200
Methods
The DREEM questionnaire, based on a Likert scale, was administered to the full class of 206 final-year Birmingham University medical students undertaking the exam module in Obstetrics and Gynaecology in 2000. All questionnaires were distributed and returned the same day of the exam, which allowed us to achieve a 100% response rate. Students were told to only comment on their recent 8-weeks experience of Obstetrics and Gynaecology. Statistical analysis was performed using Microsoft Excel and Arcus Quickstat Biomedical Statistical software, and utilised single-sample T test and One-way analysis of variance (ANOVA).
Results
The year group comprised 42% male and 58% female. The overall mean DREEM score for the study group was 139/200 (95% CL 136.1 to 141.9), or expressed as percentage of the maximal score, 70% (95% CL 68% to 71%). There was no statistically significant difference between the mean scores for the contributory DREEM domains, which were as follows: perception of learning, 34.52/48 (72%); perception of teaching, 32.05/44 (73%); academic self-perception, 19.46/32 (61%); perception of atmosphere, 34.07/48 (71%), and for social self perceptions, 18.90/28 (68%). The DREEM scores for each hospital, with comparison of all contributory elements of the DREEM inventory, are depicted in Table 2 and Figure 1.
Table 2 The DREEM domains and overall score for each hospital
HOSPITAL Number of Students LEARNING Mean Score/48 TEACHERS Mean Score/44 ACADEMIC SELF-PERCEPTION Mean Score/32 ATMOSPHERE Mean Score/48 SOCIAL Mean Score/28 OVERALL DREEM Score/200 DREEM percentage for each hospital
(total of 206) Percentage of maximum score Percentage of maximum score Percentage of maximum score Percentage of maximum score Percentage of maximum score Percentage of maximum score
BWH 53 33.77 31.89 19.77 33.40 19.32 138.15 69%
Good Hope 20 33.30 30.10 18.15 33.40 18.20 133.15 67%
B'ham Heartlands 26 34.15 32.73 18.92 32.77 19.58 138.15 69%
Walsall Manor 20 34.10 34.35 19.90 34.30 19.05 141.70 71%
City 32 35.13 28.31 19.41 34.97 17.75 135.56 68%
Wolverhampton 22 35.77 32.59 20.41 33.64 18.86 141.27 71%
Shrewsbury 13 34.77 32.85 18.69 35.00 19.15 140.46 70%
Wordsley 20 35.15 33.60 20.40 35.10 19.30 143.55 72%
Mean overall 34.52 72% 32.05 73% 19.46 61% 34.07 71% 18.90 68% 139.00 70%
Lower 95% CL 33.83 70% 30.41 69% 18.77 59% 33.33 69% 18.38 66% 136.13 68%
Upper 95% CL 35.21 73% 33.69 77% 20.14 63% 34.82 73% 19.42 69% 141.88 71%
CL Confidence Limit
Figure 1 Graphical representation of the contribution of each DREEM domain to the overall mean DREEM score
When converting the raw DREEM score to percentages, two-sided P-value single-sample Student's T test showed no statistically significant difference between hospitals by each DREEM domain, or between each DREEM domain within the same hospital. Greatest variation between hospitals occurred in the Students' Perception of Atmosphere domain, where there were four hospitals beyond the 95% Confidence Limits; this compared to three hospitals beyond 95% Confidence Limits in all other DREEM domains (Table s2). One-Way analysis of variance (ANOVA) yielded F (variance ratio) = 0.5222, P = 0.8111, which indicated no statistically significant differences between hospitals, DREEM domains, or overall DREEM scores (Table 3).
Table 3 ANOVA analysis between different hospitals for the differing DREEM domains
Percentage of maximum score for each DREEM component for each hospital
Birmingham Women's Good Hope Birmingham Heartlands Walsall City Wolver-hampton Shrewsbury Wordsley
Learning 70% 69% 71% 71% 73% 75% 72% 73%
Teachers 72% 68% 74% 78% 64% 74% 75% 76%
Academic 62% 57% 59% 62% 61% 64% 58% 64%
Atmosphere 70% 70% 68% 71% 73% 70% 73% 73%
Social 69% 65% 70% 68% 63% 67% 68% 69%
One-way analysis of variance (ANOVA) yielded F (variance ratio) = 0.5222, P = 0.8111.
Discussion
We have used the Dundee Ready Education Environment Measure (DREEM) in 'diagnosing' the educational environment of eight different teaching centres and making comparative analysis between these centres. The overall mean DREEM score was 139/200, or expressed as a percentage, 70% (95% CL 68–71%). The educational learning environment did not vary between centres. The two lowest scoring contributory domains, academic self-perception (61%) and social self-perceptions (68%), were not statistically significantly different from the other three DREEM domains or overall mean DREEM score.
This study has benefited by using an established educational measure and obtaining a 100% response rate. No students had been previously taught at the principal teaching hospital as this was solely used for Obstetrics and Gynaecology teaching. However, some of the students (surveyed to be 16/206, 8%) had previously attended the other seven teaching hospital centres due to prior clinical teaching attachments. Thus, previous experiences may have biased the teaching assessment completed by some students. Furthermore, the DREEM questions are of such a nature that it is likely that the environment of the entire curriculum was being assessed. However, by performing the DREEM survey immediately at the end of the obstetrics and gynaecology module, and emphasising reporting only the last eight weeks experience, we believe this maximised the chance that the DREEM measure assessed only the recent hospital teaching site and minimised any recall bias. Other groups [7] have highlighted the potential flaws in using means and parametric statistical tests on ordinal data from Likert scales. As there is no firmly established consensus, we adopted to use the Student's T test and ANOVA calculation to fulfil best statistical methodology.
The DREEM domains are unlikely to be independent variables, and may be less of an environment test but more of a measure of the overall motivation and learning attitude of the individual. The Course Valuing Inventory (CVI) score is made up of five domains: worthiness of learning experience, emotional awareness, personal development, cognitive enhancement and task drive. A recent study of first year medical students showed a correlation between higher Course Valuing Inventory (CVI) scores, female gender, stronger self-confidence as a learner, greater motivation to learn and higher DREEM scores [8].
There is no accepted agreement on what is an acceptable DREEM inventory score from published literature. Nevertheless, our DREEM score of 139/200 was higher than other reports. A study of final year medical students in Trinidad reported an overall mean DREEM of 109.9/200 [5]. A larger scale study, involving students from both final and earlier undergraduate training years, showed a DREEM score of 118/200 in a Nigerian medical school, and 130/200 in a Nepalese medical school [9]. Our higher score is reassuring, and is perhaps an indicator of better hospital teaching environment, the positive value of using a comprehensive course handbook, and the encouragement of formative self-assessment as guided by the course handbook and web-based package.
The non-significant differences between the DREEM domains and between hospitals were significant findings. This was conveyed to our tutors based at the various teaching centres as a positive and encouraging result. In practical terms, this meant that regardless of hospital capacity or student group size, their education delivery and environment was no different to other centres in the student's curriculum. The DREEM inventory may thus be a useful tool for educators to ensure and maintain high quality educational environments and uniformity in educational delivery despite students being placed at different teaching centres.
Competing interests
The author(s) declare they have no competing interests.
Authors' contributions
RV and ET carried out the statisitcal analysis, data interpretation, and drafted the manuscript. JKG conceived and coordinated the study, acquired the results, and made revisions of the manuscript. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
RV is funded by the MRC, UK; JKG is employed by Birmingham University; ET works voluntarily as a clinical attachment.
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Bassaw B Roff S McAleer S Roopnarinesingh S De Lisle J Teelucksingh S Gopaul S Students' perspectives on the educational environment, Faculty of Medical Sciences, Trinidad Medical Teacher 2003 25 522 526 14522676 10.1080/0142159031000137409
Till H Identifying the perceived weaknesses of a new curriculum by means of the Dundee Ready Education Environment Measure (DREEM) Inventory Medical Teacher 2004 26 39 45 14744693 10.1080/01421590310001642948
Jamieson S Likert scales: how to (ab)use them Med Educ 2004 38 1217 1218 15566531 10.1111/j.1365-2929.2004.02012.x
Sobral DT Medical students' self-appraisal of first-year learning outcomes: use of the course valuing inventory Med Teach 2004 26 234 238 15203500 10.1080/0142159042000192028
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| 15723699 | PMC553968 | CC BY | 2021-01-04 16:30:55 | no | BMC Med Educ. 2005 Feb 21; 5:8 | utf-8 | BMC Med Educ | 2,005 | 10.1186/1472-6920-5-8 | oa_comm |
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BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-6-311571323310.1186/1471-2105-6-31Research ArticleAutomated generation of heuristics for biological sequence comparison Slater Guy St C [email protected] Ewan [email protected] The Ensembl Group, EMBL – European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SD, UK2005 15 2 2005 6 31 31 15 9 2004 15 2 2005 Copyright © 2005 Slater and Birney; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Exhaustive methods of sequence alignment are accurate but slow, whereas heuristic approaches run quickly, but their complexity makes them more difficult to implement. We introduce bounded sparse dynamic programming (BSDP) to allow rapid approximation to exhaustive alignment. This is used within a framework whereby the alignment algorithms are described in terms of their underlying model, to allow automated development of efficient heuristic implementations which may be applied to a general set of sequence comparison problems.
Results
The speed and accuracy of this approach compares favourably with existing methods. Examples of its use in the context of genome annotation are given.
Conclusions
This system allows rapid implementation of heuristics approximating to many complex alignment models, and has been incorporated into the freely available sequence alignment program, exonerate.
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Background
George Box [1] once remarked that,
"All models are wrong, but some are useful."
This statement bears much relevance to biological sequence alignment, where there is no guarantee that the alignment model will accurately represent the evolutionary (or other) processes which separated the two sequences. All that is certain is that exhaustive dynamic programming (DP) algorithms (such as Smith-Waterman [2]) will yield the optimally scoring path in terms of the given model. Heuristics for sequence comparison (such as BLAST [3]) generate alignments which are valid paths through the underlying alignment model, but are not guaranteed to be optimal. Alignments generated by these heuristics can be calculated much more rapidly, and often closely match alignments which would be generated by exhaustive methods. Furthermore, many problems in the context of genome analysis consist simply of alignment of gene products (cDNA or protein) back onto the gene from which they were produced, and consequently do not require very sensitive alignment. Hence the aim here is not to attempt to develop models which are correct, but to facilitate development of models which are more useful in the context of large scale analyses.
Transformation between a finite state automata describing an alignment model and the recurrence relations used in DP is a powerful technique [4,5] as it allows modification of the alignment algorithm by direct manipulation of the alignment model. The Dynamite compiler [6] allows automated implementation of alignment algorithms from a description of the alignment model. This allows development of complex models which can be used to generate accurate alignments. However, calculation of alignments using these models always requires quadratic time, which is prohibitively slow for many large scale applications. This method can be applied to any alignment problem which can be represented by a regular grammar (or the equivalent finite state machine). This includes the simple three state model required for affine gaps in the Smith-Waterman-Gotoh algorithm [2,7], but also more complex models such as that used by EST_GENOME [8], where splice site prediction is integrated into the DP, allowing alignments to include introns. Other alignment models which may be expressed by a regular grammar include those allowing non-equivalenced regions such as the ABC model [9] for improved modelling of divergent loops regions in protein alignments, and DNA Block Aligner [10] which finds co-linear conserved blocks in the alignment of genomic sequences. This framework also allows models such as that used by PairWise [11] where the sequences are translated during alignment allowing for frameshifts, and GeneWise [12] which integrates translated alignments with modelling of introns for alignment of proteins against genomic DNA.
The availability of vast amounts of sequence data has generated a need for faster alignment algorithms [13]. Many of these approaches use fast algorithms to identify closely matching words which seed un-gapped alignments that are subsequently joined to form the final gapped alignment. For example, BLAST [3], FASTA [14], and sim4 [15] all operate by first finding matching words before building alignments. Such word finding can be done by a multitude of techniques, such as finite state machines used in BLAST [3], table-lookup used in SSAHA [16], or by suffix arrays as used in QUASAR [17]. Novel methods for word-based seeding of these alignments are not presented here, but rather a general system of joining these seeds to produce gapped alignments.
The alignment program FASTA [14], generates alignments from sets of seeds by performing DP confined to a diagonal band surrounding the initial matches [18]. In contrast, when building alignments, Gapped BLAST [19] permits gapped extension which allows the DP to be applied to an arbitrary high-scoring region surrounding the HSP seeds. Such heuristics allow very fast sequence alignment, but it is difficult to apply to more complex models. Furthermore, features such as introns cannot easily be incorporated into the resulting alignments without necessitating a large amount of DP to ensure that both very short exons and large introns can be included in alignments.
The aim here is to combine the strengths of Gapped BLAST and Dynamite, in a system which allows automated generation of heuristics in terms of the underlying model. This allows the development of heuristics for complex models such as those used in GeneWise.
Firstly, we describe bounded sparse dynamic programming (BSDP), a novel heuristic for sequence alignment, then we describe the system for automated implementation of the complex alignment algorithms required. We present proof of principle results, but this paper is primarily focussed on developing a framework for the general case.
Implementation
The basic strategy of seeding alignments used here is the same as for BLAST, in that alignments seeds are generated, and then extended to form High-scoring Segment Pairs (HSPs), which are then joined together to form the alignments. The alignments are seeded using an Aho and Corasick [20] type finite state machine (FSM) built using the word neighbourhood of the query sequence. This generates the seeds which are extended to form the HSPs. For large scale analyses, the FSM is multiplexed using word neighbourhoods from multiple sequences. This allows analysis of multiple queries during a single pass of a genomic database, in a manner similar to that used in MPBLAST [21].
However the methods for seeding HSPs are independent from those used for building the alignments, and this paper only deals with algorithms involved in the generation of gapped alignments from sets of HSPs, and not in the calculation of the HSPs themselves.
The following strategies are employed to enable alignments to be built efficiently from sets of HSPs:
• To connect the underlying alignment model to the heuristics, a portal describes a set of states in the model which correspond to a set of HSPs, a span refers to a looping state for large alignment features such as introns, and a SAR (Sub-Alignment Region) describes a rectangular region on an HSP to which DP is applied.
• To avoid DP in every SAR, upper bounds are generated for the best alignment score for each SAR, and BSDP (Bounded Sparse Dynamic Programming) exploits these bounds to yield alignments in an efficient manner.
• To perform various types of DP in these SARs, the required models are generated automati cally, including C code to produce an efficient viterbi implementation for each model.
Bounded sparse dynamic programming
Dynamic programming (for any alignment model which can be represented by a regular grammar) requires quadratic time, and hence is the most computationally expensive part of building an alignment. For pairs of sequences more than a few kilobases long, DP becomes prohibitively slow. The approach used here is similar to sparse dynamic programming [22], and the fragment chaining approaches used in the program sim2 [23], in that DP is applied to rectangular regions surrounding alignment seeds. However, there are two major differences in our approach. Firstly, DP is only applied to two small discrete regions on each HSP, as it is assumed that most of the HSP itself should appear in the alignment. These sub-alignments improve the quality of the overall alignments, and they allow complex alignment models, and large gaps such as introns to be integrated into a sparse DP framework. Secondly, as it would take too long to apply DP to every sub-alignment region (SAR), upper bounds are calculated for the DP scores for each of these SARs. This allows the sub-alignment DP to be avoided in cases where it joins HSPs which cannot feature in the final alignment, so that alignments can still be generated very rapidly even when large numbers of HSPs and SARs are involved.
Before the BSDP can be performed, a single point on each HSP is selected which will feature in any alignment generated using that HSP. This point corresponds to a pair of equivalenced symbols which must feature in any alignment to include that HSP. A point is chosen where half the HSP score is generated by equivalenced symbols on either side of it, as this is likely to be in the highest quality portion of the HSP. As shown in the example in Figure 1, this strategy is particularly beneficial where one end of the HSP has a much lower quality than the other.
The five types of region used for sub-alignments are classified in Figure 2. Each of these require a slightly different alignment algorithm. The alignment path must meet corners of the SARs that contain an HSP, so that the sub-alignments can be integrated with the HSPs to produce the final alignment. This approach has been primarily designed for local models, but BSDP may also be used for global and semi-global models, in which case constraints are added such that both the terminal regions (cases A and E in Figure 2) and the resulting sub-alignments must contain the relevant sequence ends. In the case of C and D, the two HSPs and their SARs are separated by a span, allowing large gaps or introns in the alignment. In these cases DP must be applied to the SAR before the span, and the end state scores must be integrated in an intermediate matrix before being provided as start state scores for the DP in the SAR after the span.
Regions for the sub-alignments are selected within the area between the centre points of the two HSPs to be joined, or in the case of terminal HSPs, between the HSP and the ends of the sequences. In addition, the positions of the SARs must be constrained to limit their size, and so that the HSPs correctly intersect with the corners of the SAR. In the case of overlapping HSPs, where there is a choice of positions for placement of the SAR, the position is chosen such that the highest scoring parts of the HSPs are outside the SARs.
Once the SARs have been selected, an upper bound is placed on the score for each sub-alignment. The calculation of these bounds is described in a later section describing automation of this method. The BSDP approach can then utilise these upper bounds to avoid application of DP in some SARs, as demonstrated by the example in Figure 3. In the case of a real alignment, a much greater number of HSPs will be involved, so the amount of DP avoided will be larger.
The BSDP is mediated through a set of priority queues, one of which is associated with each HSP, and will contain an entry for each partial alignment that ends at the HSP. The key for these priority queues is the highest score for any partial alignment ending in that HSP. Additionally, there is one global priority queue containing the highest scores from each of the other priority queues. The upper bound scores are confirmed by DP in the SARs in the current highest scoring putative alignment path. The highest scoring path will change as the scores are updated during this process. DP is applied to SARs in this way until the highest scoring path does not contain any bound scores, and then the alignment may be extracted. Upper bounds dictate that there can be no better alignment using these HSPs.
This algorithm is similar to A* search, (but differs in that many different points may be the start or end of the search), and retains the admissibility property of A* search, such that the result of the BSDP computation is guaranteed to be the same as if DP had been performed on every candidate SAR prior to calculation of the alignment. This is because no alignment can be extracted until all the alternative sub-alignments (which have upper bounds that indicate they could contribute to a higher-score) have been eliminated.
Suboptimal alignments
The BSDP alignment process can be iterated to generate sub-optimal alignments similar to those generated by the Waterman-Eggert algorithm [24], with only minimal recalculation of the partial alignments in the SARs. Each HSP may only appear in a single alignment, but further constraints are required to prevent overlapping alignments arising from overlapping SARs. The likelihood of this is occurring is greatly increased during translated alignments when HSPs in different reading frames may overlap each other.
After the first alignment has been reported, the scores for any SARs which have already been confirmed by DP, but which are not yet included in a reported alignment, are then considered as an upper bound. SARs are disallowed before recalculation when the region between the centre of the HSP and its SARs overlaps with a previously reported HSP, in which case, the SARs are disallowed. Otherwise the DP is recomputed for SARs which contain part of an alignment which has been reported since the DP for the SAR was last calculated.
Automated model generation
As illustrated in the previous section, BSDP becomes quite complex and requires a large number of DP algorithms for computation of the alignment through the SARs. We have build a system to facilitate implementation of these models and the integration of the sub-alignments which they produce. To allow generalisation of the BSDP, everything must be defined in terms of the underlying alignment models. The alignment models are described as finite state machines, consisting of states and transitions, similar to those used in Dynamite [6].
Briefly, to convert these models into DP implementations, each state must correspond to a score in each cell of the DP matrix, and the scores for each cell are calculated by taking the maximum of the score from transitions arriving at each cell. In addition, a topological sort is required to satisfy dependency ordering for silent states.
However, in addition to automated generation of code from alignment models, the generation of the models required for DP in the SARs is also automated, as described below.
Building simple models
Table 1 shows a few example alignment models which are generated by this system. The models are built in a modular fashion, allowing reuse of common components such as intron models and gap models. These models may be used for exhaustive alignment in quadratic time, but in order to use them for heuristic alignment, manipulations of the models are necessary to perform DP in the SARs, as detailed in the following sections.
Building the heuristic model
To enable DP in the SARs for the BSDP, a heuristic model is generated from the original alignment model. This model is not used directly for calculation of alignments, but a derived model is generated corresponding to each transition in the heuristic model. Each of these derived models correspond to a type of SAR used in the BSDP.
The model is first annotated, labelling certain states as either portal states or span states. A portal defines a group of states which can share a set of HSPs (High-scoring Segment Pairs); these are the match states. A span is a state which has sequence independent looping transitions (e.g. states for introns, or non-equivalenced regions).
The heuristic model is build using states corresponding to each portal and span state, with transitions between these states in cases where there is a valid path between the corresponding states in the original model. An example model is shown in Figure 4 for alignment of ESTs to genomic DNA. In this example, there is a portal which corresponds to the match forward and match reverse states, and the intron forward and intron reverse states are span states.
Building derived models
Derived models are produced for the DP in SARs automatically from each transition in the heuristic model. The source and destination states from each transition in the heuristic model become the start and end states in each derived model. All reachable states and transitions from these states in the original model are recursively copied to the derived model. An example of this process is shown in Figure 5. The relationships between the states and transitions in the derived model and the original model are tracked to allow conversion of the partial alignments from the derived models back to complete alignment in the original model. Terminal models (case A and E in Figure 2) are generated between portal states and a start or end state. Join models (case B in Figure 2) are generated between portal states, including from a portal state to itself. Span models (case C and D in Figure 2) are generated from a portal state to a span state, and vice versa. These allow incorporation of a large feature such as an intron into an alignment. The span models require a special end state in the model at the start of the span, and a special start start in the model at the end of a span, so that scores can be transferred from one DP matrix to the other via an intermediary score matrix.
For some cases, such as between the match forward and match reverse states, shown in Figure 4, there is no possible path, and no corresponding transition in the heuristic model, in which case, a derived model is not produced.
Ten different derived models are generated from the model shown for cDNA to genomic alignment in Figure 4, because there are two portal states and two span states in this model, and therefore, a derived model is generated for each of the five cases in Figure 2 for both the forward and reversed genes.
Building models for calculation of upper bounds
For each derived model, an additional model is created which is used for the pre-calculation of upper bounds for all possible sizes of SARs. For each transition in the model which has a position dependent score associated with it, the upper bound is also supplied. For example, in a match transition, the upper bound is the maximum value from the substitution matrix. A special model is created using these transition upper bounds, instead of the normal sequence-dependent transition weights. As this removes the sequence-dependent components of the algorithm, it allows pre-calculation of the upper bound for alignment score of any sequences up to the maximum permitted size of SARs. These bounds are then stored in a table for retrieval during the BSDP.
Results
The model used in Figure 4 was used with this system in the program exonerate for rapid comparison of cDNAs and genomic DNA. This model was used to compare a test set of genomic sequences to mRNAs extracted according to the Ensembl annotations. exonerate was compared to sim4 [15], another heuristic for comparing cDNAs to genomic DNA, and EST_GENOME [8], which is implements the exhaustive DP for essentially the same underlying model. As can be seen from the results of this comparison are shown in Table 2, similar results are produced by this method, but in much less time.
This system is used for alignment of ESTs within the Ensembl gene-building pipeline [25], and a prototype implementation of this system has been used in comparison mouse and human sequences [26]. In this analysis, 13 million raw shotgun mouse reads were aligned as part of the comparative analysis of chromosome 20.
Figure 6 shows an example the SARs generated by BSDP. Only a small proportion of these SARs will require confirmation by DP. This approach scales well – in a larger example using Titin, 27346 candidate SARs are generated, of which only 236 are confirmed and 206 appear in the final alignment. In many cases, BSDP obviates the use of DP altogether as there will be no candidate alignment with a total upper bound score over the threshold. This is especially likely to be true in cases when a high score threshold is used, such as searching for an alignment covering a certain percentage of the query, or when a dynamic score threshold is used in the search for the best few hits from a particular query to a database. Such dynamic thresholds can actually cause the search to speed up as it progresses, once good matches to the query have been found.
Another example is the alignment of the Collagen alpha 1(IX) precursor to a region of chromosome 6 containing the corresponding gene. This is a large gene, containing 38 exons over about a 90 Kb region of the genome. Using GeneWise, the alignnment required 4260 seconds (about 1 hour, 11 minutes) using exonerate to perform full DP alignment required 2700 seconds (about 45 minutes), and using exonerate with the heuristic BSDP approach required only 7 seconds, with all three methods generating identical alignments.
Conclusion
This approach represents an advance from previous methods for automatic implementation of sequence alignment algorithms [4-6], in that it is not just the generation of code from the models which is automated, but also the generation of many of the models themselves.
This has allowed development of heuristics with sub-quadratic running times. This makes their application practical to a much larger set of problems, while retaining the much of the simplicity of their implementation.
We recognise that this approach is limited to the subset of sequence comparison problems which can be represented by a regular grammar. For example, it is not possible to use this system to model stochastic context free grammars as used in some types of RNA secondary structure such as pseudo-knots [27]. This approach is also unsuitable for the types of DP algorithms used for determining optimal segmentation in gene finding algorithms [28], however for these types of problems running time of the algorithm is not an issue.
This framework has already been used for many different alignment models ranging from simple models such as Smith-Waterman-Gotoh, to more complex models such as those for protein to genome alignment as used by GeneWise [12]. It is also well suited to problems requiring comparison of very long sequences, and we are currently extending this system to accommodate syntenic pairwise comparisons of genomic sequences of the type tackled by MUMmer [29].
Although this method is useful in many cases, for some types of distantly related sequences, the method can breakdown. For example, when long regions of the correct alignment contain many gaps with intervening sequences too short to be an alignment seed, the alignment cannot be extended beyond the boundary of the SAR, and the correct alignment may be missed. These cases can be avoided by increasing the size of the SARs, but this results in higher bound scores, so DP become necessary in more of the SARs. Such gap rich alignment are the type where the gapped HSP extension used by Gapped BLAST excels, however such gapped extension necessitates DP surrounding all HSPs, which becomes time consuming when allowing both short exons and long introns during alignments of cDNAs or proteins to genomic sequences.
As this system separates the development of the underlying alignment model from the heuristics which are built on top of them, we expect that this framework will prove useful for evaluation of the quality of the heuristics, as comparison between the alignments from the two techniques can be automated, and training may be performed to select optimal parameter sets.
Availability
The implementation of the system described here is called C4 (to reflect the aim of producing something more powerful than Dynamite [6]), and is implemented in C programming language. It is available as part of the exonerate sequence alignment package available from , and in the exonerate module of the Ensembl CVS repository from . It is built using the glib portabilility library available from Both exonerate and glib are available under the GNU lesser general public license. The code has been tested extensively on Linux and OSF1/alpha but has been written to be portable to many UNIX systems.
Code generation for the DP is performed to exploit compile time optimisations such as loop un- rolling and and efficient handling of edge conditions in the viterbi matrix, which is particularly beneficial for the small DP calculations required by SARs. This generated code is typically about five times faster than using a generic viterbi implementation, but produces about a million lines of code for the current set of models used by exonerate, and hence compilation takes longer.
Acknowledgements
GSS would like to thank Ian Holmes and Roger Sayle for invaluable discussions (some several years ago), which have been a big influence on this work.
Also, we would like to thank all members of the Ensembl group, in particular Steve Searle contributed some code to an early version of exonerate.
Figures and Tables
Figure 1 Example of joining two HSPs. A single point on each HSP is chosen to be be included in the alignment. This is the centre of the HSP according to the scores, such that equivalenced bases on either side contribute to half of the HSP score. As shown in this example, if the centre of the HSPs according to their length had been chosen instead, it would not have been possible to join them to form an alignment.
Figure 2 The five types of sub-alignment region around HSPs in which dynamic programming may be applied. The start of an HSP (A), handling small gaps by joining two adjacent ends of HSPs (B), handling large gaps by joining two distant HSPs via a span (C,D) and the end of an HSP (E). Other features, such as splice sites are incorporated into the alignment within the SARs.
Figure 3 A toy example of bounded sparse dynamic programming. In this example the bounds indicate that if the overall alignment threshold is greater than 280, no sub-alignment DP is required. Otherwise, the region between A and B is confirmed first, and the bounds obviate DP between A and C. For clarity, terminal bounds are not included in this example.
Figure 4 Models for alignment of an EST to genomic sequnce. The exhaustive model for aligning ESTs to genome DNA, showing portal states and span states is shown in Figure 4 (a). The portal states are match forward and match reverse, and these share a set of HSPs. The span states are intron forward and intron reverse, and may accommodate large gaps. These portal and span states are represented in the heuristic model shown in Figure 4 (b), with each transition represented by a different derived model, labelled A to E, corresponding to the SAR types show in Figure 2.
Figure 5 Example of generation of a derived model. The original model on the left (for Smith-Waterman-Gotoh) is used to generate the derived model on the right for joining two adjacent HSPs. Extra transitions are allowed from the START state to the INSERT and DELETE states and onto the END state, as the derived model must allow gaps to open directly from the HSPs (in contrast to Smith-Waterman-Gotoh where an alignment cannot start with a gap). Additionally, the original model is local, but in this case the derived model is global, as it must start and end on the HSP.
Figure 6 Example of BSDP during alignment of a protein sequence to genomic DNA. Figure 6 (a) shows 912 candidate SARs positioned on the HSPs. Figure 6 (b) shows the 109 SARs to which DP is applied during BSDP, and Figure 6 (c) shows the 18 SARs which appear in the final alignment.
Table 1 Examples of hierarchical model building in C4. Instead of building each model from scratch, most models are created in a modular fashion by making adaptions and additions to other models. This facilitates the building of complex models.
Model Components States Transitions
ungapped substitution matrix 3 3
Needleman-Wunsch (NW) ungapped with gap penalties 3 5
Smith-Waterman (SW) NW + local alignment scope 3 5
Smith-Waterman-Gotoh (SWG) SW + affine gap costs 5 9
est2genome SWG + intron models 10 24
protein2dna SWG + translation + frameshifts 6 13
protein2genome p2d + intron model 13 33
Table 2 Results of an evaluation comparing exonerate (using the methods presented here), with sim4 and est2genome. A subset of the ensembl database was used, containing 1,827 genes, 9,917 exons and 2,387,971 bases. Times are given for alignment of all cDNAs to all contigs. The CPU time for est2genome was estimated from a smaller number of alignments. The accuracies are comparable, but the times are much faster for the heuristic methods.
Method Base Accuracy Exon Accuracy CPU Time (seconds) x-fold
est2genome 99.99% (2,387,648) 99.90% (9,907) ≅ 7.2 years (226,637,523) 1
sim4 98.97% (2,363,438) 98.12% (9,731) ≅ 60 hours (216,956) 1045
exonerate 99.88% (2,384,988) 99.98% (9,916) ≅ 46 minutes (2,577) 87946
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| 15713233 | PMC553969 | CC BY | 2021-01-04 16:02:49 | no | BMC Bioinformatics. 2005 Feb 15; 6:31 | utf-8 | BMC Bioinformatics | 2,005 | 10.1186/1471-2105-6-31 | oa_comm |
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BMC Cardiovasc DisordBMC Cardiovascular Disorders1471-2261BioMed Central London 1471-2261-5-61573331510.1186/1471-2261-5-6Case ReportAcute ST-segment elevation myocardial infarction after amoxycillin-induced anaphylactic shock in a young adult with normal coronary arteries: a case report Gikas Aristofanis [email protected] George [email protected] Kalliopi [email protected] Health Center of Salamis, Salamis, Greece2 Cardiology Department, 'Elpis' General Hospital of Athens, Athens, Greece3 Department of Allergology and Clinical Immunology, 'Laiko' General Hospital of Athens, Athens, Greece2005 25 2 2005 5 6 6 23 5 2004 25 2 2005 Copyright © 2005 Gikas et al; licensee BioMed Central Ltd.2005Gikas et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Acute myocardial infarction (MI) following anaphylaxis is rare, especially in subjects with normal coronary arteries. The exact pathogenetic mechanism of MI in anaphylaxis remains unclear.
Case presentation
The case of a 32-year-old asthmatic male with systemic anaphylaxis, due to oral intake of 500 mg amoxycillin, complicated by acute ST-elevation MI is the subject of this report. Following admission to the local Health Center and almost simultaneously with the second dose of subcutaneous epinephrine (0.2 mg), the patient developed acute myocardial injury. Coronary arteriography, performed before discharge, showed no evidence of obstructive coronary artery disease. In vivo allergological evaluation disclosed strong sensitivity to amoxycillin and the minor (allergenic) determinants of penicillin.
Conclusion
Acute ST-elevation MI is a rare but potential complication of anaphylactic reactions, even in young adults with normal coronary arteries. Coronary artery spasm appears to be the main causative mechanism of MI in the setting of "cardiac anaphylaxis". However, on top of the vasoactive reaction, a thrombotic occlusion, induced by mast cell-derived mediators and facilitated by prolonged hypotension, cannot be excluded as a possible contributory factor.
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Background
Acute myocardial infarction (MI) complicating anaphylaxis induced by drugs or other chemicals is uncommon and only sporadic cases have been reported [1-9]. The underlying pathogenetic mechanisms have not been fully elucidated. The case of a 32-year-old man with normal coronary arteries, who developed acute MI following amoxycillin-induced anaphylaxis, is the subject of this report. The underlying pathogenetic mechanisms are also discussed and the relative literature is reviewed.
Case presentation
A 32-year-old man was admitted to the emergency room of the local Health Center because of anaphylaxis, which developed 2 hours and 15 minutes after the ingestion of amoxycillin (500 mg), prescribed by his dentist. Prodromal signs of anaphylaxis (flushing, pruritus, warmth, urticaria) reportedly occurred about 15 minutes before the onset of symptoms from other systems. Ten days earlier the patient, an asthmatic since childhood, had completed a 4-day course of amoxycillin (500 mg TID) without any side effects. Apart from obesity, there were no other risk factors for coronary artery disease.
On admission the patient was in acute distress. He was complaining of dizziness, blurred vision, dyspnea and abdominal pain. Initial examination revealed an obese man (Weight = 130 kg, Body Mass Index = 38 kg/m2) with generalized erythema, angioedema, cyanosis and diffuse wheezing; the systolic blood pressure was 70 mmHg and the pulse rate 120 bpm. The patient was then connected to a cardiac monitor, which showed sinus tachycardia (approximately 140 bpm) without ST-segment and T wave abnormalities (Fig. 1A). Pulse oxymetry demonstrated an oxygen saturation (SpO2) of 90%.
Figure 1 Lead II monitor strip recorded shortly after admission (A) and at the chest pain onset (B).
Epinephrine (0.3 mg or 0.3 ml of a dilution 1:1000) was injected subcutaneously (SC); dimethindene (4 mg) and hydrocortizone (500 mg) were administered intravenously (IV) in slow infusion. Nebulized salbutamol and supplemental oxygen were given as well. Normal saline with 50 mg ranitidine hydrochloride and Ringer's solution were infused through separate intravenous lines.
Due to unsatisfactory clinical response (persistence of hypotension and tachycardia, despite improvement of the pulmonary signs) a second dose of epinephrine (0.2 mg) was given SC 20 minutes later. Almost simultaneously with the administration of the second dose of epinephrine, ST-segment elevation appeared on the monitor (Fig. 1B) and the patient complained of substernal chest pain. A 12-lead electrocardiogram (ECG) showed ST segment elevation in leads II, III, aVF, and V3 to V6 (Fig. 2). A single dose of 325 mg acetylsalicylic acid was given per os; heparin (5000 UI) and pethidine hydrochloride (25 mg) were administered IV. An hour later the patient was hemodynamically stable. The arterial blood pressure was 125/90 mmHg and SpO2 rose to 96%. However, the chest pain persisted and nitroglycerin titrated at a dose of 25 μg/min was infused IV, without complete symptom relief. At this point the patient was transferred to the Coronary Care Unit (CCU) of the nearest general hospital.
Figure 2 ECG recorded during chest pain.
Treatment in the CCU included thrombolysis with reteplase – administered according to the standard protocol – 2 hours after the onset of chest discomfort, which according to the established clinical and electrocardiographic criteria was considered successful [10]. The peak levels of serum troponin I and creatine phosphokinase were 45.5 ng/ml (normal = 0–2 ng/ml) and 575 U/L (normal = 25–195 U/L), respectively; while the MB fraction was 77 U/L (normal = 0–24 U/L). The rest of the laboratory results, including serum cholesterol, LDL, HDL, Lpa and triglycerides, were within normal limits. An ECG performed before discharge showed complete loss of potentials in leads III and aVF and partial loss of potentials with a small q wave in lead II (Fig. 3). An echocardiographic study performed on the 5th hospital day showed preserved systolic function (ejection fraction 60%) without wall motion abnormalities. Left (Fig. 4) and right (Fig. 5) coronary angiography showed no evidence of obstructive coronary artery disease; left ventriculography was normal as well. The patient recovered completely and was discharged a week after admission. He was referred to the adult Allergology Center of our area performing diagnostic work up for drug allergy.
Figure 3 ECG recorded on hospital day 4th.
Figure 4 Left coronary angiogram.
Figure 5 Right coronary angiogram.
Prick skin tests (PST) were performed using penicilloyl polylysine (PPL), and minor determinant mixute (MDM) [supplied as Allergopen by Allergopharma (Reinbeck, Germany)]; amoxycillin, ampicillin and cefamandole solutions were also used (concentration 20 mg/ml) for PST. PST were strongly positive [4+ reaction (on a scale of 1–4) i.e. wheal >5 mm in diameter with pseudopodes, with no reaction at all to the diluent control] to amoxycillin and ampicillin. Intradermal tests were performed only to PPL, MDM (dilution 1/10) and cefamandole (0.2 mg/ml and 2 mg/ml) and resulted in strongly positive reaction (4+) only to the MDM. Circulating specific IgE to penicillin V, penicillin G, amoxycillin, ampicillin and cefaclor was not demonstrable by CAP (Pharmacia, Sweden).
Discussion
Anaphylactic reactions may trigger cardiovascular events, including MI [1-9], primarily in subjects with underlying ischemic heart disease [1,2,4,7,9] and rarely in individuals with normal coronary arteries [3,5,6,8]. Cardiovascular complications in the setting of anaphylaxis rarely occur in subjects less than 35 years old [11].
Acute coronary syndromes following antibiotic-induced anaphylaxis is uncommon and only few cases have been reported so far [1,4,12-14]. To our knowledge, only one case of amoxycillin-induced anaphylaxis complicated with acute MI has been documented in a 62-year-old man with underlying coronary artery disease [4].
Evidence that the human heart is the target as well as the site of anaphylaxis is constantly growing. A number of studies by Marone et al [15,16] and others [17] have demonstrated mast cells within the human heart; they are strategically located perivascularly, in close proximity to myocytes, in the arterial intima and the shoulder region of atheromas. Cardiac mast cells express on their surface high affinity receptors for IgE (FcεRI) and also C5a receptors [18]. Mast cells activation through immunological and non-specific (non-immunological) stimuli leads to the release of a variety of vasoactive and pro-inflammatory mediators, preformed or de novo synthesized (histamine, prostaglandins, leukotrienes, enzymes, cytokines and others) [15,19]. Coronary spasm, which is proposed as the main underlying mechanism of allergy-induced coronary syndromes [5,11,13,14,20,21], can be caused, directly or indirectly, by potent vasoactive mast cell-derived mediators such as histamine, prostaglandin D2, thromboxane, cysteinyl leukotrienes [15,18,22-24]. In addition, mast cells are likely to affect coagulation and fibrinolysis at different levels through enzymes and mediators they secrete [15]. Therefore, mediators from perivascular and interstitial cardiac mast cells – as well as those reaching the heart from the pulmonary circulation – might affect coagulation, favoring platelet aggregation and thrombus formation [15,25].
In the present case, the temporal sequence of events suggests that cardiac anaphylaxis was the triggering factor of MI. We propose that prolonged coronary vasospasm induced by vasoactive and inflammatory mediators, released during anaphylaxis, was the main causative mechanism. However, in view of the persistence of chest pain following IV nitroglycerin infusion, and its complete resolution promptly after IV thrombolysis, a thrombotic vascular occlusion, on top of the vasospastic reaction, cannot be excluded. The latter is also supported by the prolonged systemic hypotension, which, as it has been emphasized in previously reported cases [26], probably caused further reduction of the myocardial perfusion, thus, favoring in situ thrombus formation and subsequent coronary artery occlusion. In acute MI cases, presenting to centers with cardiac catheterization facilities, urgent coronary arteriography appears as the management strategy of choice for the final diagnosis. However, in the setting of acute anaphylaxis, this might not be the wisest choice, particularly in cases, like the present one, in which the offending allergen had been administered orally. Moreover, continuous or delayed allergen absorption could further aggravate anaphylaxis, and a late phase reaction represents a potential risk in all anaphylactic reactions. Finally, no bibliographical data are available concerning the tolerability of IV administered contrast agents in patients who have suffered a recent episode of severe systemic anaphylaxis.
The administration of epinephrine – a life saving agent in cases of anaphylaxis – has been implicated as a cause of acute MI in a limited number of reports [27,28]. In the present case, however, it appears unlikely that exogenous epinephrine was the initiating event for the following reasons: a) the first epinephrine dose (0.3 mg) was rather low to induce significant vasoconstriction in a subject with a body weight of 130 kg, b) the mode of administration (SC) is considered the safest in this regard, c) the second dose (0.2 mg), administered at a safe interval (20 min) after the first one, does not seem to be involved since its injection coincided with the onset of chest pain, before any anticipated drug absorption. On the contrary, in the above quoted cases [27,28] the epinephrine-induced MI developed 5–15 minutes post injection. Moreover, the possibility of inadvertent intravenous administration of epinephrine appears improbable, since no blood was withdrawn in the syringe before drug injection. However, one might argue that the exogenous epinephrine could have aggravated a pre-existing coronary spasm, induced by mast cell-derived mediators. Hence, the precise effect of epinephrine in this clinical setting remains a matter of speculation.
The allergological evaluation performed in our patient showed strong sensitivity to amoxycillin and to the minor determinants, which are the allergenic epitopes associated with systemic anaphylaxis. The negative CAP results do not negate the in vivo findings, since it is well established that the in vitro techniques are not as sensitive as skin tests. Furthermore, CAP detects antibodies against the major determinants of penicillins (involved pathogenetically in penicillin-induced urticaria) and not to the minor ones, which are implicated in systemic anaphylaxis. For technical reasons, relating to both the Allergology Center and the patient's professional obligations as well as residence distance, the allergological work up was completed 6 months later; such a delay might also have contributed to the negative in vitro findings.
Conclusion
Acute ST-elevation MI is a rare but potential complication of anaphylactic reactions, even in young adults with normal coronary arteries. Physicians should be alert for such a complication in order to diagnose it early and treat properly.
It is fairly well established that that human heart can be both, the site and the target of severe anaphylaxis; in this setting cardiac mast cells – activated and releasing multiple vasoactive mediators – play an important role in pathogenesis of cardiac complications [16-18].
In the above case, mediator-induced coronary artery spasm was the main, but probably not the exclusive causative mechanism of anaphylaxis-related MI. The thrombotic vascular occlusion, induced by inflammatory mediators and facilitated by prolonged hypotension, cannot be excluded as a possible contributory factor.
Abbreviations
ECG = electrocardiogram; IV = intravenously; MDM = minor determinant mixute; MI = myocardial infarction; NV = normal values; PPL = penicilloyl polylysine; PST = prick skin tests; SC = subcutaneously.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
AG was responsible for the initial evaluation and management of the patient, GL was involved in the cardiological evaluation and management and, KK-F performed the allergological evaluation. All authors have equally contributed in the preparation and revision of the manuscript. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We thank the physician K. Lidatakis and the nurses A. Koulouri and I. Lountzi for their contribution in the first-hour management of the patient and the documentation of the case. We thank the patient for giving us a written consent for publishing his case.
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| 15733315 | PMC553970 | CC BY | 2021-01-04 16:30:06 | no | BMC Cardiovasc Disord. 2005 Feb 25; 5:6 | utf-8 | BMC Cardiovasc Disord | 2,005 | 10.1186/1471-2261-5-6 | oa_comm |
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BMC Endocr DisordBMC Endocrine Disorders1472-6823BioMed Central London 1472-6823-5-11573723210.1186/1472-6823-5-1Research ArticleMeasurement of fractionated plasma metanephrines for exclusion of pheochromocytoma: Can specificity be improved by adjustment for age? Sawka Anna M [email protected] Lehana [email protected] Amiram [email protected] Mitchell [email protected] William F [email protected] Department of Internal Medicine, St. Joseph's Healthcare, Hamilton, Ontario, L8N 4A6, Canada2 Division of Endocrinology and Metabolism, McMaster University, Hamilton, Ontario, L8N 3Z5, Canada3 Centre for Evaluation of Medicines, St. Joseph's Healthcare, Hamilton, Ontario, L8N 1G6, Canada4 Department of Clinical Epidemiology and Biostatistics, McMaster University, Hamilton, Ontario, L8N 3Z5, Canada5 Division of Endocrinology, Metabolism, Nutrition, and Internal Medicine, Mayo Clinic, Rochester, MN, 55905, USA2005 28 2 2005 5 1 1 4 11 2004 28 2 2005 Copyright © 2005 Sawka et al; licensee BioMed Central Ltd.2005Sawka et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Biochemical testing for pheochromocytoma by measurement of fractionated plasma metanephrines is limited by false positive rates of up to 18% in people without known genetic predisposition to the disease. The plasma normetanephrine fraction is responsible for most false positives and plasma normetanephrine increases with age. The objective of this study was to determine if we could improve the specificity of fractionated plasma measurements, by statistically adjusting for age.
Methods
An age-adjusted metanephrine score was derived using logistic regression from 343 subjects (including 33 people with pheochromocytoma) who underwent fractionated plasma metanephrine measurements as part of investigations for suspected pheochromocytoma at Mayo Clinic Rochester (derivation set). The performance of the age-adjusted score was validated in a dataset of 158 subjects (including patients 23 with pheochromocytoma) that underwent measurements of fractionated plasma metanephrines at Mayo Clinic the following year (validation dataset). None of the participants in the validation dataset had known genetic predisposition to pheochromocytoma.
Results
The sensitivity of the age-adjusted metanephrine score was the same as that of traditional interpretation of fractionated plasma metanephrine measurements, yielding a sensitivity of 100% (23/23, 95% confidence interval [CI] 85.7%, 100%). However, the false positive rate with traditional interpretation of fractionated plasma metanephrine measurements was 16.3% (22/135, 95% CI, 11.0%, 23.4%) and that of the age-adjusted score was significantly lower at 3.0% (4/135, 95% CI, 1.2%, 7.4%) (p < 0.001 using McNemar's test).
Conclusion
An adjustment for age in the interpretation of results of fractionated plasma metanephrines may significantly decrease false positives when using this test to exclude sporadic pheochromocytoma. Such improvements in false positive rate may result in savings of expenditures related to confirmatory imaging.
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Background
Pheochromocytoma is a rare tumor of the adrenal medulla or sympathetic ganglia, which can secrete excessive catecholamines [1]. Signs and symptoms of pheochromocytoma may include hypertension, pain (including headache, flank pain, abdominal pain, or chest pain), hyperhidrosis, anxiety or panic attacks, cardiac arrythmias, or sudden death [1-6]. A pheochromocytoma may also be detected as an asymptomatic incidental adrenal mass seen on abdominal imaging [7]. Metabolites of norepinephrine and epinephrine, specifically normetanephrine and metanephrine, may be measured in the plasma by high performance liquid chromatography with electrochemical detection, as described by Lenders et al. [8]. Measurement of fractionated plasma metanephrines has been called "the best test for excluding or confirming pheochromocytoma" and some investigators have recommended that such measurements "should be the test of first choice" [9]. In a recent systematic review of the world literature, we have observed that measurement of fractionated plasma metanephrine measurements have a high sensitivity ranging of 96 to100% and a variable specificity ranging from 82% to 100% [10]. The specificity of fractionated plasma metanephrines in excluding pheochromocytoma appears lowest in populations without known genetic predisposition to disease (those in whom sporadic disease is sought), with a false positive rate of up to 18% in such patients [9]. We have previously observed that the normetanephrine fraction is elevated in the majority of false positive test results and that false positives are associated with increasing age [11]. Indeed, investigators from the National Institute of Health have agreed that, "measurements of plasma normetanephrine and metanephrine provide a highly sensitive test for diagnosis of pheochromocytoma, but false positive results remain a problem" [12]. False positive biochemical test results may result in needless imaging procedures and generate excessive healthcare expenditures in detection of sporadic pheochromocytoma [13].
Reasons for false positive fractionated metanephrine test results have been explored and alternatives for further evaluation of patients with positive test results have been proposed. It is known that acetaminophen may interfere with measurements of fractionated plasma metanephrines using the Lenders' method [8], so this drug has traditionally been avoided prior to testing. Eisenhofer and colleagues have also suggested that that tricyclic antidepressants and phenoxybenzamine, respectively, may result in false positive tests [12]. Of note, in a recent Mayo study, 13% of subjects with false positive fractionated metanephrines used tricyclic antidepressants [11] but 9% of subjects who had normal fractionated metanephrine measurements also used these drugs. Thus, tricyclic use did not seem to explain the majority of false positives seen at the Mayo Clinic. Furthermore, given that phenoxybenzamine is rarely used in patients without known pheochromocytoma, use of this drug does not explain why there are so many false positive fractionated metanephrine results observed in clinical practice. As a method to distinguish false positives from true positives, Eisenhofer and colleagues have recommended clonidine-suppression testing in patients with positive fractionated plasma metanephrine measurements [12]. Eisenhofer and colleagues have recommended that plasma norepinephrine and normetanephrine levels be measured three hours after a dose of 0.3 mg clonidine in such patients [12]. We have proposed an alternative strategy to deal with false positive test results in patients without known genetic predisposition to disease [13]. As normetanephrine is the fraction responsible for the majority of false positive results, we have proposed that 24-hour urinary measurements of fractionated catecholamines and metanephrines be performed in patients with normetanephrine elevations that are approximately one and two times the upper limit of the normal range, to confirm the biochemical presence of sporadic pheochromocytoma [13]. Of note, in the case of high risk patients with known genetic predisposition to pheochromocytoma, the pre-test probability of disease may be sufficiently high that measurement of fractionated plasma metanephrines, without confirmatory biochemical studies may be reasonable [11]. Thus, the issue of lack of specificity of fractionated plasma metanephrine measurements is applicable primarily to non-genetically predisposed individuals in whom sporadic pheochromocytoma is sought.
Our objective was to determine whether an adjustment of fractionated plasma metanephrines for age may improve the specificity of interpretation of these measurements when biochemically excluding sporadic pheochromocytoma, and thus result in costs savings in confirmatory imaging studies.
Methods
Study populations
The age-adjusted fractionated plasma metanephrine score was derived from a previously described dataset of 349 subjects (including 33 people with pheochromocytoma) who underwent measurement of fractionated plasma metanephrines as part of an evaluation of suspected pheochromocytoma (also known as the derivation set) [11]. The diagnostic efficacy of this logistic-regression derived prediction rule was then tested in a second dataset of 158 subjects (including 23 with sporadic pheochromocytoma) who had measurements of fractionated plasma metanephrines performed at the Mayo Clinic Rochester the following year (the validation dataset). None of the patients in the validation set had known genetic predisposition to pheochromocytoma. All patients with pheochromocytoma had histologic confirmation of the diagnosis and those without pheochromocytoma had an alternative diagnosis assigned at the completion of their evaluation based on a combination of other biochemical test results (such as normal 24-hour urinary fractionated metanephrine and catecholamine measurements with or without normal imaging of the adrenals in the form of computerized tomography scanning [CT] or magnetic resonance imaging [MRI]). All data were obtained by retrospective chart review. The Institutional Review Board at Mayo Clinic Rochester approved the study.
Measurement of fractionated plasma metanephrines
The technique of Lenders (by high performance chromatography and electrochemical detection) was used to measure fractionated plasma metanephrines [8]. The traditional criterion for positivity is a metanephrine fraction greater than or equal to 0.5 nmol/L or a normetanephrine fraction greater than or equal to 0.9 nmol/L, based on a 95% reference range derived by Mayo Medical Laboratories. Subjects were advised to avoid acetaminophen for 48 hours prior to measurement of fractionated plasma metanephrines. Fractionated plasma metanephrine measurements were taken in the sitting position, with no indwelling venous cannula, and no dietary restrictions prior to testing. The lower limit of detection for the normetanephrine and metanephrine fractions was 0.20 nmol/L. Therefore, normetanephrine and metanephrine fractional measurements reported as being below the detection limit were given the value of 0.19 nmol/L for use in the logistic regression formula. Subjects who had "interfering substances" reported by the laboratory on measurement of fractionated plasma metanephrines were excluded from analyses but the number of such subjects was recorded.
Statistical methods
Clinical characteristics of subjects in the derivation set who did not have pheochromocytoma but had measurements of the normetanephrine or metanephrine fraction above the upper reference limits (false positive tests using traditional positivity criteria) were compared to those without pheochromocytoma who had true negative tests (χ2 was used for categorical variables and Student's t-test for independent samples was used for continuous variables). Variables which were different between both groups at a significance level of 0.1 were then entered into a multivariable logistic regression model predicting pheochromocytoma. Age was the only variable of statistical significance distinguishing true positive from false positive fractionated plasma metanephrine measurements in the derivation set. Thus, we forced age with measurement values of normetanephrine and metanephrine fractions in a multivariable logistic regression model predicting pheochromocytoma in the derivation set (SPSS 10.0, Chicago, ILL). The formula for this age-adjusted fractionated plasma metanephrine score is shown below:
-4.188 + -0.07(age) + 4.516(metanephrine) + 3.129(normetanephrine).
Age was in years, metanephrine fraction in nmol/L and normetanephrine fraction in nmol/L in this formula. In the derivation set, the Hosmer and Lemeshow test had χ2 = 4.73, df = 8, p = 0.79 and the Cox and Snell r2 = 0.38 showing significant model goodness-of-fit. A positivity cut-off age-adjusted metanephrine value of = -1.4752 was chosen as it carried an acceptable sensitivity of = 90.9% (30/33 patients, 95% CI = 76.4%, 96.9%) and specificity of 96.8% (300/310 patients, 95% CI = 94.2%, 98.2%) in the derivation set. The sensitivity level of over 90% was chosen because such a sensitivity level was believed to be clinically reasonable and at this level, specificity was still acceptable. We were aware that the lower the cut-off, the higher the sensitivity, but this would be at the expense of specificity.
The formula for adjustment of age was applied to fractionated plasma metanephrine measurements in the second dataset (validation dataset) for testing of sensitivity and specificity in a population in whom sporadic pheochromocytoma was sought. In the validation set, individuals with known genetic predisposition to pheochromocytoma were excluded. Sensitivities were calculated by division of subjects with true positive test results by all the subjects with pheochromocytoma, and specificities were calculated by division of subjects with true negative test results divided by all subjects without pheochromocytoma. For sensitivities, specificities, and likelihood ratios, 95 percent confidence intervals (CI) were calculated using Wilson's method (except the Score Method was used for calculation of 95% CI of likelihood ratios when a zero cell was noted) [14]. The specificity of the age-adjusted metanephrine score were compared (at the same level of sensitivity) to traditional interpretation of fractionated plasma metanephrine measurements using McNemar's test [15].
Economic evaluation (decision analysis)
We investigated whether use of an age-adjusted fractionated plasma metanephrine measurement could result in cost savings in imaging expenditures, compared to use of fractionated plasma metanephrine measurements interpreted in a conventional fashion, for detection of sporadic pheochromocytoma in a hypothetical tertiary care hypertensive population. We thus performed a decision analysis, with resource implications defined by costs of confirmatory imaging (CT and MRI), interpreted from a third party payer perspective. In the decision analysis, we compared algorithm "A" in which biochemical testing consisted of measurement of fractionated plasma metanephrines with these measurements interpreted relative to the 95% reference range (defined by a normetanephrine fraction above 0.9 nmol/L or a metanephrine fraction above 0.5 nmol/L) to algorithm "B", in which fractionated plasma metanephrine measurements were interpreted by using the age-adjusted metanephrine score. The sensitivity and specificity of biochemical tests was based on data from the validation set. In each algorithm, all patients with positive biochemical testing would undergo confirmatory imaging. The imaging protocol for patients with positive biochemical tests in either strategies began with computerized tomography ([CT] with and without intravenous contrast) of the abdomen, then if negative, I-131 or I-123 metaiodobenzylguanidine (MIBG) scintigraphy (efficacy for I-131 and costs for I-123 shown). The horizon (endpoint) of the analyses was positive diagnosis or exclusion of pheochromocytoma, for hypothetical hypertensive patients subjected to each strategy. The outcome of the analyses was the number of patients with pheochromocytoma expected to be detected by each strategy. The costs of false positive biochemical tests were reflected only in the costs of subsequent imaging and not in potential costs of needless surgery or its possible complications.
The diagnostic efficacy of imaging studies was based on respective estimates from the literature: The sensitivity of CT imaging of the abdomen was assumed to be 98% with a specificity of 70% [16]. The sensitivity of MIBG in detecting benign sporadic pheochromocytoma was assumed to be 87.4% with a specificity of 98.9% [17].
All costs were reported in 2002 US dollars. Costs of imaging investigations were obtained from the Mayo Clinic Rochester Business Office. For the purpose of the decision analysis model, the prevalence of pheochromocytoma in the hypertensive population that would typically be screened was assumed to be 0.5% [18].
Results
Findings in the derivation set
The derivation set (from which the age-adjusted score was developed) consisted of 349 consecutive subjects (including 33 people with pheochromocytoma) who underwent fractionated plasma metanephrine measurements as well as 24-hour urinary total metanephrine measurements with or without 24-hour urinary catecholamine measurements in testing for pheochromocytoma at Mayo Clinic Rochester. In the derivation set, 8 of the 33 individuals had clinically-diagnosed genetic syndromes predisposing to pheochromocytoma (three familial malignant paraganglioma, two von Hippel-Lindau, one had multiple endocrine neoplasia 2a, one had multiple endocrine neoplasia 2b, and one had familial multiple benign paraganglioma). The 316 individuals in the derivation set who did not have pheochromocytoma underwent such testing the following reasons: refractory hypertension (174, 55%), spells (periodic episodes of symptoms such as palpitations, headache, or sweating, 124, 39%), adrenal mass (45, 14%), previous pheochromocytoma or known genetic predisposition to pheochromocytoma (24, 8%). The mean age of subjects with pheochromocytoma was 48 years (SD 18 years, range 16 to 60 years), whereas the mean age of subjects without pheochromocytoma was 52 years (SD 15 years, range 10 to 73 years). Six of the 316 subjects without pheochromocytoma in the derivation set did not have a plasma metanephrine fraction recorded secondary to "interfering substances" and therefore were excluded from the analyses. In the derivation set, the sensitivity of traditionally interpreted fractionated plasma metanephrine measurements (using 95% reference ranges) was 93.9% (95% CI, 80.4, 98.3) (31/33 subjects), with a specificity of 85.2% (95% CI, 80.8, 88.7) (264/310 subjects). Baseline characteristics of individuals in the derivation set without pheochromocytoma were compared for individuals who had true negative fractionated metanephrine measurements (n = 264) to those who had false positive results (n = 46) (Table 1). The individuals with false positive fractionated plasma metanephrine measurements in the derivation set were significantly older than those with true negative measurements (p = 0.007), whereas blood pressure, antihypertensive medication use, and rates of obstructive sleep apnea were not significantly different between these groups. Thus, age was chosen as an important variable to adjust for in interpretation of fractionated plasma metanephrines and an age-adjusted metanephrine score was developed from the derivation set data using logistic regression (as described in the Methods). At a cut-off value of -1.4752, the sensitivity of the age-adjusted metanephrine score was 90.9% (30/33 patients, 95% CI, 76.4%, 96.9%), with a specificity of 96.8% (300/310 patients, 95% CI, 94.2%, 98.2%). In this derivation set, which included individuals genetically predisposed to pheochromocytoma, one individual with a dopamine-secreting paraganglioma, another patient with a von Hippel-Lindau disease (diagnosed clinically), and a third patient with sporadic pheochromocytoma had false-negative age-adjusted metanephrine scores. The efficacy of the age-adjusted metanephrine score was then validated in the validation set.
Table 1 Clinical characteristics of subjects without pheochromocytoma from the derivation set
Clinical characteristic True negative Measurements of fractionated plasma metanephrines (n = 264) False positive Measurements of fractionated plasma metanephrines (n = 46) Significance testing results
Gender (females/n) 142/264 26/46 P = 0.73 (χ2 = 0.12, df = 1)
Age (mean, SD years) 50.7 (15.03) 57.3 (14.6) P = 0.007 (t = 2.81, df = 63)
Systolic blood pressure (mean, SD mmHg) 147 (26) (n = 260) 153 (32) P = 0.24 (t = 1.19, df = 56)
Diastolic blood pressure (mean, SD mmHg) 87 (12) (n = 260) 89 (15) P = 0.39 (t = 0.87, df = 57)
Number of antihypertensive agents currently used (mean, SD) 1.4 (1.5) 1.6 (1.3) P = 0.28 (t = 1.09, df = 70)
Known diagnosis of obstructive sleep apnea 15/264 4/46 P = 0.43 (χ2 = 0.62, df = 1)
Findings in the validation set
In the validation set of 158 subjects, 23 patients had histologically-proven sporadic pheochromocytoma (17 adrenal, 6 extra-adrenal, 8 malignant). Of the patients with pheochromocytoma, none were known to be genetically predisposed to pheochromocytoma and 14/23 were women (61%). The mean age of subjects with pheochromocytoma was 50 years (SD 16 years, range 16 to 83 years), whereas the mean age of subjects without pheochromocytoma was 55 years (SD 16 years, range 7 to 86 years). Of the 135 subjects without pheochromocytoma, 83 (62%) were women. Reasons for measurement of fractionated plasma metanephrines in the subjects without pheochromocytoma were as follows: hypertension (55, 41%), spells with or without hypertension (44, 33%), an incidentally discovered adrenal mass (20, 15%), and previously surgically cured pheochromocytoma (16, 12%).
In the validation set, the sensitivity of the age-adjusted metanephrine score was the same as the traditional interpretation of fractionated plasma metanephrine measurements at 100% (23/23, 95% CI, 85.7%, 100%). The specificity of the traditional interpretation of fractionated plasma metanephrine measurements was 83.7% (113/135 patients, 95% CI, 76.6%, 89.0%) and the specificity of the age-adjusted plasma metanephrine score was 97.0% (131/135 patients, 95% CI, 92.6%, 98.8%). Thus, the false positive rate with traditional interpretation of fractionated plasma metanephrine measurements was 16.3% (22/135, 95% CI, 11.0%, 23.4%) and with the age-adjusted score it was significantly lower at 3.0% (4/135, 95% CI, 1.2%, 7.4%) (Figure 1) (p < 0.001 using McNemar's test).
Figure 1 Percentage of false positive test results (and 95% confidence interval) at 100% sensitivity in using a traditional interpretation of fractionated plasma metanephrine measurements or an age-adjusted metanephrine score. Legend – Pmet(s), fractionated plasma metanephrine measurements; traditional fractionated plasma metanephrine measurements are considered positive if the metanephrine fraction is greater than or equal to 0.5 nmol/L or the normetanephrine fraction is greater than or equal to 0.9 nmol/L; an age-adjusted metanephrine score is positive if it is greater than -1.4752. The difference between false positive rates is statistically significant with p < 0.001 using McNemar's test.
Imaging cost implications of screening strategies for pheochromocytoma
In the decision analysis, biochemical testing by measurement of fractionated plasma metanephrines (traditional versus age-adjusted interpretation) was followed by CT imaging for all positive biochemical tests and if CT imaging was negative, then MIBG (I-123 or I-131) would be performed (Figure 2). For the purpose of the economic evaluation, in all three screening strategies, a 0.5% prevalence of pheochromocytoma was assumed in a target hypertensive population, so 500 patients with pheochromocytoma would be expected in a sample of 100,000 hypertensive subjects (Figure 2). Mayo Clinic Rochester charges for diagnostic studies were used: CT scan of the abdomen (with and without contrast) $1460, I-123 MIBG scan (with and without spect) $1875.
Figure 2 Decision analysis: Testing algorithm for pheochromocytoma in 100,000 hypothetical hypertensive subjects (including 500 individuals with pheochromocytoma)
If 100,000 subjects with hypertension would be screened using algorithm "A" (beginning with biochemical testing by measurement of fractionated plasma metanephrines, traditional interpretation), 499/500 patients with pheochromocytoma (overall sensitivity 99.8%) would be expected to be detected (1 patient expected to have false negative CT and MIBG imaging); furthermore 94,510/99,500 of subjects without pheochromocytoma would be reassured with a negative diagnosis (overall specificity 95.0%). In algorithm "A", 16,718 individuals would undergo CT scanning and 11,363 individuals would undergo I-123 or I-131 MIBG imaging. The total cost of imaging for algorithm "A" would be estimated to be 45.7 million dollars.
If algorithm "B" (biochemical testing using the age-adjusted metanephrine score) would be used in 100,000 subjects with hypertension, 499/500 patients with pheochromocytoma (99.8%) would be expected to be detected and 98592/99,500 individuals without pheochromocytoma would be reassured with a negative test result (overall specificity of 99.1%). In algorithm "B", 3,485 individuals would undergo CT scanning and 2,100 individuals would undergo I-123 or I-131 MIBG imaging. The cost of imaging for algorithm "B" would be approximately 9.0 million dollars. Thus, use of the age-adjusted plasma metanephrine score for biochemical testing for sporadic pheochromocytoma in a hypothetical population of 100,000 tertiary care hypertensive patients could result in a cost savings of 36.7 million dollars with equal detection of pheochromocytoma cases, relative to using the same biochemical testing but interpreting fractionated plasma metanephrine measurements in a traditional fashion.
Discussion
We agree with observation by Eisenhofer and colleagues that when it comes to measurement of fractionated plasma metanephrines for exclusion of pheochromocytoma, "false-positive results remain a problem" [12], particularly when attempting to exclude sporadic disease. Originally, it was the hope was that measurement of fractionated plasma metanephrines could result in cost savings because of avoidance of multiple biochemical tests [19]. However, investigators from the National Institute of Health have recommended that clonidine-suppression tests need to be done in order to distinguish true positives from false positives [12]. An alternative to clonidine-suppression testing may be measurement of 24-hour urinary metanephrines and catecholamines in patients with mild to moderate elevations of the normetanephrine fraction (for normetanephrine values approximately one to two times the upper limit of the normal range) [13]. In our study, we have provided a unique alternative approach for improving specificity of interpretation of measurements of fractionated plasma metanephrines. By adjusting the metanephrine score for age, we have shown that it may be possible to improve specificity of interpretation of fractionated plasma metanephrines, with no loss of sensitivity in detection of sporadic pheochromocytoma, and potential savings in imaging expenditures.
Of note, the sensitivity of the age-adjusted metanephrine score was superior in the validation set (100%) to that observed in the original dataset from which it was derived (91%). An explanation for this finding may be that the validation set included only people who were at risk for sporadic pheochromocytoma (in other words, non-genetically predisposed individuals), whereas genetically predisposed individuals were included in the original derivation dataset. We have previously observed that fractionated plasma metanephrine measurements may be normal in genetically-predisposed individuals with small pheochromocytomas [11]. Moreover, the physiologic cause for the observed relationship of normetanephrine measurements with age is unclear. Of note, Raber et al have noted exaggerated increases in plasma normetanephrine after exercise in hypertensive individuals with type 2 diabetes, compared to normotensive individuals with or without diabetes [20]. Furthermore, Raber et al have suggested that the excessive response of plasma normetanephrine to exercise may serve as a marker of exaggerated sympathoadrenal function in hypertensive type 2 diabetics [20]. Fractionated plasma metanephrine measurements were performed only at rest in our study and we did not examine any potential relationship with diabetes. Systolic and diastolic blood pressures were not significantly different between individuals with false positive fractionated metanephrine measurements and those with true negative measurements in the derivation set in our study.
Our study is subject to several limitations. Firstly, limited clinical data on each studied individual were collected so variables that could be of interest such as: body mass index, creatinine-clearance, and rates of diabetes mellitus were not recorded. Furthermore, without autopsy confirmation, one cannot be absolutely certain that individuals labelled as not having a pheochromocytoma did not have an occult paraganglioma or pheochromocytoma. However, we believe that reasonable clinical criteria were used in excluding pheochromocytoma in our study. Another limitation is that we used an assay for measurement of fractionated plasma metanephrines that may be have been subject to interference with acetaminophen [8], whereas other assays, such as the one described by Roden et al, could have been preferable due to lack of acetaminophen interference [21]. The cut-off that we chose for positivity of the age-adjusted metanephrine score was also arbitrary and use of a lower cut-off could have resulted in improved sensitivity, albeit with likely some expense of specificity. Finally, our findings have not been validated outside a single institution.
Is calculation of an age-adjusted metanephrine score practical for use in daily clinical practice? In this day of palm pilots and desktop computers, we believe that it may be feasible for clinicians to enter the formula for age-adjustment into standard desktop spreadsheet software (such as Excel, Microsoft) and perform such adjustments in the clinic. Alternatively, laboratories can provide age adjusted values to physicians when reporting test results. Thus, we do believe that calculation of an age-adjusted score is feasible to assist physicians in interpretation of fractionated plasma metanephrine measurements. Indeed, such calculations may be less cumbersome and may generate fewer healthcare expenditures than alternative procedures such as supplemental clonidine-suppression testing or collection of 24-hour urinary measures.
Our observations should, however, be validated in another population outside of Mayo Clinic. Of particular note, our findings are applicable only to the screening of pheochromocytoma in low risk subjects who do not have known genetic predisposition to disease. In high risk, genetically predisposed individuals, mild elevations of normetanephrine or metanephrine measurements may be clinically significant and should prompt imaging.
Conclusion
An adjustment for age in interpretation of results of fractionated plasma metanephrine measurements may significantly improve the high false positive rate seen with this test when aiming to exclude sporadic pheochromocytoma. This improvement in specificity may result in savings in expenditures related to confirmatory imaging. Additional research is needed to investigate the generalizability of these findings in other clinical centres.
Competing interests
Dr. Sawka is a Skeletal Health Scholar funded, in part, by the Canadian Institutes of Health Research. Dr. Sawka was also a Fellow in Health Economics at McMaster University, partly funded by an unrestricted educational grant from Hoffmann-La Roche.
The other co-authors have no competing interests to declare.
Authors' contributions
All co-authors reviewed the manuscript and made suggestions for revisions. The project idea was conceived by A.M. Sawka. Analyses were performed by A.M. Sawka, with input from Dr. Thabane. The manuscript was written and revised by Dr. Sawka.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
A.M. Sawka has received an unrestricted educational grant from Hoffmann-La Roche for a Research Fellowship in Health Economics.
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| 15737232 | PMC553971 | CC BY | 2021-01-04 16:27:55 | no | BMC Endocr Disord. 2005 Feb 28; 5:1 | utf-8 | BMC Endocr Disord | 2,005 | 10.1186/1472-6823-5-1 | oa_comm |
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BMC Pregnancy ChildbirthBMC Pregnancy and Childbirth1471-2393BioMed Central London 1471-2393-5-51572768310.1186/1471-2393-5-5Study ProtocolIndividual patient data meta-analysis : Cervical stitch (cerclage) for preventing pregnancy loss in women Tudur-Smith Catrin [email protected] Andrea L [email protected] Zarko [email protected] Paula R [email protected] Centre for Medical Statistics and Health Evaluation, University of Liverpool, Liverpool, L69 3BX, UK2 University Department of Obstetrics and Gynaecology, Liverpool Women's Hospital, Liverpool, UK2005 23 2 2005 5 5 5 19 1 2005 23 2 2005 Copyright © 2005 Tudur-Smith et al; licensee BioMed Central Ltd.2005Tudur-Smith et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Cervical cerclage is a surgical procedure involving suturing the cervix with a purse type stitch to keep it closed during pregnancy. This procedure has been used widely in the management of pregnancies considered at high risk of preterm delivery. Several observational studies into the efficacy of cervical cerclage have claimed high rates of successful pregnancy outcome in women with a poor obstetric history attributed to cervical incompetence. However, a recent aggregate data Cochrane review found no such conclusive evidence from seven included randomised studies. Current data suggests that cervical cerclage is likely to benefit women considered to be 'at very high risk' of a second trimester miscarriage due to a cervical factor, however identifying such women remains elusive and many women may be treated unnecessarily. Undertaking an individual patient data (IPD) meta-analysis of the studies will allow us to investigate whether treatment is more effective in particular subgroups. Such an analysis will also provide a more powerful analysis of the predictors of preterm delivery and pregnancy loss, including ultrasound measurement of cervical length, and will allow a more complete analysis of 'time to event' outcomes.
Methods/Design
The analysis will include data from randomised trials comparing the intervention of elective cerclage versus no cerclage or bedrest to prevent miscarriage or pre-term labour. A specific list of data will be requested for each trial, including demographic and obstetric history data. The primary outcomes of interest will be neonatal mortality/morbidity. Attention will also be given to secondary outcomes such as time from randomisation to delivery, preterm delivery before 32 weeks and maternal morbidity. An intention to treat analysis will be performed, with attention paid to assessing clinical and statistical heterogeneity. Multilevel models with patients and trials as the two levels will be explored to investigate treatment effect on various outcomes. Patient-level covariates will be incorporated into the models in an attempt to account for statistical heterogeneity as well as to investigate interactions with treatment effect.
Discussion
Predictive models generated from our analysis should lead to more effective counselling of women at risk and a more cost effective use of cerclage.
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Background
Cervical cerclage is a surgical procedure carried out during pregnancy. The operation involves suturing the neck of the womb (cervix) with a purse type stitch to keep the cervix closed. This surgical procedure has been used widely in the management of pregnancies considered to be at high risk of preterm delivery.
Several observational studies in the last 50 years have claimed high rates of successful pregnancy outcome in women that had a poor obstetric history attributed to cervical incompetence. However, a recent Cochrane review found no conclusive evidence from seven included randomised studies that inserting a cervical stitch in women perceived to be at risk of preterm birth or second trimester pregnancy loss attributed to cervical factors, reduces the risk of pregnancy loss, preterm delivery or morbidity associated with preterm delivery (Drakeley 2003)[1].
In the Cochrane review, the data for important clinical outcomes including preterm delivery and maternal infection showed significant heterogeneity due to inconsistency in clinical definitions used, including the cut off gestational age defining preterm delivery, and different patient populations studied.
Practically, methods of undertaking a meta-analysis of several studies may involve collecting either aggregate data, or data on each patient individually. The advantages of the latter approach, described as the 'yardstick' (Chalmers 1993)[2] include (i) a more complete analysis of 'time of event' outcomes and (ii) a more powerful analysis of whether treatment is more or less effective in particular subgroups (Stewart 1993)[3].
One of the main concerns regarding current evidence related to cervical cerclage and other interventions for preventions of preterm delivery is a possibility that the 'primary outcomes' may have been selected to give results in greatest accord with the a priori beliefs of the authors. The evidence to support this phenomenon of within-study selective reporting comes from empirical research, which demonstrates discrepancies between research protocols and subsequent publications (Hahn 2002 [4], Williamson 2005 [5], Chan 2004 [6]). Individual patient data (IPD) meta-analysis has the capacity to overcome these problems.
Currently available data suggest that cervical cerclage is likely to be of benefit for women considered 'at very high risk' of second trimester miscarriage due to a cervical factor e.g. greater than two second trimester losses or progressive shortening of the cervix on ultrasound. However, predicting those women who will miscarry due to a cervical factor remains elusive and many women may be treated unnecessarily. The use of IPD will allow us to investigate predictors of preterm delivery including ultrasound measurement of cervical length and other woman-cerclage interactions.
IPD meta-analysis will allow an investigation of the hypothesis that the effect of cerclage is greater on extreme preterm delivery. In addition, an IPD meta-analysis has greater power than a single trial for examining subgroups. The efficacy of a treatment may depend on several factors. For aggregate data, a meta-analysis stratifying by the absolute risk in the control group may be the only method possible for accounting for these multiple factors simultaneously. This analysis is 'flawed and produces seriously misleading results' (Sharp 1996)[7]. A regression analysis of IPD allows the relation between treatment effect and risk score, derived from these multiple risk factors, to be investigated thereby avoiding these problems.
Methods/Design
Objectives
The aim of this project is to undertake an IPD meta-analysis of randomised trials of cervical cerclage. Specific objectives are as follows.
1. To estimate the effect of cervical cerclage on gestational age at delivery.
2. To investigate whether cervical cerclage is more likely to prevent extreme prematurity (<28 weeks) or delivery at later gestations.
3. To investigate risk factors for preterm delivery.
4. To investigate interactions between risk factors and cervical cerclage.
5. To model the effect of cervical cerclage and other risk factors on neonatal and maternal morbidity.
Criteria for considering studies for this IPD meta-analysis
The types of studies considered for inclusion in the analysis will be all randomised trials comparing cervical cerclage with expectant management or no cerclage during pregnancy. The previous Cochrane review (Drakeley 2003) [1] identified eight eligible trials with 2,513 randomised women (Rust 2000 [8], Althuisius 2000 [9], Althuisius 2001 [10], Rush 1983 [11], Lazar 1984 [12], Dor 1982 [13], MRC/RCOG 1988 [14], Meekai To et al. [15]). We have agreement in principle to provide IPD from six of these trials (Meekai To et al. [15], MRC/RCOG 1988 [14], Rust 2000 [8], Althuisius 2000 [9], Althuisius 2001 [10], Rush 1983 [11]), accounting for 1919(78%) of all women randomised. The remaining trialists and trialists of any further trials identified as eligible will be approached at the start of the project and we anticipate their willingness to collaborate. Already, two further trials have been identified (Berghella 2004 [16], Ezechi 2003 [17]) and the authors have agreed to provide IPD from these trials.
The data collected in the studies will relate to women with confirmed, or suspected of having, cervical incompetence who desire future pregnancies and women who present as an emergency and are thought to have a diagnosis of cervical incompetence. The intervention investigated in the studies will be elective cerclage by whichever method (Shirodkar technique, McDonald technique, transabdominal and transvaginal methods), versus no cerclage or bed rest as interventions to prevent miscarriage or pre-term labour as defined in the original Cochrane review (Drakeley 2003)[1].
Search strategy for identification of studies
The methods of trial identification described in the original Cochrane review (Drakeley 2003)[1] (see below) will be adopted and updated to December 2004.
The original review has drawn on the search strategy developed for the Pregnancy and Childbirth Group. The full list of journals and conference proceedings as well as the search strategies for the electronic databases, which are searched by the Group on behalf of its reviewers, are described in detail in the 'Search strategies for the identification of studies section' within the editorial information about the Cochrane Pregnancy and Childbirth Group. Briefly, the Trials Search Coordinator searches on a regular basis MEDLINE, the Cochrane Controlled Trials Register and reviews the Contents tables of a further 38 relevant journals received via ZETOC, an electronic current awareness service.
In addition, handsearches will be performed on congress proceedings of the International and European society meetings of feto-maternal medicine, recurrent miscarriage and reproductive medicine. Whenever possible, investigators will be contacted to ask about any additional studies potentially eligible for inclusion.
Trial eligibility and methodological quality assessment
Two reviewers will independently assess eligibility of identified randomised controlled trials for inclusion in the review. Any difference of opinion will be resolved by discussion. The methodological quality of each trial will be assessed by summarising the method of generation of randomisation list, method of allocation concealment, and potential impact of losses to follow-up. Quasi-randomised studies in which allocation was transparent (e.g. use of alternative allocation or medical record numbers) were excluded in the original review.
Data collection
The following data for each woman/infant pair will be requested from all trials: date of randomisation and gestational age, maternal demographics and obstetric characteristics at randomisation including cervical length on ultrasound, fibronectin and bacterial vaginosis data if available, treatment allocated, complications during pregnancy including ruptured membranes, maternal pyrexia or chorioamnionitis, date of delivery, gestational age at delivery and all neonatal data including birthweight, length of stay at NICU and morbidity related to prematurity.
The following methodological data will also be requested for all trials: method of generation of randomisation list, method of concealment of randomisation, stratification factors and blinding methods.
Data will be accepted either in electronic (floppy disk/CD/internet) or paper form. A desired format and coding will be specified but trialists may supply data in the most convenient way open to them, providing details of coding are sent with the data.
Data validation strategy
A copy of the original data sent (before checking) will be held in a separate file. The following procedures will then be performed and documented for all trial data supplied. Trial details will be crosschecked against any published report of the trial. Range and consistency checks will be applied – missing data, errors and inconsistencies will be followed up with a nominated individual. The chronological randomisation sequence will be reviewed. The balance of prognostic factors will be checked, taking into account of factors stratified for in the randomisation procedure.
Outcome measures
The primary outcome of interest will be neonatal mortality/morbidity. Choice of primary outcome is about what should determine clinical decision-making. However it is recognised that trials to date may have insufficient power and there is a need to consider secondary outcomes of time from randomisation to delivery, preterm delivery before 32 completed weeks (<32+0 weeks) and maternal morbidity as defined in the Cochrane Review (Drakeley 2003)[1]. We will aim to obtain all neonatal and maternal morbidity outcome data collected in each trial and not just those reported in publications.
Reporting of these outcomes in the original trial report is not an eligibility requirement for this review.
Data analysis
Data on all randomised patients will be requested to perform an intention-to-treat analysis as far as possible. Clinical heterogeneity will be assessed by reviewing the differences across trials in characteristics of randomised patients.
Initially, an aggregate data analysis will be undertaken although treatment effect estimates will be obtained from the individual patient data. Binary outcomes will be summarised in terms of odds ratios or relative risks, depending on the degree of heterogeneity observed. Time-to-event outcomes will be summarised in terms of the log (hazard ratio). The I square statistic and chi-square test for statistical heterogeneity will be applied to these summary data.
Regression models, stratified by trial, will be used to explore the effects of treatment, risk factors and treatment-covariate interactions on the various outcomes of interest. These will include Cox and accelerated life models for time-to-event outcomes (Tudur-Smith 2004 [18], Williamson 2002 [19]) and logistic regression models with trial indicator variables for binary outcomes (Whitehead 2002) [20]. Factors other than treatment to be investigated are gestational age at randomisation, maternal demographics, obstetric characteristics including obstetric history, cervical length on ultrasound, fibronectin, bacterial vaginosis, multiple pregnancy.
Two-level multilevel regression models will be fitted with patients corresponding to level one units and trials as level two units for the various outcomes of interest, adopting the relevant approach for continuous, binary, categorical and time to event outcomes as applicable. Trial effects will be represented by fixed effects whilst treatment effects will be represented by random effects in an attempt to reflect the assumed similar (but not identical) treatment effect across trials. Patient-level covariates (as listed above) will then be incorporated into the model in an attempt to account for some of the remaining statistical heterogeneity. An attempt will be made to incorporate these covariates first of all by assuming their effect to be constant across all trials and subsequently by assuming some heterogeneity in the covariate effect across trials by modelling them either as fixed or random effects. Finally, treatment-covariate interactions will be investigated by including additional variables and adopting a similar approach.
If IPD are not available for some trials, the potential for bias will be investigated as follows. The reasons for not being able to obtain the data will be assessed for the potential for bias. Results using aggregate data from these trials will be compared with results using aggregate data from trials where IPD have been supplied, and any difference investigated. The analysis plan will be reviewed in light of the availability of IPD but prior to any comparative analyses.
Discussion
Predictive models generated by our analysis should allow more effective counselling of women at risk of preterm delivery and thus more cost effective use of cerclage.
Competing interests
ZA and PRW were authors of a paper that will be included in the IPD meta-analysis (To, 2004). ZA was an author of the non-IPD systematic review on this topic (Drakeley, 2003)[1]. The authors declare that they do not have any other competing interests.
Authors' contributions
PRW conceived the idea, CTS drafted the initial protocol, and all authors commented on and approved this final version.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The authors would like to thank the trialists who have kindly agreed to provide IPD data from their trials.
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Stewart LA Parmar MKB Meta-analysis of the literature or of individual patient data: is there a difference? Lancet 1993 341 418 422 8094183 10.1016/0140-6736(93)93004-K
Hahn S Williamson PR Hutton JL Investigation of within-study selective reporting in clinical research: follow-up of applications submitted to an LREC Journal of Evaluation in Clinical Practice, 83 (August) 2002
Williamson PR Gamble C Identification and impact of outcome selection bias in meta-analysis Statistics in Medicine 2005
Chan A-W Hrbjartsson A Haahr H GtzscheGotzsche PC Altman DG Empirical evidence for selective reporting of outcomes in randomised trials: Comparison of protocols to publications Journal of the American Medical Association 2004 291 2457 2465 15161896 10.1001/jama.291.20.2457
Sharp SJ Thompson SG Altman DG The relation between treatment benefit and underlying risk in meta-analysis British Medical Journal 1996 313 735 738 8819447
Rust OA Atlas RO Jones KJ Benham BN Balducci J A randomised trial of cerclage versus no cerclage among patients with ultrasonographically detected second trimester preterm dilation of the internal os American Journal of Obstetrics and Gynecology 2000 183 830 5 11035321 10.1067/mob.2000.109040
Althuisius SM Dekker GA van Geijn HP Bekedam DJ Hummel P Cervical incompetence prevention randomized cerclage trial (CIPRACT): study design and preliminary results American Journal of Obstetrics and Gynecology 2000 183 823 29 11035320 10.1067/mob.2000.108874
Althuisius S Dekker G Hummel P Bekedam D van Geijn H CIPRACT (cervical incompetence prevention randomized cerclage trial): final results [abstract] Am J Obstet Gynecol 2001 184 S2
Rush R Isaacs S Prophylactic cervical cerclage and gestational age at delivery Proceedings of the 2nd Conference on Priorities in Perinatal Care; South Africa 1983 132
Lazar P Gueguen S Multicentred controlled trial of cervical cerclage in women at moderate risk of preterm delivery British Journal of Obstetrics and Gynaecology 1984 91 731 5 6380565
Dor J Shalev J Mashiach S Blankstein J Serr DM Elective cervical suture of twin pregnancies diagnosed ultrasonographically in the first trimester following induced ovulation Gynecologic and Obstetric Investigation 1982 13 55 60 7056503
Anonymous Interim report of the medical research council/royal college of obstetricians and gynaecologists multicentre randomized trial of cervical cerclage. MRC/RCOG working party on cervical cerclage British Journal of Obstetrics and Gynaecology 1988 95 437 45 2900021
To MS Alfirevic Z Heath VCF Cicero S Cacho AM Williamson PR Nicolaides KH Cervical cerclage for prevention of preterm delivery in women with short cervix: randomised controlled trial The Lancet 2004 363 1849 1853 15183621 10.1016/S0140-6736(04)16351-4
Berghella V Odibo AO Tolosa JE Cerclage for prevention of preterm birth in women with a short cervix found on transvaginal ultrasound examination: A randomized trial American Journal of Obstetrics and Gynecology 2004 191 1311 1317 15507959 10.1016/j.ajog.2004.06.054
Ezechi OC Kalu BKE Nwokoro CA Prophylactic cerclage for the prevention of preterm delivery Int J Gynecol Obstet 2004 85 283 284 10.1016/j.ijgo.2003.11.015
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| 15727683 | PMC553972 | CC BY | 2021-01-04 16:32:05 | no | BMC Pregnancy Childbirth. 2005 Feb 23; 5:5 | utf-8 | BMC Pregnancy Childbirth | 2,005 | 10.1186/1471-2393-5-5 | oa_comm |
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BMC Cell BiolBMC Cell Biology1471-2121BioMed Central London 1471-2121-6-81571792410.1186/1471-2121-6-8Research ArticleNovel hepatocyte growth factor (HGF) binding domains on fibronectin and vitronectin coordinate a distinct and amplified Met-integrin induced signalling pathway in endothelial cells Rahman Salman [email protected] Yatin [email protected] Jacqueline [email protected] Kirti V [email protected] Rushika [email protected] Michael [email protected] Errol S [email protected] Coagulation Research Laboratory, Division of Cardiovascular Medicine, GKT School of Medicine, St. Thomas' Hospital, London, UK2 Division of Vascular Surgery, University of Washington School of Medicine and VA Puget Sound Health Care System, Seattle WA, USA2005 17 2 2005 6 8 8 9 8 2004 17 2 2005 Copyright © 2005 Rahman et al; licensee BioMed Central Ltd.2005Rahman et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The growth of new blood vessels in adult life requires the initiation of endothelial cell migration and proliferation from pre-existing vessels in addition to the recruitment and differentiation of circulating endothelial progenitor cells. Signals emanating from growth factors and the extracellular matrix are important in regulating these processes.
Results
Here we report that fibronectin (FN) and vitronectin (VN) modulate the responses of endothelial cells to HGF (Scatter Factor), an important pro-angiogenic mediator. Novel binding sites for HGF were identified on both FN and VN that generate molecular complexes with enhanced biological activity and these were identified in the supernatants of degranulated platelet suspensions implicating their release and formation in vivo. In the absence of co-stimulation with an ECM glycoprotein, HGF could not promote endothelial cell migration but retained the capacity to induce a proliferative response utilising the Map kinase pathway. Through promoting Met-Integrin association, HGF-FN and HGF-VN complexes coordinated and enhanced endothelial cell migration through activation of the PI-3 kinase pathway involving a Ras-dependent mechanism whereas a Ras-independent and attenuated migratory response was promoted by co-stimulation of cells with HGF and a non-binding partner ECM glycoprotein such as collagen-1.
Conclusions
These studies identify a novel mechanism and pathway of HGF signalling in endothelial cells involving cooperation between Met and integrins in a Ras dependent manner. These findings have implications for the regulation of neovascularization in both health and disease.
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Background
The generation and repair of blood vessels in adult life requires the regulation of endothelial cell survival, migration, proliferation and their differentiation from lineage-committed progenitors by the coordinated action of several classes of vaso-active agents including growth factors, cytokines, and the extracellular matrix (ECM) [1-4]. Elucidating the molecular mediators of these signals and their mechanism of action is vital to understanding the fine regulation of neo-vessel development and maintenance.
There is growing evidence pointing to a close collaboration between growth factors and the ECM in several biological processes including vasculogenesis and post-natal revascularization. Studies have shown that the response of cells to growth factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) are potentiated by integrin ligation to specific ECM glycoproteins [5-8]. In a previous report, we showed that VEGF-induced endothelial cell migration was augmented by fibronectin (FN) [9]. We also presented evidence that the VEGF/VEGFR-2 pathway is coupled to the integrin α5β1 through a mechanism involving the promotion of an integrin α5β1-VEGFR-2 signalling moiety generated as a consequence of receptor ligation by a VEGF-FN complex. These events promoted the sustained activity of Erk kinase, which was coupled to the migratory response. More recently, we presented data demonstrating that FN significantly enhanced VEGF-mediated migration of CD34+ cells and their differentiation into endothelial cells [10]. In addition to the VEGF pathway, in vitro studies have highlighted the importance of hepatocyte growth factor (HGF) as a pro-angiogenic mediator. HGF, also termed scatter factor, has a well-established role in tumourogenesis but may be an important mediator of neovascularization since studies show that HGF induces the expression of VEGF in endothelial cells in vitro and that HGF synergises with VEGF to promote capillary-tube assembly in collagen matrices [11,12]. In addition, neovascularization in the rat cornea was also elevated by co-administration of HGF and VEGF compared to either growth factor in isolation [11]. The emerging significance of HGF as a pro-angiogenic mediator was further highlighted by a recent study of a large cohort of patients (1090 patients, CAPTURE trial) with acute coronary syndromes and identified serum levels of HGF as a positive indicator of patients' prognosis associated with a significantly lower event rate and increased collateralization of the target vessel [13]. Although the pro-angiogenic effects of HGF are known, the detailed mechanism of HGF action on the vascular cells, including the identity of intracellular mediators remains poorly understood.
In the present work we show that HGF forms a specific physical complex with FN and VN and that these complexes are present in degranulated platelet suspensions implicating a putative role in vivo. Significantly, we show that HGF-FN and HGF-VN molecular complexes induce a unique and enhanced intracellular signal employing Ras, thereby highlighting an important mechanism of growth factor receptor tyrosine kinase and integrin cooperation in promoting pro-angiogenic responses.
Results
Identification of novel HGF binding domains on FN and VN
We recently identified binding domains on FN for VEGF, which played an important role in promoting the activity of VEGF [9]. Since HGF is also an important angiogenic factor, experiments were designed to establish whether HGF had specific ECM binding partners. Using a solid phase assay, we measured the binding of 125I-labelled HGF to a variety of ECM molecules immobilized on plastic wells. As shown in Fig. 1A, 125I-labelled HGF bound to both FN and VN specifically with residual binding observed to either collagen-1 or laminin. Further experiments were performed to locate the HGF binding site on the FN molecule using purified FN proteolytic fragments immobilised onto the polystyrene microtiter wells. In these experiments 125I-labelled HGF bound to the 70 kDa N-terminal fragment and the 40 kDa C-terminal fragment. No significant binding was observed to the 120 kDa fragment that harbours the internal cell binding domain (Fig. 1B). To further analyse the association between HGF and FN, the interaction of HGF with the FN fragments was measured in real time by surface plasmon resonance analysis (SPR). As shown in Fig. 1C &1D, HGF bound to the 70 kDa N-terminal FN fragment immobilized on the sensor chip in a specific and saturable manner with a Kd of approximately 300 ± 93 nM for a one-site model. The data shown in Fig. 1D could be applied to a two-site model with equal probability showing Kd values for the high and low affinity sites of 15 nM ± 2 nM and 4 μM respectively. HGF binding to the 40 kDa fragment could not be measured directly by SPR, as immobilization of the 40 kDa fragment on the sensor chip appeared to mask the HGF binding site (data not shown).
Figure 1 Identification of HGF Binding Domains on FN and VN and the presence of HGF-FN and HGF-VN complexes in platelet supernatants. Panel A-Plastic wells coated with various matrix proteins or BSA (10 μg/ml) were incubated with 125I-labelled HGF to equilibrium and then washed and counted in a γ-counter. Panel B-Plastic wells were coated with FN and FN derived proteolytic fragments or BSA. 125I-labelled HGF was incubated in these wells to equilibrium binding, washed and the bound HGF levels determined using γ-counter. Panels C &D-Binding of HGF to FN 70 kDa fragment in real time by SPR. Panel C-Real time binding isotherms of increasing concentrations of HGF (0, 16.5, 31.25, 62.5, 125, 188, 250, 315, 375, 500 nM)with association and dissociation phases for a single representative experiment with the arrows indicating injection start and finish. Panel D-The data from panel C is plotted as a function of HGF concentration (30–500 nM). Inset shows the data analysed by the method of Scatchard showing a single binding site with a Kdapp = 300 nM. The data could also fit equally well to a two-site model (see text for details). n = 3. Panel E-Platelet suspensions (20.0 × 108/ml) were stimulated with either saline (-) or 1 U/ml thrombin (+) for 5 min at room temperature to allow degranulation. Cellular and membranous material was cleared by centrifugation (100,000 × g) and the supernatants were immunoprecipitated with monoclonal antibodies to FN or an isotype matched control reagent (control IgG). Immune complexes were analysed by SDS-PAGE and Western blotting probing with an antibody to HGF (Santa Cruz). The blot was stripped and re-probed with antibodies to FN to confirm the specificity of the primary immunoprecipitation step. Panel F-Supernatants derived from thrombin-stimulated platelets were immunoprecipitated with antibodies with specificity for either FN, VN or an isotype matched control reagent. The immune complexes were analysed as in panel E. Lower panel shows the same blot stripped and re-probed with a monoclonal antibody to VN to confirm the primary precipitation. Blots were developed by chemiluminescence. The data shown are representative blots of experiments repeated three times.
Platelets release HGF complexed to FN and VN
To establish whether HGF-FN and HGF-VN molecular complexes occur in vivo we examined platelets, a rich source of growth factors, for the presence of these complexes. Washed human platelet suspensions were stimulated with thrombin (1 U/ml) to promote degranulation and the derived supernatants were immunoprecipitated with antibodies directed to FN or VN. The resulting immune complexes were analysed for co-precipitation of HGF (Fig. 1E &1F). Immunoprecipitation of FN from thrombin-stimulated platelet supernatants resulted in significant co-precipitation of HGF (Fig. 1E). In contrast, minimal levels of HGF was observed in samples derived from unstimulated platelet supernatants or from samples derived from thrombin-stimulated platelet supernatants when an isotype-matched control antibody was employed in the experiment. Probing of the same blot with antibodies to FN confirmed that the primary precipitation of FN was responsible for the co-precipitation of HGF (Fig. 1E, lower panel). In a parallel experiment, immunoprecipitation of VN also co-precipitated HGF to a similar if not greater extent than FN (Fig. 1F). These experiments demonstrate that HGF is released from platelets and is found in the form of soluble molecular complexes with both FN and VN, confirming the results of the ligand binding studies in vitro.
HGF-Induced endothelial cell migration is dependent upon co-stimulation with ECM
We next sought to determine whether the responses of endothelial cells to HGF could be modulated by its ECM binding partners. In cell migration assays, human microvessel endothelial cells (HMVEC) were incubated with an optimal concentration of HGF alone or in combination with fixed concentrations of FN, VN or collagen-1 (Fig. 2A). Significantly, little or no endothelial cell migration above basal levels (control) was observed when cells were stimulated with HGF (10 ng/ml) in the absence of ECM. A moderate migratory response of endothelial cells to HGF was observed in the presence of collagen-1 (non-HGF binding ECM), which was less than 2-fold above basal levels. When HGF was co-administered with either FN or VN, endothelial cell migration was significantly enhanced by 4–5 fold. The differences in magnitude of the migration in the presence of these ECM glycoproteins was not related to variable degrees of cell adhesion upon the transwell filters as HGF-stimulated endothelial cells adhered equally well to ECM glycoprotein-coated transwells (Fig. 2B). The migratory response to HGF was dose responsive with a maximal response observed at a concentration of 10–20 ng/ml (data not shown). In addition, a negligible migratory response was observed when HMVEC were stimulated with these ECM molecules in the absence of HGF consistent with our previous report (data not shown, ref .9).
Figure 2 Effect of ECM molecules on HGF induced Endothelial cell migration. Panel A-Dye-loaded HMVEC suspensions (9 × 104/ml) were treated with HGF (10 ng/ml) in the presence or absence of ECM molecules, FN or VN or collagen-1 in a modified Boyden chamber assay. Migration of cells was measured at 3 hours. The data is presented as relative migration with the migratory response presented as ratio to the basal migration in the absence of stimulus. Panel B-The levels of cell adhesion to Fluorblok transwell filters coated with either poly-L-lysine (PLL) or the various ECM glycoproteins was determined. Dye loaded HMVEC (105 cells) were treated with HGF (10 ng/ml) for 30 min prior to application to upper chamber of the transwell. Following extensive washes the number of adherent cells was determined using a fluorescence plate reader. The role and identity of integrins mediating the migration response in cells stimulated with HGF and FN (panel C) and HGF and VN (panel D) was demonstrated by the pre-treatment of HMVEC suspensions with anti-integrin monoclonal reagents (10 μg/ml) with specificity for integrins α5β1 (JBS5), αvβ3 (LM609) and αvβ5 (LM142) for 30 min at room temperature prior to application into the upper transwell chamber. The data is presented as specific migratory response in fluorescence units with the basal migration subtracted from the total migratory response. (n = 2).
To further characterize the degree and identity of integrin involvement in the observed migratory response, we investigated the consequences of blocking integrin receptors on HMVEC with specific integrin antibodies prior to HGF-ECM stimulation. Antibodies directed to the integrin α5β1 completely inhibited HGF-FN-induced endothelial migration (Fig. 2C). In contrast, an antibody with specificity for the αv-subunit (LM142) had no inhibitory effect on endothelial cell migration. However, antibodies to the αvβ3 integrin (LM609) did inhibit endothelial cell migration to HGF-FN by 20% suggesting an ancillary role for this integrin in mediating HGF-FN responses. When endothelial cell migration was induced by HGF-VN complexes, the integrin dependence shifted as expected (Fig. 2D). Under these conditions endothelial cell migration was predominantly dependent on αv-integrins for mediating the migratory signal with some apparent involvement of the integrin α5β1 (approximately 30%). This latter effect may be a consequence of integrin signal cross-talk (transdominant integrin regulation), as reported previously [14,15]. These experiments demonstrate that for HMVEC, HGF induced cell migration is dependent upon the ligation of integrins by ECM molecules.
Met associates with αvβ3 and α5β1 integrins
Previous work [5-7,9] has demonstrated that the physical association of growth factor receptor tyrosine kinases and integrins promote enhanced cellular responses. We, therefore, postulated that the elevated cell migration induced by HGF-FN and HGF-VN in the present study could be due to a signalling mechanism involving the physical association between Met and integrins on endothelial cells. As shown in Fig. 3A, endothelial cell lysates derived from samples exposed to collagen-1, FN or VN, in the presence of HGF, when immunoprecipitated with antibodies to integrins α2β1, α5β1 and αvβ3 respectively, predominantly co-precipitated Met with the integrins α5β1 and αvβ3. In contrast, Met co-precipitation with the integrin α2β1 was minimal for lysates derived from cells stimulated with HGF and collagen-1. The level of Met expression in these samples was not altered by treatment of the cells with various combinations of HGF and ECM molecules (Fig. 3A lower panel) discounting the possibility that the differences in the level of Met co-precipitation was due to differences in the expression levels of its antigen. In the absence of HGF, co-precipitation of Met with the integrins α5β1 and αvβ3 was minimal despite the presence of the ECM glycoprotein, indicating that ligation of the integrin with its cognate ligand was not sufficient to induce an association with Met.
Figure 3 Association of Met with integrins is driven by HGF-FN and HGF-VN complexes. Panel A-HMVEC (1 × 106 /ml) were seeded onto collagen-1, FN or VN coated plastic wells and were stimulated with either saline or HGF (10 ng/ml) for 60 min at room termperature. Cells were lysed and the corresponding lysates (500 μg protein) were immunoprecipitated with antibodies to integrins α2β1 (lanes 1 & 4), αvβ3 (lanes 2 & 5) and α5β1 (lanes 3 & 6). Immunocomplexes were analysed by SDS-PAGE and Western blotting probing with an anti-Met antibody using chemiluminescence detection (top panel). The bottom panel shows the antigen levels of Met in the corresponding cell lysates after analysis by SDS-PAGE (40 μg/lane) and Western blotting probing with an antibody to Met were essentially unaltered during the duration of the experiment. Panel B-HMVEC (5 × 106) were stimulated with HGF or HGF-FN or HGF-VN complexes at the indicated time periods at room temperature. Cells were lysed and immunoprecipitated with a polyclonal antibody to phosphotyrosine Met and the immune complexes analysed by SDS-PAGE and Western blotting using a monoclonal antibody to Met. The Met antigen was detected using chemiluminescence. Panel C-HMVEC (5 × 106) were stimulated with HGF or HGF-FN or HGF-VN complexes for 15 minutes at room temperature. Cells were lysed and the lysates immunoprecipitated with monoclonal antibodies to the integrins α5β1 and αvβ3 and the immune complexes analysed by SDS-PAGE and Western blotting probing with a polyclonal antibody to Met.
To elucidate the role of Met activation in the formation of the Met-integrin signalling complex, endothelial cells were treated with HGF in the absence of ECM glycoprotein and with HGF-FN and HGF-VN complexes and the kinetics of Met tyrosine phosphorylation investigated (Fig. 3B). These experiments demonstrated that HGF in the absence of ECM glycoprotein could activate Met transiently with a strong signal present at 15 min but absent at 1 hour. In contrast, cells stimulated with HGF-FN and HGF-VN showed strong activation of Met at 15 min, which was sustained at 1 hour and was evident, although reduced, at 2 hours post-stimulation. Cell lysates derived from samples stimulated for 15 mins were also assessed for the presence of a Met-integrin complexes. As shown in Fig. 3C, HGF in the absence of FN or VN did not promote a significant association of Met with the integrins α5β1 or αvβ3. However, cells treated with HGF-VN and HGF-FN for 15 min contained significant levels of Met in a physical association with these integrins. These studies show that Met activation by HGF is insufficient to promote a physical association with integrins.
HGF binding domains on FN and VN promote enhanced intracellular signals
We next investigated whether the association of Met with integrins modulated HGF/ECM-induced intracellular signalling, focussing on the ERK and the PI-3 kinase pathways. Analysis of the phosphorylation kinetics of Erk-1/2 in response to HGF alone or HGF/ECM combinations showed distinct patterns of activation (Fig. 4A). With HGF alone, Erk 1/2 phosphorylation showed kinetics with a peak signal at 60 min post-stimulation and significant reduction by 120 min although phosphorylation was still apparent. A distinct activation profile was observed when cells were stimulated with HGF and collagen-1 (non-HGF binding ECM), with Erk 1/2 levels peaking at 30 min and returning to near basal phosphorylation levels by 120 min. However, stimulation of endothelial cells with either HGF-FN or HGF-VN complexes promoted a rapid but sustained phosphorylation of Erk 1/2 with levels near maximal at 120 min post-stimulation. Analysis of the activation of the PI-3 kinase pathway was assessed by measurement of the phosphorylation status of Akt/PKB on Ser473 (Fig. 4B). Interestingly, both distinct levels and kinetics of Akt phosphorylation were observed in these samples. When endothelial cells were stimulated with HGF in the absence of ECM co-stimulation, little phosphorylation of Akt above basal levels was observed. However, when cells were treated with HGF plus collagen-1, Akt phosphorylation was rapidly detected at 5 min and peaked at 30 min with significant reduction by 120 min. In contrast, with cells treated with either HGF-FN or HGF-VN complexes, Akt phosphorylation kinetics appeared to mirror Erk 1/2 phosphorylation kinetics implying a common regulatory mechanism for both pathways. As with Erk 1/2 phosphorylation, Akt phosphorylation peaked by 30 min post stimulation and this level of activation was sustained even at 120 min. Significantly, Akt phosphorylation levels in these samples were elevated approximately 3-fold, (assessed by densitometry) compared with the levels in observed in cells stimulated with HGF plus collagen-1 (data not shown).
Figure 4 HGF-FN and HGF-VN complexes augment HMVEC migration via the PI3 kinase pathway. Panel A-HMVEC suspensions (1 × 106/ml) were stimulated with HGF (10 ng/ml) in the absence of ECM molecules or in the presence of collagen-1, FN or VN (2 μg/ml) for varying time intervals (2–120 min) at room temperature. The reaction was stopped by rapid centrifugation and lysis of the cell pellet. Samples of cell lysate were analysed by SDS-PAGE and Western blotting probing with an antibody with specificity for phosphorylated Erk 1/2 (top panel). The blots were reprobed with an antibody to Erk 2 (bottom panel). Panel B-The samples from panel A were also probed simultaneously with an antibody with specificity for phosphorylated Akt (top panel) and these blots were stripped and re-probed with an antibody to Akt. Visualization was by chemiluminescence. Panel C &D-Effect of inhibitors on HMVEC migration in response to HGF-FN and HGF-VN respectively. The data is a representative experiment using triplicate samples which was performed three times giving essentially similar results.
HGF/ECM-Induced endothelial migration is coupled to the PI-3 kinase pathway
To determine the intracellular pathway(s) that were coupled to the migratory response, HMVECs were treated with specific inhibitors of MEK and PI-kinase. In cell migration assays, LY294002 but not U1026 inhibited endothelial cell migration induced by HGF-FN (Fig. 4 C) and HGF-VN (Fig. 4 D), clearly demonstrating that the PI-3 kinase pathway was predominantly coupled to the migratory response and not the Map kinase pathway. Other inhibitors of potential down stream effectors were also tested. HGF-FN stimulated cells pre-treated with PP1, U73122, and piceatannol showed maximal migratory responses indicating that Src, PLCβ and Syk were not components of the migratory signal (data not shown).
HGF-Induced endothelial proliferation is coupled to the Erk-pathway
The effect of the co-administration of ECM molecules with HGF on endothelial cell proliferation was also investigated. In contrast to cell migration, HGF, in the absence of ECM molecules, induced a significant proliferative response (Fig. 5A). However, in the presence of FN or VN, HGF-induced endothelial proliferation was enhanced compared to HGF alone or in combination with collagen-1. As with the migratory response, endothelial cell proliferation was dose responsive to HGF with an observed maximal response at a concentration of 10–20 ng/ml (data not shown). Chemical inhibitors were then used to determine the signalling pathways involved in HGF-induced endothelial cell proliferation. In these studies, the MEK inhibitor, U1026 significantly impaired HGF-induced endothelial proliferation (50–80% inhibition) irrespective of co-stimulation with or without ECM molecules. This suggests that unlike migration, which was shown above, to be PI-3 kinase dependent, the Erk-pathway plays an important role in mediating HGF-induced endothelial cell proliferation. While both LY294002 and FPT-III blocked HMVEC proliferation, this appeared to be due to apoptosis (data not shown). This observation is consistent with the role of PI-3 kinase in promoting cell survival and a role for Ras in regulating PI-3 kinase in these cells [16,17].
Figure 5 HGF induced HMVEC proliferation requires the Map kinase pathway. Panel A-HMVEC in MCDB-131 medium was plated on poly-D-lysine coated 48-well plates at a density of 2.5 × 103 cells/well and were stimulated with HGF (10 ng/ml) in the presence and absence of ECM molecules FN, VN or collagen-1. Basal proliferation was measured with cells treated with non-supplemented medium. Cell numbers were quantified 48 hours post-stimulation using CyQuant reagent. Data is presented as cell numbers with the increase of proliferation of HGF-FN and HGF-VN treated samples significant compared with cells treated with HGF alone as determined by a one-way ANOVA (p < 0.05) n = 3. Panel B-HMVEC were plated onto poly-D-lysine coated wells (1 × 104/well) overnight in supplemented medium. Cells were then incubated with medium comprising 0.1% FBS plus HGF (20 ng/ml) in the presence or absence of ECM proteins (10 μg/ml) as shown containing no inhibitor (white bars) or the MEK inhibitor U1026 (10 μM, black bars). Cells numbers were quantified after a further 48 h using CyQuant reagent.
Ras is a specific, upstream regulator of Erk and PI-3 kinase pathways in cells stimulated with HGF-FN and HGF-VN complexes
The data shown above indicate that HGF-induced endothelial cell migration and proliferation were mediated by PI-3 kinase and Erk pathway respectively. We next investigated the role of Ras in regulating these two pathways induced by HGF-FN and HGF-VN complexes. Since Ras is a well-documented regulator of p85 PI-3 kinase and Erk and as well as a down stream effector of both the Met and integrin receptors, we assessed the activation status of Ras by measuring the comparative levels of GTP-loaded Ras after endothelial cells were stimulated with HGF in the presence and absence of ECM molecules (Fig. 6A &6B). Endothelial cells stimulated with HGF alone showed high levels of GTP-Ras at 60 min post-stimulation and this was sustained even at 120 min (Fig. 6A). In contrast, cells co-stimulated with HGF and collagen-1 showed activation of Ras at 60 min post-stimulation but to a significantly lower degree (approx 50% compared to HGF alone Fig. 6B), with the signal diminished by 120 min. With HGF-FN and HGF-VN co-stimulation, GTP-Ras levels were more than two-fold higher than observed when cells were co-stimulated with HGF-collagen-1 (Fig. 6B). Significantly, GTP-Ras levels were sustained at 120 min consistent with the observations of the activation profiles for the MAP kinase and PI-3 kinase pathways. These studies suggested that inhibiting Ras in cells stimulated with HGF-FN and HGF-VN complexes would exhibit reduced migration responses. To test this hypothesis, cells were treated with the membrane permeable farnesyltransferase inhibitor FPT-III, which inhibits Ras function as a consequence of the loss of membrane localization in the absence of farnesylation. Upon stimulation with HGF-FN, endothelial cells treated with FPT-III (100 μM) showed little activation of Ras following HGF-FN stimulation compared to basal levels in unstimulated cells (Fig. 7A). In comparison, pre-treatment of cells with the geranylgeranyl transferase inhibitor GGTI (2 μM) had little inhibitory effect on HGF-FN induced Ras activation. The effect of these inhibitors was tested in endothelial migration assays. Endothelial cells pre-treated with FPT-III displayed a profound reduction in cell migration of 50% and 73% when stimulated with HGF-FN and HGF-VN complexes respectively compared to cells pre-treated with GGTI (Fig. 7B). In contrast, FPT-III had little inhibitory effect on migration induced by HGF plus collagen-1 indicating that Ras has no significant role in the regulation of the migratory signal with this stimulus.
Figure 6 Enhanced and sustained activation of Ras by HGF-FN and HGF-VN Complexes Panel A-Kinetic GTP-Ras pull down analysis. HMVEC suspensions (3 × 106/ml) were stimulated with HGF (10 ng/ml) in the absence and presence of ECM molecules (2 μg/ml) as shown for 5, 60 and 120 min at room temperature. Lane Rest. represents resting (unstimulated) levels of GTP-Ras. Cells were pelleted and lysed in cold MLB buffer (see methods). Samples of cell free lysates were incubated with of RBD-Sepharose and then analysed by SDS-PAGE and Western blotting probing for Ras. Visualization was by chemilumininescence using a Kodak imaging station. Panel B-The levels of GTP-Ras at the 60 min time point from the gel shown in panel A were quantified by densitometric analysis using ImageQuant software (Kodak).
Figure 7 HGF-FN and HGF-VN induced HMVEC migration is Ras dependent. Panel A-GTP-Ras pull down assay showing the affects of FPT III (100 μM) and GGTI (2 μM) inhibitors upon GTP-Ras levels. HMVEC suspensions (3 × 106/ml) were pre-treated with FPT III and GGTI inhibitors for 45 min at room temperature prior to stimulation with HGF-FN complexes for 60 min at room temperature. Samples were analysed as mention in the legend to Fig 2. Panel B-Effect of Ras inhibition upon cell migration. Calcein M loaded HMVEC suspensions (1 × 105/ml) were pre-treated with the inhibitors as mentioned above prior to application to the top chamber of the transwell filter. Cells were stimulated with HGF plus ECM proteins (placed in the bottom chamber) as shown. Cell migration was measured at 3 hours post stimulation using a fluorescence plate reader. The data is shown as relative migration and is the combined data from two independent experiments with sample wells in triplicate.
To further characterise the role of Ras in regulating endothelial cell responses to HGF/ECM, the effect of the FPT-III inhibitor on the phosphorylation levels of Erk 1/2 and Akt was investigated. In cells stimulated with HGF alone, Erk 1/2 was activated and significantly inhibited by the FPT-III inhibitor and to a lesser extent by GGTI (Fig. 8A). A similar inhibitory profile was observed for cells stimulated with HGF-FN (Fig. 8A) and HGF-VN (not shown). In contrast, cells stimulated with HGF and collagen-1 showed no apparent reduction in Erk 1/2 phosphorylation levels when pre-treated with the FPT-III inhibitor suggesting little involvement of Ras in the activation of Erk 1/2. These samples were also assessed for Akt phosphorylation as an indication of PI-3 kinase activity. Consistent with our observations, stimulation of HMVEC with HGF in the absence of ECM did not lead to a significant activation of Akt (Fig. 8B). However, in the presence of collagen, Akt activation was observed but this was not affected by pre-treatment of the cells with FPT-III implying that Ras was not an upstream regulator of the activation of PI-3 kinase. In contrast, HGF-FN complexes promoted a 3-fold enhancement of Akt phosphorylation and this was inhibited by approximately 50 % by treating the cells with FPT-III (Fig. 8B). These observations suggest that the inhibition of Ras activation reduces the activation of PI-3 kinase for cells stimulated with HGF-FN complexes and not cells stimulated with HGF and collagen-1. The data would therefore predict that when HMVEC are stimulated with HGF-FN and HGF-VN complexes, specific integrins are utilized to recruit Ras, which in turn would regulate the activation of PI-3 kinase. To test this hypothesis, we immunoprecipitated integrins α5β1 and α2β1 from cells stimulated with HGF-FN and HGF with collagen-1 respectively and analysed these integrin immune complexes for the co-precipitation of Ras. High levels of Ras was specifically associated with α5β1 immune complexes and this appeared be independent of HGF stimulation. Little or no Ras was co-precipitated with the integrin α2β1 (Fig. 8C).
Figure 8 HGF-FN Induced Activation of Map Kinase and PI-3 Kinase Pathways is Ras dependent Panel A &B-HMVEC suspensions were pre-treated with buffer (no inhib.) or inhibitors FPT III (100 μM) and control GGTI (2 μM) for 45 min at room temperature prior to stimulation with HGF with no ECM proteins, or HGF plus collagen-1 or HGF-FN complexes for 60 min at room temperature. Cells were pelleted and lysed in an ice-cold lysis buffer. Samples were analysed by SDS-PAGE and Western blotting probing for phospho Erk 1/2 (panel A) and phospho AKT (panel B). The relative band intensities measured by image analysis software have been placed above each band as a ratio of the signal obtained in the sample where no inhibitor was present. Blots were stripped and re-probed with antibodies to Erk 2 and Akt to confirm equal loadings. Panel C-HMVEC suspensions (5 × 106/ml) were incubated with either FN or collagen in the presence or absence of HGF as shown for 60 min at room temperature. Cells were pelleted and lysed in lysis buffer. Immunoprecipitation analyses of the lysates were performed with antibodies to integrins α5β1 and α2β1 and the immune complexes analysed by SDS-PAGE and Western blotting probing with antibodies to Ras. Visualization was by chemiluminescence.
Discussion
The major finding of the present report is that HGF-induced endothelial cell responses are significantly augmented through the formation of molecular complexes between this growth factor and the ECM glycoproteins FN and VN. The significance of this finding is highlighted by the observation that HGF-induced endothelial cell migration, a PI-3 kinase coupled response, did not occur in the absence of additional signals originating from the ECM. However, HGF-induced endothelial cell proliferation was evident in the absence of signals emanating from the ECM. These observations have led us to propose a model for the mechanisms of HGF-induced responses in endothelial cells (Fig. 9). This model shows that HGF alone can induce endothelial cell proliferation through its receptor Met via activation of the Ras-Erk kinase pathway (Fig. 9A). However, this signal is insufficient to promote significant cell migration for which an additional signal(s) from the ECM via specific integrin ligation appears necessary for activation of the PI-3 kinase pathway (Fig. 9B &9C). Uniquely, in cells stimulated with HGF-FN or HGF-VN complexes, which promotes the association of Met with integrins, an enhanced and unique intracellular signal is generated by the recruitment and sustained activation of Ras, which presumably, concomitantly activates both p85 PI-3 kinase and Raf (Fig. 9C). This is in contrast to the mechanism of activation of the PI-3 kinase pathway induced by HGF in the presence of collagen-1, which is Ras independent (Fig. 9B).
Figure 9 Mechanisms of HGF induced cellular responses in endothelial cells. Panel A-HGF in the absence of ECM molecules can activate the Map kinase pathway through the Met receptor tyrosine kinase leading to a proliferative response. Panel B-HGF and co-stimulation with collagen induces activation of both the Map kinase and PI-3 kinase pathways through an unknown mechanism presumably through integrin ligation, which is Ras independent. Panel C-HGF-FN (and by analogy HGF-VN) complexes promote enhanced cellular responses by promoting the association of integrins with the Met receptor leading to the recruitment and enhanced activation of Ras, Erk 1/2 kinases and PI-3 kinase promoting both elevated proliferative and migratory responses.
The model in Fig. 9C is supported by the following observations. The enhanced responses of HMVEC to HGF-FN and HGF-VN complexes is consistent with the observation that in these cells the activity of Ras, PI-3 kinase (AKT phosphorylation) and Erk 1/2 phosphorylation were sustained, and in the case of Ras and Akt, were 2–3 fold higher than observed in cells stimulated with HGF and collagen-1 (Fig. 6). The distinct signalling mechanisms induced by the co-activation of endothelial cells with HGF in the presence of a binding and non-binding ECM glycoprotein partner was also supported by the observation that treatment of cells with the inhibitor of Ras farnesylation, FPT III, reduced the phosphorylation of both Erk 1/2 and Akt kinases in cells stimulated with HGF-FN but not HGF plus collagen-1 (Fig. 8A &8B). Furthermore, Ras co-precipitated with the integrin α5β1 derived from endothelial cell lysates stimulated with HGF-FN complexes but not with the integrin α2β1 derived from cells stimulated with HGF and collagen-1 (Fig. 8C). These results are consistent with the pioneering work by Rodriquez-Viciana and colleagues who demonstrated the regulation of p85 PI-3 kinase by Ras via direct molecular interaction. It is now known that the regulatory subunit of all type 1, PI-3 kinases contain a Ras binding domain that associates with activated Ras (GTP-Ras) [17-20]. Therefore, our data and model demonstrating the sustained activation of Ras and PI-3 kinase by stimulation of endothelial cells with HGF-FN and HGF-VN complexes is consistent with previous work showing Ras to be a key regulator of PI-3 kinase. The identity of the Ras subtypes mediating the regulation of PI-3 kinase in our cell system is currently under investigation.
The results of the present study both support and extend our previous observations of the enhanced endothelial cell migration induced by VEGF-FN molecular complexes [9]. In that study, VEGF binding domains identified on FN drove the formation of VEGF-FN complexes that upon receptor ligation promoted the association of the integrin α5β1 with VEGFR-2. This co-receptor activation gave rise to a sustained activation of the Erk kinase activity, which promoted an enhanced migratory response. Similarly, the present work has shown that HGF-FN and HGF-VN molecular complexes induce the formation of Met-integrin signalling complexes promoting the transduction of a unique Ras-dependent signal. Several studies have illustrated the significance of the cooperation between integrins and growth factor receptor tyrosine kinases in mediating cellular responses. For example, the proliferation and migration of fibroblasts in response to PDGF-BB was enhanced in the presence of VN and was accompanied by the physical association of the αvβ3 integrin with the PDGF-β receptor [5,7]. Furthermore, it was recently demonstrated that HGF in combination with FN prolongs the survival of GM-colony-forming cells [21] and enhanced the adhesion and motility of MTLn3 breast carcinoma cells [22]. In addition, integrins αvβ3 and αvβ5 were shown to be necessary for mediating FGF-2 and VEGF mediated angiogenesis respectively by the differential regulation of components of the Erk kinase pathway [23]. However, the present study extends these observations and is, to our knowledge, the first description of a distinct signalling pathway employed by the activity of growth factor-ECM molecular complexes as opposed to growth factors and ECM proteins functioning independently through ligation of their respective receptors. The identification of a Ras-dependent pathway in endothelial cells specifically activated with HGF-FN and HGF-VN complexes as opposed to HGF in the presence of collagen-1 is significant and correlates with Met-integrin association. Although the precise nature of the interaction between the Met tyrosine kinase and integrins was not elucidated, the role of Ras in this system appears important for the sustained and enhanced activation of the PI-3 kinase and Erk kinase pathways.
In contrast to the migratory signals promoted by VEGF-FN molecular complexes [9], HGF-FN and HGF-VN complexes induce a response in endothelial cells characterized by a tight coupling of the PI-3 kinase pathway to cell migration. Several additional pro-angiogenic mediators such as sphingosine 1-phosphate and NO, or the activation of CD40 and Eph B4 receptors by their counter ligands, promote endothelial cell migration through activation of the PI-3 kinase pathway [24-28]. In addition, HGF on its own was shown to stimulate smooth muscle cell migration in a PI-3 kinase dependent manner [29]. However, the lack of a significant migratory response, coupled with the absence of Akt phosphorylation observed in the present study, suggests that in primary endothelial cells the Met receptor is unable to activate PI-3 kinase without cooperative signals from the ECM/integrins. This observation is intriguing bearing in mind that Met has been shown to activate PI-3 kinase in epithelial cells via recruitment and activation of Gab-1, which directly interacts with the p85 subunit [30]. Consistent with our observation of an integrin dependency for signal transduction, Trusolino et al showed that in carcinoma cell lines Met induced signals were considerably amplified as a consequence of its constitutive association with the integrin α6β4. Intriguingly, in this system the authors showed that the role of the integrin α4 subunit was independent of extracellular integrin ligation since a truncated α4 construct lacking its extracellular portion could mediate HGF/Met responses and signals to downstream effectors provided that its ability to recruit the adaptor Shc was not affected [31]. In contrast, our studies using primary endothelial cells showed that integrin ligation was essential for generating a significant migratory signal via PI-3 kinase and in the case of HGF-FN and HGF-VN complexes, for promoting the association of Met with the integrins α5β1 and αvβ3 respectively. Indeed, Met association with the integrins α5β1 and αvβ3 was dependent upon the activation of both Met and integrins through ligation of their cognate ligands since tyrosine phosphorylation of Met by HGF alone could not induce integrin association (Fig. 3C). These observations support the contention of a signalling mechanism requiring the formation HGF-ECM molecular complexes as a prerequisite for Met-integrin association and consequent signal amplifiation as proposed in Fig. 9C. However, the importance of integrin cytoplasmic domains in recruiting Ras and Ras-binding partners appears to reflect a common mechanism of HGF signal transduction between these cellular systems.
Conclusions
The results of the present work demonstrate an important mechanism by which integrins collaborate with growth factor receptor tyrosine kinases on endothelial cells and predict that HGF binding domains on both FN and VN may play a significant role in promoting wound healing and post-natal neovascularization. In support of this contention, HGF-FN and HGF-VN complexes were identified in the supernatants derived from degranulated platelet suspensions indicating that these complexes do exist in vivo and may be deposited at sites of vessel perturbation or injury. This observation is similar to the identification of VEGF-FN molecular complexes in platelet supernatants in our previous report [9] and suggests that HGF and VEGF may act synergistically in vivo. Indeed, recent studies have shown that HGF synergises with VEGF to promote capillary-tube assembly in collagen matrices and neovascularization in the rat cornea [11]. Furthermore, HGF positively regulates VEGF expression and down regulates TSP-1, an inhibitor of angiogenesis, thereby promoting angiogenesis [32]. It is noteworthy that the HGF binding domains for FN were located in the same proteolytic fragments as those of VEGF, namely the N-terminal 70 kDa and C-terminal 40 kDa fragments. Further studies involving the fine mapping and characterization of the binding domains for VEGF and HGF on FN and VN should help decipher the mechanism of interplay between these important pro-angiogenic mediators.
Methods
Solid phase assay and Surface Plasmon Resonance Analysis (SPR)
ECM proteins and FN peptides were purchased from Sigma and Gibco and were further purified by gel filtration and ion exchange chromatography. The assay was performed as described previously [9]. 125I-HGF (NEN) in binding buffer (PBS containing 2% BSA) were added to the microtitre plates and incubated for 30 min at room temperature (RT) before washing and counting to determine bound radioactivity. SPR analysis was performed on the BIAcore X (Biacore Herts UK) as described previously [9]. HGF (30–500 nM) was injected across the FN 70 kDa fragment immobilised on a CM5 chip in HEPES saline (pH 7.4) supplemented with 1 mM MgCl2, 2 mM CaCl2 and the sensograms recorded. The data was analysed by the ASSAY programme (Biosoft, UK) in order to determine the EC50 value and Kd.
Migration and proliferation assays
Human dermal microvessel endothelial cells (HMVEC) were maintained in EBM-2 growth medium (Clonetics Corp). Migration studies were carried out essentially as described previously [9] using serum starved Calcein AM-loaded HMVEC in a modified Boyden chamber assay using Fluorblok transwell chambers (BD Bioscience) as described by the manufacturer. Cell migration was detected by fluorescence measurement (within the lower chamber compartment). Membranes of transwell chambers were coated with either FN or VN or collagen-1 (10 μg/ml) overnight at 4°C and preliminary experiments were performed to assess the optimal dosage of both HGF and ECM protein. With antibody inhibition studies, the transwell chamber was coated with poly-L-lysine (Sigma) to facilitate cell attachment to the filters as opposed to adhesion using ECM glycoproteins. HMVEC were pre-treated with αvβ3 and α5β1 integrin blocking antibodies for 30 min at room temperature prior to application to the upper transwell chamber. The levels of cell adhesion to ECM-coated transwell filters were determined by allowing HGF-stimulated HMVEC to adhere to transwells (coated overnight with ECM glycoprotein (10 μg/ml) and then blocked by incubation in 3.5 mg/ml BSA in basal culture medium) for 1 hour at room temperature followed by extensive washes with basal culture medium. The remaining cells were measured using a fluorescence plate reader (measuring fluorescence in the upper transwell compartment). For proliferation experiments, cell division was measure by fluorescence labeling of DNA (CyQuant, Molecular Probes). HMVEC was plated on poly-D-lysine coated 48-well plates and cultured overnight in MCDB-131 medium containing 5% FBS. After washing plates with PBS, endothelial cells were then cultured in MCDB-131 medium + 0.1% FBS containing HGF (10 ng/ml) in the presence or absence of VN, FN or Collagen-1 (10 μg/ml). Cells incubated for 48 h and HGF/ECM was added every 24 hours. Cell proliferation was quantified using a fluorescence plate reader.
Phosphorylation analysis and ras activation
HMVEC were assessed for the activation profiles of Erk1/2 and Akt using phosphospecific antibodies (Cell Signalling Technology) to Erk (Thr202/Tyr204) and Akt (Ser473) respectively by Western blotting. These studies were performed with both cells in suspension and with adherent populations. Cells were grown to 80% confluence and serum starved for 2 hours prior to harvesting. Cells were resuspended in serum-free MCDB-131 medium (BioWhittaker) supplemented with 0.1% BSA (resuspension buffer) at a concentration of 1–5 × 106 cells /ml. The cell suspensions were challenged 10 ng/ml HGF supplemented with 2 μg/ml collagen-1, or FN or VN for various durations ranging from 2 to 120 min at room temperature. Cells were harvested by centrifugation at 4°C and lysed in 10 mM Tris pH 7.4, 145 mM NaCl supplemented with 0.1% Triton X-100 and protease inhibitors. For inhibitor studies, serum-starved HMVEC suspensions were pre-treated with the inhibitor for 45 min prior to stimulation with HGF and ECM molecules for a further 60 min at room temperature. The cells were pelleted, washed in ice-cold resuspension buffer without BSA and lysed in a lysis buffer containing 1% (v/v) Triton X-100. Cell lysates were analysed by Western blotting using protocols specific for the phosphospecific antibodies according to the manufacturer's recommendations. Blots were cut along appropriate marker divides and probed with antibodies to phopho Erk 1/2 and Akt (Ser473) (Cell Signaling Technologies) simultaneously. For GTP-Ras pull down assays, serum-starved HMVEC were stimulated with HGF and ECM molecules for a desired time point and the cells were spun down and washed in ice-cold resuspension buffer without BSA. Cell pellets were lysed in MLB buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 1 % Igepal CA-630, 10% Glycerol, 25 mM NaF, 10 mM MgCl2, 1 mM EDTA, 1 mM sodium orthovanadate and protease inhibitor cocktail) and 500 μg of cell lysate was mixed with a 10 μl suspension of RBD-Sepharose (Upstate Biotechnology) for each reaction at 4°C for 60 min. Sepharose beads were spun down and washed in MLB prior to solubilization and analysis by Western blotting probing for Ras using a monoclonal antibody (Upstate Biotechnology). For Ras inhibition studies cells were pre-incubated with FPT-III (100 μM) and GGTI (2 μM) (approx 40 × IC50 values) for 45 min at room temperature prior to cell stimulation for 60 min with HGF and ECM.
Met-Integrin immunoprecipitation
Human microvessel endothelial cells (HMVEC) in serum-free MCDB-131 medium (BioWhittaker) supplemented with 0.1% BSA were plated on collagen, FN and VN coated petri dishes in the absence or presence of HGF (50 ng/ml) for 15 min to 1 hour at room temperature. Cells were then harvested as described previously [9] and integrin immunoprecipitation was performed with monoclonal antibodies (Chemicon) to α2β1 (clone JBS2), α5β1 (clone JBS5) and αvβ3 clone (LM609). After analysis by SDS-PAGE and protein transfer, the blot was then probed with a monoclonal to Met (clone DL-21, Upstate Biotechnology) and developed by chemiluminescence. For Met tyrosine phosphorylation analysis, cells were stimulated with HGF alone or HGF-FN and HGF-VN complexes for various time points ranging from 15 mins to 2 hours at room temperature. Lysed samples were immunoprecipitated with a polyclonal anti-phosphoMet antibody (Cell Signalling) and the immune complexes analysed by SDS-PAGE and Western blotting using a monoclonal ant-Met antibody (Upstate). Met was visualised using chemiluminescence technology (Pierce).
Immunoprecipitation of FN-HGF and VN-HGF complex from platelet supernatants
Supernatants from non-stimulated and thrombin-stimulated washed platelet suspensions were prepared as previously described [9]. Supernatants were immunoprecipated with an antibody to FN or VN (Chemicon) or an isotype matched control reagent (IgG). Following SDS-PAGE and immunoblotting, HGF was detected with a polyclonal antibody (Santa Cruz) by chemiluminescent development.
Abbreviations
HGF, hepatocyte growth factor, FN fibronectin, VN vitronectin, Col-1, collagen-1, HMVEC, human microvessel endothelial cells.
Authors' contributions
SR, study design, HMVEC migration studies, SPR ligand binding analysis, HMVEC signalling studies, co-immunoprecipitation studies (integrin-Ras), manuscript preparation. ESW, study design, HMVEC proliferation studies, solid-phase ligand binding studies, co-imunoprecipitation (integrin-Met), manuscript preparation. YMP, study design, co-imunoprecipitation studies (HGF-FN/VN). JM, protein purification and preparation, KVP, GTP-Ras pull-down assays, RS, technical support. MS, study design.
Acknowledgements
SR and YP thank Grifols UK and Centeon for sponsorship respectively. This study was funded in part by a grant from the American Heart Association to ESW
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| 15717924 | PMC553973 | CC BY | 2021-01-04 16:39:11 | no | BMC Cell Biol. 2005 Feb 17; 6:8 | utf-8 | BMC Cell Biol | 2,005 | 10.1186/1471-2121-6-8 | oa_comm |
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BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-6-161571003910.1186/1471-2164-6-16Research ArticleA class of models for analyzing GeneChip® gene expression analysis array data Fan Wenhong [email protected] Joel I [email protected] James M [email protected] Najma [email protected] Lue Ping [email protected] Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, WA 98109, USA2 Clinical Research Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, WA 98109, USA2005 14 2 2005 6 16 16 28 7 2004 14 2 2005 Copyright © 2005 Fan et al; licensee BioMed Central Ltd.2005Fan et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Various analytical methods exist that first quantify gene expression and then analyze differentially expressed genes from Affymetrix GeneChip® gene expression analysis array data. These methods differ in the choice of probe measure (quantification of probe hybridization), summarizing multiple probe intensities into a gene expression value, and analysis of differential gene expression. Research papers that describe these methods focus on performance, and how their approaches differ from others. To better understand the common features and differences between various methods, and to evaluate their impact on the results of gene expression analysis, we describe a class of models, referred to as generalized probe models (GPMs), which encompass various currently available methods.
Results
Using an empirical dataset, we compared different formulations of GPMs, and GPMs with three other commonly used methods, i.e. MAS 5.0, dChip, and RMA. The comparison shows that, on a genome-wide scale , different methods yield similar results if the same probe measures are chosen.
Conclusion
In this paper we present a general framework, i.e. GPMs, which encompasses various methods. GPMs permit the use of a wide range of probe measures and facilitate appropriate comparison between commonly used methods. We demonstrate that the dissimilar results stem primarily from different choice of probe measures, rather than other factors.
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Background
Microarray experiments are routinely conducted to assess associations of experimental factors (or disease outcomes) with gene expression profiles. The Affymetrix GeneChip® gene expression analysis array, one of most commonly used microarray technologies, uses multiple oligonuleotides (25-mers) to measure expression abundance of a single gene. Recognizing that non-specific hybridization could significantly alter the accurate quantification of transcript abundance, Affymetrix designs the array to contain two types of probes. Probes that are perfectly complementary to the target sequence, called Perfect Matches (PM), are intended to measure mainly specific hybridization. A second set of probes identical to PM except for a single nucleotide in the center of the probe sequence (the 13th nucleotide), called Mismatches (MM), are intended to quantify non-specific hybridization [1]. A PM and its corresponding MM constitutes a probe pair, and multiple probe pairs, i.e. a probe set, are summarized to measure transcript abundance for a particular gene. "Probe measure" is used in this paper to refer to the manner in which probe hybridization is quantified based on a pair of PM and MM intensity values. For example, PM-MM is a probe measure, and PM only is another probe measure.
A number of methods have been developed to quantify gene expression abundance from GeneChip® expression analysis array data using different probe measures and summary schemes. Among them, Microarray Suite 5.0 (MAS 5.0) [1], dChip [2] and robust multiple-array average (RMA) [3] are the best known.
Prior to MAS 5.0, the probe measure used in MAS 4.0 was PM-MM [4]. The problem arises when a significant proportion of MM values, (~33% in the HuGeneFL array and ~25% in the Human Genome U133A array), is greater than the corresponding PM values, which makes PM-MM negative. To resolve this anomaly, in MAS 5.0, Affymetrix computes an "ideal mismatch" (IM) based on missing data theory such that PM-IM is always greater than zero [1]. Then, all probe pairs are used to estimate a gene expression value based on Tukey's Biweight algorithm. However, even with the use of IM, the variation among probes could be greater than between samples.
Li and Wong modelled probe level data to generate model based expression index (MBEI) and implemented it in the dChip software [2]. Noting that probe specificity is significant, highly reproducible and predictable, Li and Wong used a hybridization rate parameter to account for the hybridization specificity for a probe. For a probe pair, hybridization rates are different for PM and MM; the former is always greater than the latter, and both are greater than zero. The rate was fixed for the same probe across all the samples. Both PM and MM together or PM only, can be used in the Li and Wong model.
Another approach, RMA, available from Bioconductor [5], summarizes probe intensities into a gene expression measure based on an additive model on the logarithmic scale of a background corrected PM (PMrma) [3]. RMA estimates a common mean non-specific hybridization background (for an entire chip) from PM using a convolution model and then subtracts this background from PM to generate the PMrma.
The gene expression obtained from either MAS 5.0 or dChip or RMA can then be used to associate the gene expression values with experimental factors using an algorithm of the users' choice. Three main factors affect the analytical results of differential gene expression analysis: the probe measure chosen, the algorithm used to summarize probe level data into gene expression (called summary algorithm in this paper), and the model used to associate gene expression with the experimental factors (called association model). Direct comparisons of the various approaches proposed for analyzing GeneChip® gene expression data are complicated considering these three factors. Generalizing the various algorithms into one framework would facilitate comparisons.
In this paper we propose a class of generalized probe models (GPMs) that includes various analytical approaches for GeneChip® gene expression analysis array data as special cases. Using an empirical dataset, we assess the impact of different processes on the analytical results by comparing different formulations of GPM as well as GPMs with three other methods, MAS 5.0, dChip, and RMA.
Results
We applied GPM to the analyses of data obtained from a study investigating gene response to ATRA (all trans retinoic acid) or drug diluent (ETOH, ethyl alcohol). Briefly, at twenty-four hours after treatment, total RNA was extracted from cells, processed and hybridized to the HuGeneFL GeneChip®. The dataset consists of ten samples from the ATRA treatment group and ten samples from the control group (ETOH treated) in four medulloblastoma cell lines. We are interested in identifying genes that are differentially expressed between the two treatment groups.
We used three different probe measures: PM-IM, PM only and PMrma, and compared the performance of different methods using standardized coefficients, defined as the estimated coefficient divided by its standard error. The reason for using this index is that the standardized coefficients, usually known as Z-score test statistics, are independent of scale, and may be used to make statistical inference.
GPM-1 (2), GPM-2 (3) and GPM-3 (4) can be derived from the full GPM model (1) by making different statistical modeling assumptions. GPM-1 takes summarized gene expressions and associates them with experimental factors; GPM-2 and GPM-3 directly associate probe level data with experimental factors without first summarizing gene expressions (see Methods).
Comparison of GPMs with three other commonly used methods
We compared GPMs with three commonly used methods, i.e. MAS 5.0, dChip, and RMA. Each of these methods dictates its own specific probe measure, i.e. PM-IM in MAS 5.0, PM-MM, or PM only in dChip and PMrma in RMA. We found that all the methods were similar when the same probe measure was used, and the dissimilarity between the MAS 5.0 and other PM based approaches most likely stems from the different probe measure used. We first computed the gene expression using the software available for MAS 5.0, dChip (using the PM only option) and RMA, and then estimated the standardized coefficients for each gene with the association model GPM-1. We refer to these analytical options as MAS 5.0 PM-IM and hereafter (in MAS 5.0 PM-IM we omitted the term GPM-1 which indicates the association model used since GPM-1 is the only one in GPMs that handles gene expression values), dChip PM and RMA PMrma, respectively. Figure 1 shows the pair-wise comparisons among MAS 5.0 PM-IM, dChip PM, and RMA PMrma. For each pair-wise comparison, we plotted the standardized coefficients for each pair in a XY plot. To assess the similarity between two methods, we computed the correlation coefficients (R) between the standardized coefficients generated from the two methods. In addition, we computed the mean squared error (MSE) between the two standardized coefficients, i.e. , where N is total number of genes, Zj1 and Zj2 are the standardized coefficients for j th gene, for two methods, respectively. When two methods are similar, the XY plot of their standardized coefficients will lie closely along the diagonal line. Correspondingly, the correlation coefficient will be closer to one and the MSE will be closer to zero. In Figure 1, we see smaller R and larger MSE in the comparisons of MAS 5.0 PM-IM versus dChip PM, and MAS 5.0 PM-IM versus RMA PMrma compared to dChip PM versus RMA PMrma.
Figure 1 Comparison among MAS 5.0, dChip and RMA. Gene expression was computed from MAS 5.0, dChip, and RMA using the probe measure dictated in the methods. Standardized coefficients for each method were estimated using the association model GPM-1 and were plotted pair-wise.
Next, using probe measures of PM only and PM-IM, we directly (in a single step) estimated standardized coefficients with GPM-2 (referred as GPM-2 PM and GPM-2 PM-IM. We omitted the term which indicates the summary algorithm since GPM-2 and GPM-3 directly associate the probe level data with the experimental factors without first summarizing across all probes) and GPM-3, respectively. Figure 2 shows the pair-wise comparisons among MAS 5.0 PM-IM, GPM-2 PM, GPM-3 PM, GPM-2 PM-IM, and GPM-3 PM-IM. We see greater similarity between MAS 5.0 PM-IM and GPM-2 PM-IM or GPM-3 PM-IM (Figure 2, second row), than between MAS 5.0 PM-IM and GPM-2 PM or GPM-3 PM (Figure 2, first row). In the latter comparisons, only the probe measure is different indicating that the probe measure plays a more significant role than the combined effect of the summary algorithm and the association model.
Figure 2 Impact of probe measure on analytical results. Gene expression from MAS 5.0 was used to estimate standardized coefficients using the association model GPM-1. Either PM-IM or PM only was used directly in GPM-2 or GPM-3 models. Standardized coefficients were plotted pair-wise.
Comparison among the GPMs
We also compared the results from GPM-1 (2), GPM-2 (3) and GPM-3 (4), to evaluate their differences, and found similar results when using the same probe measure. We selected the top eight candidate genes from the results of MAS 5.0 PM-IM and used them to compare the performance of GPM-2 PM-IM and GPM-3 PM-IM. In Table 1, for the eight selected genes we list estimated coefficients, their standard errors and standardized coefficients, estimated under the three GPM models. From Table 1, for these eight selected genes, the statistics generated from the three GPMs formulation are similar using probe measure of PM-IM. Next, to compare the standardized coefficients on a genome-wide scale, Figure 3 panel A shows the pair-wise comparisons using probe measure of PM-IM. Figure 3 panel B shows the pair-wise comparisons using PM only and PMrma. The six plots in Figure 3 demonstrate the similarity of standardized coefficients on a genome-wide scale among variants of GPMs when the same (or similar, in the case of PM versus PMrma) probe measures are used in the analyses.
Table 1 Estimated parameters for eight candidate genes from GPMs
Probe set ID Y00291_at L13738_at D79990_at M13666_at X02158_rna1_at X84002_at L19605_at M60503_at
Parameters β SE Z β SE Z β SE Z β SE Z β SE Z β SE Z β SE Z β SE Z
GPM-1 PM-IM 3.17 0.48 6.65 0.80 0.18 4.50 3.04 0.68 4.47 1.07 0.25 4.24 -1.07 0.25 -4.24 0.63 0.15 4.18 0.51 0.12 4.16 -2.05 0.50 -4.13
GPM-2 PM-IM 2.79 0.36 7.74 0.65 0.13 4.89 2.92 0.67 4.3 1.11 0.24 4.6 -0.92 0.24 -3.86 0.33 0.12 2.63 0.51 0.09 5.53 -1.56 0.35 -4.44
GPM-3 PM-IM 3.05 0.35 8.60 0.71 0.12 5.69 3.10 0.63 4.89 1.31 0.22 5.93 -1.40 0.25 -5.73 0.25 0.11 2.26 0.52 0.09 6.02 -2.25 0.38 -5.96
Estimated coefficients (β ), their standard errors (SE ) and standardized coefficients (Z ) for eight candidate genes from GPM-1 (using MAS 5.0 expression measure), GPM-2 and GPM-3 using PM-IM
Figure 3 3A:Comparison among GPMs using PM-IM. Gene expression from MAS 5.0 was used to estimate standardized coefficients using the association model GPM-1. Standardized coefficients from GPM-1, GPM-2 and GPM-3 with same PM-IM probe measure were plotted pair-wise. 3B: Comparison of GPM-2, GPM-3, dChip and RMA using PM. Gene expression from dChip, and RMA were used to estimate standardized coefficients using the association model GPM-1. Standardized coefficients from GPM-2, GMP-3, dChip and RMA with probe measures of PM or PMrma were plotted pair-wise.
In summary, we conclude that the GPMs are similar to MAS 5.0, dChip and RMA on a genome-wide level when using the same probe measures and that the choice of probe measure may be more important than the summary algorithms to obtain the gene expression or models used to compute the coefficients.
Discussion
In this paper, we have described a general framework that can be used to compare various methods and evaluate their similarities and differences. We found that various methods tend to generate similar results, on a genome-wide scale, when the same probe measure is chosen, and probe measure seems to have greater impact on the analytical results than other factors.
In Figure 1, we compared the standardized coefficients estimated with GPM-1 using gene expression computed from MAS 5.0, dChip and RMA with their own dictated probe measures. Since we consistently used GPM-1 as the association modeling machinery for each analysis, we assessed the combined impact of probe measure and summary algorithm. We found that the results obtained from dChip PM and RMA PMrma were similar to each other, but different from those obtained from MAS 5.0 PM-IM. Although dChip PM and RMA PMrma use different summary algorithms, their analytical results are similar due to the PM based probe measures used in both analyses.
In Figure 2, we see again, that on a genome-wide scale, results from MAS 5.0, GPM-2 and GPM-3 are more similar when same probe measure is used than when the probe measures are different, indicating that the probe measure plays a key role in determining the similarity of results from two methods. Our preliminary analyses suggest that the choice of probe measure has bigger impact on the results than summary algorithm and association modeling.
For the three variants of GPMs, we compared the standardized coefficients from GPM-2 or GPM-3 with those from GPM-1 using the gene expression values computed in MAS 5.0, dChip and RMA. From the high R values (and correspondingly, low MSEs) in the six plots shown in Figure 3, we infer that the standardized coefficients obtained from variants of GPMs are similar when they used the same probe measure.
For seven of the eight candidate genes selected by GPM-1 using gene expression values generated by MAS 5.0, the gene-specific regression coefficients were similar among the MAS 5.0 PM-IM, GPM-2 PM-IM and GPM-3 PM-IM. This indicates that for these seven genes it makes little difference between using summary measures or modeling directly at the probe level data in GMP-2 or GPM-3, when the same probe measure is used.
In addition to the three factors we mentioned (i.e. choice of probe measure, summary algorithm and association modeling) that have an impact on analytical results, data pre-processing/normalization could also affect the analytical results. Some researchers combine the probe measure and pre-processing normalization together. Normalization matters the most when the arrays in an experiment are not comparable to each other. In such cases, normalization process could significantly impact on the result. In our case, we normalized the data in the GPMs using a regression-based approach [6], either at probe level in GPM-2 and GPM-3, or on gene expression level in GPM-1. The expression measures obtained from dChip and RMA were normalized by their own normalization schemes. However, even with the different normalization schemes, probe measure appears to be the primary factor to impact the results in our data set.
An important feature of the framework presented in this paper is that it accommodates various probe measures (see Table 2) to quantify the abundance of the transcript. A question arises: how does one combine results from analyses using different probe measures. This is the dilemma we face when we analyze thousands of genes simultaneously. On the one hand, microarray technology is still imperfect and it is prudent to evaluate a number of exploratory approaches. On the other hand, by the very nature of the problem, it is unlikely that a single approach will be equally appropriate for each gene. The reality is that microarrays afford a rapid preliminary assessment of thousands of genes for future experimental validation. Ultimately, any scientific validation has to be drawn from further bench experiments.
Table 2 A list of selected probe measures
Scenario Calculation Annotation
1. MAS4.0-equivalent Zjik = (yji1k - yji0k ) Direct difference between PM and MM
2. MAS5.0-equivalent Zjik = (yji1k - ) is the Idealized Mis-match (IM)
3. PM only Zjik = yji1k Ignore MM
4. RMA-equivalent Zjik = log(yji1k - ) is the mean background estimated from PM for the kth chip
5. Log ratio Zjik = ln(yji1k / yji0k ) Difference on the logarithmic scale
6. Log difference Zjik = ln(yji1k - ) Difference on the logarithmic scale
7. Log PM Zjik = ln(yji1k ) PM only on the logarithmic scale
8. Box-Cox on PM Zjik = ( - 1) / ω Box-Cox transformation on PM only
9. Box-Cox on PM-IM Zjik = [(yji1k - )ω - 1] / ω Box-Cox transformation on PM-IM
To facilitate the evaluation and use of GPMs, we have developed a software program, called ProbePlus that implements our GPMs. This program will be made available to academic researchers through the website .
Conclusions
In this paper we describe a general framework to analyze GeneChip® gene expression analysis array data. This framework is flexible to permit comparisons of different methods with respect to the choice of probe measure and analytical models used. We found that different methods yield similar result when probe measures are the same.
Methods
The generalized probe model
Consider an experimental study with K chips. Each chip is engineered to assess levels of J gene expressions. Each gene has I probe pairs. Now let yjilk denote the intensity value for the jth gene (j = 1,2...J), the ith probe pair (i = 1,2...I), PM (l = 1) or MM (l = 0), and the kth sample (k = 1,2...K). Table 3 displays the notation for a typical microarray dataset. The probe intensity yjilk , quantifying the abundance of the RNA hybridized on a probe, is treated as a random variable, influenced by the effects of probe-specific hybridization, gene-specific hybridization, non-specific hybridization and random noise. In this paper we use Zjik to denote the quantification of the signal of the ith probe, in the jth gene from the kth sample. Zjik could be based on any probe measure, such as PM only or PM-IM (some other selected probe measures are listed in Table 2).
Table 3 A typical probe-level data generated from GeneChip® gene expression analysis array
Sample ID Probe PM (1) 1 2 ... k ... K
Covariate ID MM(0) x1 x2 ... xk ... xK
ORF1 1 1 y1111 y1112 y111k ... y111K
1 0 y1101 y1102 y110k ... y110K
2 1 y1211 y1212 y121k ... y121K
2 0 y1201 y1202 y120k ... y120K
...
N 1 y1N 11 y1N12 y1N 1k ... y1N1K
N 0 y1N01 y1N 02 y1N0k ... y1N 0K
...
ORFj 1 1 yj111 yj 112 yj11k ... yj 11K
1 0 yj101 yj 102 yj10k ... yj 10K
2 1 yj211 yj 212 yj21k ... yj 21K
2 0 yj201 yj 202 yj20k ... yj20K
...
i 1 yji11 yji12 yji1k ... yji1K
i 0 yji01 yji02 yji0k ... yji0K
...
N 1 yjN11 yjN12 yjN1k ... yjN1K
N 0 yjN01 yjN02 yjN0k ... yjN0K
...
ORFJ 1 1 yJ111 yJ112 yJ11k ... yJ11K
1 0 yJ101 yJ102 yJ10k ... yJ10K
2 1 yJ211 yJ212 yJ21k ... yJ21K
2 0 yJ201 yJ202 yJ20k ... yJ20K
...
N 1 yJN11 yJN12 yJN1k ... yJN1K
N 0 yJN01 yJN02 yJN0k ... yJN0K
In a typical experiment as described above, it is frequently of interest to discover genes that are significantly associated with one or more experimental covariates xk . For example, consider an experiment to discover genes that are differentially expressed between two groups, xk takes a binary values: xk = 0 for the control group and xk = 1 for the treatment group. To achieve the scientific objective, the analytic procedure is to assess associations of = (Zj1k , Zj2k ,...,ZjNk )' with covariates xk via the distribution function f ( | xk ). In essence, Zjik are treated as vectors of multivariate correlated outcome variables, and used to identify the probes/genes that are differentially expressed. Recognizing the high dimensionality of multiple probes and multiple genes, we propose to apply a marginal model that uses marginal means to describe relationship of probes/genes with the covariates without the necessity of specifying the full distribution f ( | xk ). Our framework directly associates experimental factors with probe intensities and is referred to as the generalized probe model or GPM. We propose the following (1) to describe relationship between and xk ,
where (δk , λk ) are chip-specific heterogeneity factors for k th chip [7], τji are gene- and probe-specific parameters quantifying the mean intensity value for the ith probe of the jth gene, βji quantifies the gene- and probe-specific parameters quantifying the difference between treated and control groups, and vjik quantifies expression values for individual probe pairs. Lastly, (ξj1k , ξj2k ,...,ξjNk ) represents a vector of gene- and probe-specific random variations across K independent samples. Since probe pairs are selected to target the jth gene and are spatially arranged by a pre-selected design to eliminate common artifacts, they may be correlated because of cross-hybridizations or spatial dependencies. From the biological perspective, specifying a joint distribution for (ξj1k , ξj2k ,...,ξjNk ) would be difficult, if not impossible. It is thus preferable to leave it unspecified.
The above GPM (1) includes a range of more simplified models based on specific assumptions. First, under the assumption that all probe-specific parameters are the same, i.e., τji = τj and βji = βj , the general model (1) simplifies to the following model:
and is equivalent to using a summarized gene expression to associate with the experimental factors [7]. For simplicity and comparison with other special models, we refer this model as GPM-1.
If one postulates that all probe-specific parameters are not the same, but follow an additive probe model, then general model (1) under modeling assumption that βji = βj , with probe-specific values (τj1, τj2,...,τjN ) may be written as
in which estimating βj is of primary interest. This variation of the general model is referred to as GPM-2.
On the other hand, the probe parameters may follow a multiplicative model (in the spirit of Li and Wong's model), then the third model, referred to as GPM-3, is derived under the assumption that τji ≈ φji τj and βji ≈ φji βj , and may be written as
where φji denote the multiplicative probe-specific effects and can be uniquely determined by constraining the mean to be one.
Estimation and inference
Our estimation procedures do not require any assumptions with respect to the error distribution, since any distributional assumptions, which may be appropriate for some genes, are likely to be violated for other genes. To ensure the robustness of statistical inference, we propose to use generalized estimating equation theory, which has been fully described in a seminal paper by [8]. In the current context, we choose the "working independence" assumption for modeling dependencies between probes [8,9]., to avoid making any assumptions on dependence structures. The asymptotic variance matrix is estimated with the usual "sandwich" estimator [8]. Diagonal elements in the variance matrix are estimates of marginal variances for all estimated parameters, and are denoted by for the estimated parameters in the model. Both estimates can be used to construct test statistics, such as the ratio of over , known as Wald-statistic. Under the null hypothesis, each statistic has an asymptotic normal distribution when the sample size is sufficiently large, and can therefore be used for making statistical inferences. When the sample size is small, this quantity is treated as a standardized regression coefficient.
List of abbreviations
ATRA: All Trans Retinoic Acid
ETOH: Ethyl Alcohol
GPM: Generalized Probe Model
IM: Ideal Mismatch
MAS: Microarray Suite
MM: Mismatch
ORF: Open Reading Frame
PM: Perfect Match
RMA: Robust Multiple Array average
Authors' contributions
WF carried out analyses and prepared the manuscript. JIP conducted microarray experiments. JMO conceived the study. NK: prepared the manuscript. LPZ: conceived the study, developed the GPM algorithms and prepared the manuscript.
Acknowledgements
We wish to thank Andrew Strand for his helpful discussion during the preparation of this manuscript. This work was supported by grants from National Institutes of Health.
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| 15710039 | PMC553974 | CC BY | 2021-01-04 16:39:52 | no | BMC Genomics. 2005 Feb 14; 6:16 | utf-8 | BMC Genomics | 2,005 | 10.1186/1471-2164-6-16 | oa_comm |
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BMC Palliat CareBMC Palliative Care1472-684XBioMed Central London 1472-684X-4-31572535010.1186/1472-684X-4-3Research ArticleTransitions in care during the end of life: changes experienced following enrolment in a comprehensive palliative care program Burge Frederick I [email protected] Beverley [email protected] Patrick [email protected] David [email protected] Department of Family Medicine, Dalhousie University, Halifax, NS, Canada2 Active staff, Timmims and District Hospital, Timmons, ON, Canada2005 22 2 2005 4 3 3 10 11 2004 22 2 2005 Copyright © 2005 Burge et al; licensee BioMed Central Ltd.2005Burge et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Transitions in the location of care and in who provides such care can be extremely stressful for individuals facing death and for those close to them. The objective of this study was to describe the distribution of transitions in care experienced by palliative care patients following admission to a comprehensive palliative care program (PCP). A better understanding of these transitions may aid in reducing unnecessary change, help predict care needs, enhance transitions that improve quality of life, guide health care system communication links and maximize the cost-effective utilization of different care settings and providers.
Methods
Transition and demographic information pertaining to all patients registered in the PCP at the Queen Elizabeth II Health Sciences Centre (QEII), Halifax, Nova Scotia, Canada between January 1, 1998 and December 31, 2002 and who died on or prior to December 31, 2002 was extracted from the PCP database and examined. A transition was defined as either: (1) a change in location of where the patient was cared for by the PCP or, (2) a change in which clinical service provided care. Descriptive analysis provided frequencies and locations of transitions experienced from time of PCP admission to death and during the final two and four weeks of life, an examination of patient movement and a summary of the length of stay spent by patients at each care location.
Results
Over the five year period, 3974 adults admitted to the QEII PCP experienced a total of 5903 transitions (Mean 1.5; standard deviation 1.8; median 1). Patients with no transitions (28%) differed significantly from those who had experienced at least one transition with respect to survival time, age, location of death and diagnosis (p < 0.0001). The majority of patients were admitted to the PCP from various acute care units (66%). Although 54% of all transitions were made to the home, only 60% of these moves included care provided by PCP staff. During the last four weeks of life, 47% of patients experienced at least one transition; 36% during the final two weeks of life. Shorter stays in each location were evident when care was actively provided by the PCP.
Conclusion
A relatively small number of patients under the care of the PCP at the end of life, made several transitions in care setting or service provider. These particular patients need closer scrutiny to understand why such transitions take place so that clinical programs may be designed or modified to minimize the transitions themselves or the impact transitions have on patients and families.
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Background
For individuals facing death, and for those close to them, transitions in the location of care and in who provides care can be extremely stressful.[1] Such transitions include moving from home to hospital or to long-term care facilities, from ward to ward within hospitals, or in and out of care directed by particular care providers (such as specialists). With or without good continuity of information transfer, patients and caregivers may, at each transition, need to retell their story, renegotiate the goals of care and redefine their relationships with health professionals. At each point, new communication channels must be established and new trusts formed.
Much of the health service research literature in end-of-life care focuses on issues either in hospital or at home. [2-5] We believe it is also important to examine the changes or transitions patients make in where they are cared for and by whom during the end-of-life.
The ultimate goal of understanding these transition issues better is to reduce unnecessary changes, help to predict care needs, enhance transitions that improve patient and caregiver quality of life, guide communication links within the health care system and maximize the cost-effective utilization of different care settings and providers.
The primary purpose of this study was to describe the distribution of transitions in care experienced by palliative care patients during the time subsequent to admission to a comprehensive palliative care program. We also report the proportion of transitions as death becomes imminent (the last two and four weeks of life) and describe the length of stay in each location of care or care setting.
Methods
Subjects
Subjects included all adult patients registered in the palliative care program (PCP) at the Queen Elizabeth II Health Services Centre (QEII) in Halifax, Nova Scotia, Canada between January 1, 1998 and December 31, 2002 with a recorded date of death on or prior to December 31, 2002. The PCP includes multidisciplinary care for the dying, an in-patient acute care unit, an in-hospital consultation service, a home consultation service and an oncology outpatient clinic consultation service. The team consists of physicians, nurses, social workers, pharmacists, spiritual care and volunteers. There is no free standing hospice facility in Nova Scotia. The PCP has existed since 1988 and, by 1997, had at least one contact with over 62% of those who die annually of cancer in the Halifax Regional Municipality (population approximately 350,000).[6]
Data
Individual level information extracted from the PCP database included demographics (sex, date of birth, date of death, postal code), diagnoses, the relationship of the primary caregiver to the patient (for example, spouse, daughter, son, friend), reason for referral, location of death and program transition data. The program transition information provided the date of each transition and locations the patient had been moved to or from (for example, home, an acute care facility, long-term care) as well as the clinical service providing care. For example, for inpatients the service might be the Palliative Care Unit, or a medical or surgical service; for outpatients it might be the Nova Scotia Cancer Centre (NSCC) PCP clinic, the PCP Home Support Service, or the family doctor. The service indicator field also provided a record of whether patients were 'actively' being cared for by PCP staff or whether their care had been transferred to the staff of the NSCC or family doctor (either locally or elsewhere in the province).
Ethical approval for this research was provided by the Nova Scotia Capital District Health Authority research ethics board.
Measures
A transition in this study is defined as either: (1) a change in location of where the patient was cared for by the PCP or, (2) a change in which clinical service provided care. For example, a transition might be a move to or from the home, a specific acute care unit or a long-term care facility. A transition would also occur if the patient stayed in a single location, for example at home, but the care being provided was transferred from PCP staff ('actively cared for' by the PCP) to their family physician or home care nurse ('non-active' and no longer 'actively' cared for by PCP staff) or vice versa. This 'transfer of care' transition scenario is illustrated below:
Length of stay was calculated as the number of days a patient stayed in a single location while receiving the same form of care. For example, a patient might be admitted to the PCP from home and stay in the home for a total of 86 days at which time the patient was admitted to an acute care inpatient unit. After spending 10 days in acute care the patient was sent home where they died 25 days later.
In this scenario the patient experienced two transitions (1. from home to acute care; 2. from acute care to home) but had three stay periods (two at home and one in acute care).
Survival in this study was defined as the number of days between date of the initial admission to the PCP and death. Cancer is the most prevalent disease among PCP patients, in particular lung cancer. To reflect this we created a diagnostic summary with three categories: lung cancer, all other cancers and other disease only (no cancer). Lung cancer was separated from other cancers as it is the most common cancer affecting both sexes in Nova Scotia, has a short prognosis and often has PCP involvement.
Analysis
The analysis focused on providing a description of the number and location of transitions experienced by patients over the five-year study period. For each patient, the total number of transitions occurring from the date of initial admission to the PCP to the date of death and during the final two and four weeks of life were counted and described. Locations of admission, death, care and service provision are summarized as well as an examination of patient movement from location to location or 'site movements'. Summary statistics are provided to describe the length of stay (LOS) or number of days spent by all patients at each care location.
Differences between patients who experienced no transitions versus those who experienced at least one transition were assessed using contingency tables with chi-square techniques and logistic regression.
Results
In total, 3972 adult patients were admitted to the QEII PCP between January 1, 1998 and December 31, 2002 and had died on or prior to December 31, 2002. There was a slight preponderance of male patients (52%); patients tended to be older (mean 68.5 years, standard deviation [SD] 13.7) and diagnosed with cancer (90%) (Table 1). Lung was the major cancer site accounting for 29% of all cancer diagnoses. Survival time, the number of days between program admission and death, was highly variable, ranging from 0 days (died same day as admitted to the PCP) to 1688 days, with an average of 100.6 (SD 163.2) and median of 45 days. As recorded in Table 1, 40% of patients survived less than 15 days and 24% survived 121 days or more. Eighty five percent of patients admitted to the PCP survived 6 months or less.
Table 1 Characteristics of patients admitted to the Queen Elizabeth II Palliative Care Program between Jan 1, 1998 and Dec 31, 2002 and died during the same period
Characteristic Number of patients* (%)
Sex
Female 1925 (48.5)
Male 2047 (51.5)
Age, years
< 60 992 (25.1)
60–69 873 (22.1)
70–79 1208 (30.6)
≥ 80 878 (22.2)
Year of admission to PCP
1998 883 (22.2)
1999 824 (20.8)
2000 876 (22.1)
2001 771 (19.4)
2002 618 (15.6)
Year of death
1998 645 (16.2)
1999 820 (20.7)
2000 858 (21.6)
2001 824 (20.8)
2002 824 (20.8)
Survival (days)
0–30 1605 (40.4)
31–60 723 (18.2)
61–90 432 (10.9)
91–120 251 (6.3)
121+ 960 (24.2)
Location of death
Hospital death (not in PCP unit) 2009 (50.6)
Inpatient PCP unit 635 (16.0)
Home 1218 (30.7)
Long-term care facility 110 (2.8)
Diagnoses, summarized
Lung cancer 1025 (26.1)
All other cancers 2525 (64.3)
Other disease, no cancer 375 (9.6)
Caregiver relationship
Spouse 2165 (57.8)
Child 1017 (27.2)
Parents / other relations 448 (12.0)
Other 116 (3.1)
Primary reasons for referral to PCP
(responses are not exclusive)
Pain 1630 (41.0)
Other symptoms 1868 (47.0) 1729 (43.5)
Patient support 1729 (43.5)
Family support 1620 (40.8)
Staff support 317 (8.0)
Home consultation 1067 (26.9)
Terminal care 345 (8.7)
Respite care 5 (0.1)
Grief 19 (0.5)
* N = 3,972. Smaller n by characteristic due to missing values.
Figure 1 illustrates the distribution of the number of transitions experienced by patients over the five-year study period. The number of transitions totalled 5903, ranging from 0 to 21, with an average of 1.5 (SD 1.8) per patient (median: 1). Overall, 28% did not experience a transition, 41% experienced one transition and 31% experienced two or more. Patients with no transitions experienced no change in the care provided or the location of care from the point of PCP admission to death. However, this group differed significantly from patients who experienced at least one transition, at the 0.001 level of significance, with respect to survival time, age, location of death and diagnosis. Compared to those who had at least one transition during the time period between admission to the PCP and death, patients with no transitions were much more likely to have a survival time of fourteen days or less (64% versus 10%), to be aged 80 years or older (32% versus 19%), to experience a hospital death (68% versus 44%) and to have a diagnosis other than cancer (19% versus 6%).
Figure 1 Total transitions over a five year period among all patients admitted to a comprehensive Palliative Care Program (PCP) (N = 3972)
The majority of patients (66%) were initially admitted to the PCP as inpatients of various acute care units (n = 2619). Of this inpatient group, only 65 patients (2.5%) were admitted directly into the inpatient unit of the PCP. PCP admissions to care at home accounted for 34% of patients (n = 1335). Care services provided to home patients were provided upon admission by either PCP home care staff (51%) or on an outpatient basis through the NSCC (49%). Very few were admitted to the service from a long-term care facility (n = 17).
Overall, 54% of all transitions were made to the home, 27% to other acute care inpatient units within the same hospital, 17.5% to the PCP inpatient unit, and almost 2% to a long-term care facility. PCP staff members, however, were not always actively providing the care provided within each of these locations. For instance, in transitions to home only 60% involved care being 'actively' provided by PCP home care staff or through the NSCC PCP outpatient clinic. The reasons why the PCP was not involved in care provision are varied and include improvements in health status (for example their original symptoms have abated, or they were thought to be dying, but improved unexpectedly), changes in their personal caregiver situation or movement out of the PCP coverage area etc. In contrast, few transitions involving acute care units were due to a change in care from active care provision by PCP staff to non-active PCP care (4%). Table 2 illustrates the number of transition changes from one location or care site to another. For example, from the table we show that 32% of transitions originating from the home were to the PCP inpatient unit; 65% of transitions from the PCP inpatient unit were to the home.
Table 2 Location or site / care movements: a summary of the number of transitions made by all patients by location change
Location / care changing FROM Location / care changing TO Number of all transitions (%)
PCP inpatient unit All other acute care units Home Home with NSCC care Long term care Total
PCP inpatient unit 1201 (26.1) 28 (6.1) 301 (65.4) 0 11 (2.4) 460 (7.8)
All other acute care units 184 (7.2) 3502 (13.7) 1899 (74.5) 73 (2.9) 44 (1.8) 2550 (43.2)
Home 628 (32.1) 897 (45.8) 1993 (10.2) 206 (10.5) 29 (1.5) 1959 (33.2)
Home (with NSCC care) 93 (10.4) 290 (32.3) 504 (56.1) 93 (1.0) 2 (0.22) 898 (15.2)
Long term care 3 (9.1) 10 (30.3) 14 (42.4) 0 63 (18.2) 33 (0.6)
Total 1028 (17.4) 1575 (26.7) 2914 (49.4) 288 (4.9) 92 (1.6) 59004 (100)
Site/care movements from and to the same location were evident.
1 The patient was admitted under the PCP but placed in a bed elsewhere in the hospital while waiting transfer to the inpatient PCP unit. The movement in this situation reflects a physical location change from within the acute care facility to the PCP inpatient unit. It does not represent a change in care.
2Site movements across different acute care units are captured here.
3Each of these same site movements reflect a change in 'active' care status to 'non-active' care status or vice versa. 'Active' care is defined as care provided to a patient by PCP staff. 'Non-active' care refers to care being provided by individuals not associated with the PCP program.
4Three transition records were missing site information
During the last four weeks of life, 47% of patients experienced at least one transition. The majority of transitions were to an acute care facility (26% to the PCP inpatient unit, 35% to other acute care units), followed by the home (37%) and long-term care (2%). Similar patterns were evident when moves associated with the 2 weeks prior to death were examined. At least one transition was experienced by 36% of patients during these final 2 weeks of life. Moves to an acute care facility accounted for 68% (29% to the PCP inpatient unit; 39% to other acute care units), to the home 30% and 2% to long-term care.
The location of death among PCP patients followed a pattern similar to that found at admission. Sixty-seven percent (n = 2644) of deaths occurred within an inpatient acute care unit, 16% of these deaths occurred in the PCP inpatient unit (n = 423). Home deaths were experienced by 31% of all admissions (n = 1218), while 3% (n = 110) of patients died as a long-term care resident.
Table 3 summarizes the median length of stay (LOS) in days associated with each transition by location or care setting and the PCP's role in providing care. Medians are reported due to the very wide, skewed distributions associated with each LOS. We have split the LOS by PCP role in care to illustrate how LOS tended to be longer during transitions where PCP health care providers did not provide care. The median length of stay spent by patients who transitioned to acute care units without PCP care was 27.5 days. In contrast, transitions to the PCP inpatient unit and other acute care units providing active care by PCP health care providers was much shorter, with median days stayed of seven and six days respectively. The median length of stay spent at home while under the care of PCP home care providers was 28 days, 10 days less than that experienced at home when care was provided by others not associated with the PCP.
Table 3 Median length of stay spent in each care setting by PCP role in care1
Location / care setting Median number of days by PCP role in care (range)
'Active' care 'Non-active' care
Acute care:
PCP inpatient unit 7 (0 – 175) -
All other inpatient units 6 (0 – 390) 27.5 (0 – 1139)
Home2 28 (0 – 845) 38 (0 – 1658)
Home, followed by NSCC 30 (0 – 1295) 44 (2 – 901)
Long-term care 18 (0–112) 63 (3 – 1079)
1 'Active' care is defined as care provided to a patient by PCP staff.
'Non-active' care refers to care being provided by individuals others not associated with the PCP program.
2 Home is defined as when a patient is not required to leave their home for contact with health care providers.
Home, followed by NSCC indicate the patient leaves the home to go to the PCP clinic at the Cancer Centre
Discussion
Patients followed by the QEII PCP are primarily elderly (75% 60 years of age or older), urban dwellers, with cancer and survive less than six months from time of initial program admission (80%). This age and sex distribution is similar to that experienced among all Nova Scotians who died due to cancer between 1992 and 1998.[2] The average number of transitions experienced in care settings by this group of elderly was 1.5. Two or more transitions were experienced by 31% of patients. We were surprised that the vast majority of patients (69%) had fewer than two transitions. We had expected the number to be somewhat higher. Our clinical experience of providing care for these patients is perhaps skewed by the challenges faced by the minority of patients with multiple transitions.
Patients who had no transitions beyond entry to the PCP appear to be a different population than those with one or more transitions. This group may be a much sicker population since they tended to have much shorter survival times, were older and more likely to die in hospital. They were also more likely to die of disease other than cancer. This population warrants special attention in program planning and service delivery given their potentially higher institutional based needs.
The most common transition identified was from an acute care hospital unit "to" the home. This fact, in and of itself, provides evidence for the substantial focus that must take place in hospital on discharge planning for those at the end of life. Attention to the multiple discharge issues for this unique group of patients is likely the single biggest transition issue facing the acute care units and the consulting QEII PCP. Issues of symptom control, drug supplies, home equipment needs, family and professional caregiving needs in the home, advance planning for routine follow up and crisis management as well as the psychological supportive issues all need planning. The acute care teams must do this planning along with the QEII PCP consult team in collaborative fashion. These transitions present challenges in coordination and information transfer in order to facilitate continuity of care for patients and families.
The next most common category of transitions was from home "to" the hospital, either to an acute care unit or to the PCP inpatient unit. Just as the previous transition needs to be planned and coordinated so does the home to hospital one. Unfortunately we have no data on how this latter transition occurs. Most often it may be due to a symptom crisis in the home, a lack of caregiver capacity in the home, or a lack of financial resources to bring adequate care to the home.[7] Given the substantial pressure on acute hospital beds in Canada today, many of these admissions take place via the emergency department. Such environments may be appropriate in acute symptom stabilization before admission but the emergency department could be bypassed with planned and coordinated direct admission to the inpatient unit concerned. More information is needed on the "route" taken to hospital and the issues that require admission, the goals of admission and whether or not hospital based care versus respite / long-term care would best meet patient care needs.
Most patients came to the care of the PCP from within the acute care system. This may be a reflection of the lateness of referral for a substantial number of individuals (40% dying within 15 days of referral). These individuals, by the time of referral, may be quite ill in hospital. This initial entry to PCP (also a transition of care but one we did not explore) may need to be a focus of concern to better understand it. We need to understand the timing of referrals, what can be done to identify and meet patients' needs when required and whether these needs could be met outside of the hospital setting.
Once under the care of PCP the most common transitions were "to" home. Only a very small number of patient move "to" or "from" long term care (LTC). The fact that the majority of transitions were to home reflects the goals and expertise of the PCP in emphasizing the home location by coordinating the services needed for care at home. The fact that long term care is a rare transition also needs exploration. Policies may exist or wait times for admission may be such that transition to these facilities for people with short prognosis is difficult to achieve. At the same time, few patients transition "from" long term care. Some local LTC facilities have intramural palliative care programs designed to specifically meet the needs of their residents and their goals of staying in LTC as death approaches, avoiding hospital transfers.
The results of this study indicate almost half of patients have a transition in the last month of life and 36% in the last 2 weeks of life. As death approaches these transitions are more likely to be "to" hospital (i.e. 62% of those in the last 4 weeks and 68% of those in the last 2 weeks). Defining the "appropriateness" of these late transitions is difficult. As stated before, they may be due to acute symptom crises or caregiver inability to meet all of the care needs. We have also heard that patients are admitted when they appear in emergency departments with these issues as they have not been able to access professionals to assess and problem solve during nights and weekends.[7] Therefore, some late admissions are entirely appropriate and others may have been avoidable if more resources were available to patients and families at home.
As to the generally longer stays for patients when not under "active PCP" care, it may be that they are much less sick with other chronic, non-palliative problems and their prognosis is longer. One might also postulate that for the in-patients, the active involvement of PCP may facilitate shorter stays and transitions to the home. Such facilitation could include more rapid control of symptoms or more expeditions discharge planning. The phenomena may be similar in both the home and long-term care settings.
Limitations
One substantial limitation is the loss of information pertaining to patients who are transitioned to non-active care permanently. Although we do have a record of their death, once a patient is moved to non-active care and ceases further involvement with the PCP, we do not have a record of who has taken responsibility for their care or where this care has been received. Our results and our clinical experience suggest this group may be quite different from those who remain actively cared for by the PCP. We have begun efforts to collect information about this group in order to understand them better.
Conclusion
In conclusion, a small number of patients under the care of the PCP very near the end of their lives, make several transitions in care setting or service provider. These particular patients need much closer scrutiny in order to understand why such transitions take place. We will then be able to design or modify clinical programs to minimize the transitions themselves or the impact the transitions have on patients and families. Possible negative impacts of multiple transitions include discontinuity of care, poor coordination of care, financial burden and psychological stress that each move may bring to patients and their families.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
FB and BL participated in the conceptualization and design of the project, the analysis and interpretation of the data, first-drafted the majority of the article, and incorporated co-authors' comments into the final draft. PC participated in the conceptualization and design of the project, interpretation of data, and revising of the manuscript. DM participated in the analysis and interpretation of data and revising each draft for critical content. All authors gave approval to the final version.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The study was supported by a grant from the Capital District Health Authority, Halifax, NS. Dr. Burge is supported by a Senior Clinical Research Scholar Career Award from the Faculty of Medicine, Dalhousie University. At the time of this study, P. Critchley was a Palliative Care physician with the QEII Palliative Care Program, Halifax, NS.
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Addington-Hall J Altmann D McCarthy M Which terminally ill cancer patients receive hospice in-patient care? Soc Sci Med 1998 46 1011 1016 9579752 10.1016/S0277-9536(97)10021-1
Axelsson B Christensen SB Place of death correlated to sociodemographic factors: A study of 203 patients dying of cancer in a rural Swedish county in 1990 Palliat Med 1996 10 329 335 8931069
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Burge F Johnston G Lawson B Dewar R Cummings I Population based trends in referral of the elderly to a comprehensive palliative care program Palliat Med 2002 16 255 256 12047004 10.1191/0269216302pm550xx
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| 15725350 | PMC553975 | CC BY | 2021-01-04 16:30:52 | no | BMC Palliat Care. 2005 Feb 22; 4:3 | utf-8 | BMC Palliat Care | 2,005 | 10.1186/1472-684X-4-3 | oa_comm |
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BMC BiotechnolBMC Biotechnology1472-6750BioMed Central London 1472-6750-5-71570748010.1186/1472-6750-5-7Research ArticleThe case for well-conducted experiments to validate statistical protocols for 2D gels: different pre-processing = different lists of significant proteins Meleth Sreelatha [email protected] Jessy [email protected] Helen [email protected] Biostatistics and Bioinformatics Unit, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA2 Department of Pharmacology, University of Alabama at Birmingham, Birmingham, AL 35924 USA3 Department of Pharmacology, University of Alabama at Birmingham, Birmingham, AL 35294, USA2005 11 2 2005 5 7 7 15 4 2004 11 2 2005 Copyright © 2005 Meleth et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The proteomics literature has seen a proliferation of publications that seek to apply the rapidly improving technology of 2D gels to study various biological systems. However, there is a dearth of systematic studies that have investigated appropriate statistical approaches to analyse the data from these experiments.
Results
Comparison of the effects of statistical pre-processing on the results of two sample t-tests suggests that the results of 2D gel experiments and by extension the conclusions derived from these experiments are not independent of the statistical protocol used.
Conclusions
This study suggests that there is a need for well-conducted validation studies to establish optimal statistical techniques to be used on such data sets.
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Background
The effort to produce an index of all human proteins (the human protein index, or HPI) began over twenty years ago. This project pre-dates the human genome project by more than a decade. However, the complexity of the task of creating this index was underestimated and the relative simplicity of the human genome with four known nucleic acids arranged in a linear coding order allowed the process of the sequencing of the human genome to progress exponentially [1]. The successful completion of the human genome project is now putting the focus back on proteins. The emergence of new and improved protein technologies from re-engineered two-dimensional (2D) gel systems to mass spectrometry has made the mapping and identification of the entire proteome of a cell (tissues) a much more accessible goal. Over the past few years a number of databases documenting the protein content of a single organism, organ or organelle have been created [2-6], and a number of papers describing results of experiments using these new and improved techniques have been published.
The advantages of 2D gel technology
Two-dimensional electrophoresis is an extremely powerful tool for the analysis of complex protein mixtures. Proteins carry both positive and negative charges. The pH of the medium they are in determines their net charge. The pH that gives a zero net charge is the isoelectric point of the protein (pI). In Isoelectric Focusing (IEF), protein mixtures are electrophoresed in a gel containing a pH gradient. The proteins in the mixture migrate according to charge density until they reach the part of the gel that corresponds to their pI. At this point, their net charge is zero, and migration stops. This is the first dimension of separation in a 2D gel experiment. The electrophoresed gel is then layered on top of a polyacrylamide gel and electrophoresed once again. The proteins now move from top to bottom depending on molecular weight. The distance covered by a protein is inversely proportional to its size. This is the second dimension in a 2D gel. 2D is an effective method for identifying qualitative and quantitative differences between proteins expressed in various tissues or between tissues exposed to different experimental treatments. Although the number of proteins displayed by 2D is much lower than the estimated number of genes in a particular tissue, 2D is currently the only available technique that enables the isolation and separation of thousands of the individual proteins that constitute a tissue proteome [7]. Anderson et al [1] point out that although writing obituaries for 2D gels has become a popular past time, the supply of unrelated parameters applicable to protein separation is limited and nearly all other combinations have been explored in the past.
Database for statistical analysis
Images of 2D gels are acquired into a database using an image scanner. Image analysis software converts the gel image into a digitised image in a computer, matches gels and spots on gels across the different groups and creates a database with information about spot intensity and spot location. As mentioned above, the two variables – the pI representing net charge of the protein and the molecular weight of the protein – are not correlated. In geometric terms this suggests that the two dimensions are orthogonal to each other. The two dimensions in a two-dimensional gel thus can be thought of as the two axes in a two dimensional graph. The coordinate on the x-axis is a measure of the isoelectric point (pI) of the protein, and the coordinate on the y-axis is a measure of the molecular weight of the protein
The information in the database includes a gel identification variable, a spot identification variable, the x and y coordinates of a protein spot and its intensity measured by the amount of light transmitted by the spot. Depending on the software package, one can obtain other parameters in the database, including a measure of the quality of the spot to various measures associated with spot intensity, such as volume, area, peak height, etc.
The rationale for this study
The intensity of a protein spot is assumed to be directly related to the amount of protein in the particular tissue under investigation at that given time point. Changes in protein intensity are therefore approximated by changes in the intensities of protein spots in gel images. Changes in protein structure associated with post-translational modifications such as phosphorylation, oxidative modification or glycosylations may result in changes in the pI or molecular weight of the protein and are manifested in the gel by a change in the vertical or horizontal position. The object of 2D gel experiments is to detect differences in protein intensity/complexity between two groups of gels.
A number of recent publications [8-10] have used statistical models generally known as classifiers to detect differences in protein intensity/complexity between two groups of gels. Classifiers are increasingly being used in the analysis of hi-dimensional data sets derived from gene and protein expression experiments. These models help one to determine if the changes in protein intensity /complexity are specific enough to enable a clear separation of the gels into the right groups. They can also be used to provide a good visual demonstration of the differences between groups.
Classifiers
Classification is the process of assigning objects to a category. An interest in classification permeates many scientific studies [11]. There are two broad categories of classification problems. In the first, e.g. discriminant analysis, one has data from known groups. Information that distinguishes these groups (i.e. differences in protein intensity/complexity) collected from an experiment is used to assign samples (gels) to these known groups. In the second case, e.g., cluster analysis, one has the information but no preset classification. The data is mined to see if there are naturally occurring clusters. These clusters are then investigated to identify commonalities within and differences between clusters. Stein and Zvelebil (12) and Patel et al (13) describe using 2D gel data sets to build supervised and unsupervised classifiers
Both types of classification problems have three stages, input, algorithm and output. Most published literature concentrates on the second of these. However, careful thought about what variables to use and how to characterize or summarize them as inputs into an algorithm are very important issues [11]. It is evident that the reliability and reproducibility of a classification is a function of the input, which in turn depends upon the process of data normalization, data reduction, and variable selection, i.e. the pre-processing of data. This paper focuses on the effects of preprocessing on the selection of variables that enters a classifier.
Pre-processing
In order to conduct a systematic analysis of 2D gel data, one has to pre-process the data set. Pre-processing in the case of 2D gel analysis includes: 1) normalizing intensities to remove effects of differential loading and staining; 2) transformation of outcome variables to normally distributed variables; and 3) imputing values for missing spot intensities. We are not aware of any other study that has looked systematically at the effect of pre-processing 2D gel data on the results of subsequent statistical analysis of the data. In this paper, we present a protocol for the analysis of 2D gel data and examine the effect of statistical pre-processing of 2D gel datasets.
The effect of using two different formulas for normalizing versus not normalizing, log-transformation versus no log-transformation and single value imputation versus multiple value imputation, and averaging spot intensities across replicates versus keeping the replicate information separate are compared using the results of two sample t-tests. We also compare the results of two-sample t-tests provided by the image analysis software PDQUEST to results obtained after following the protocol described in Figure 1. PDQUEST allows the user to normalize the data but has no facility for testing the distribution of the outcome variable and transforming it to fit a normal distribution. To the best of our knowledge, PDQUEST replaces missing spot intensities with a zero.
The experiment
The data used in this study is from an experiment that looked at the effect of a diet enhanced with grape seed extract on the proteome of whole brain homogenates of Sprague-Dawley rats [10]. There were five treated animals and five control animals. Due to sample availability and other issues related to the creation of 2D gels, each biological replicate had different numbers of technical replicates. The maximum number of replicates was four, and the minimum was two. A number of changes in proteins that were attributed to treatment differences in this study have been identified with Matrix Assisted Laser Desorption Ionization – Time Of Flight (MALDI-TOF) Mass Spectrometry. These changes have also been confirmed in later experiments with transgenic mice. Thus the protein changes detected by the statistical protocol used in this study have been shown to be biologically valid and relevant to the systems being studied.
Results
Variability in the resolution of protein spots in 2D gels
The resolution of protein spots in a 2D gel is highly variable. It can differ considerably between technical replicates of the same biological sample. Samples 6, 7, 8, 9, and 10 were the five biological replicates in the treatment group. Samples 22, 23, 24, 25, and 26 were the five biological replicates in the control group. Table 1 demonstrates the breakdown of the resolved protein spots in the different samples and its replicates. Biological sample 7, for instance, had 546 protein spots resolved in at least one of its four technical replicates. 169 (31%) proteins occurred in all the four replicates. An additional 130 (26%) were present in at least three replicates out of four. A further 97(18%) were present only in two replicates out of four, and 150 (27%) were present only in one of the four replicates.
Low correlation between technical replicates
Table 2 displays the range of Pearson's correlation coefficients and Kappa coefficients between the technical replicates of the same sample. The correlation coefficient here is a measure of the association between the spot intensities on technical replicates. The correlation between technical replicates is not very high. The correlation coefficients range from a high of 0.93 to a low of 0.47. The r-square ranges from 87% to 22%. The Kappa coefficient measures the degree of agreement between the spots present on two replicate gels of the same sample. A zero indicates no agreement and one indicates perfect agreement. If the confidence interval spans zero then the hypothesis that there is no agreement between the replicate gels cannot be rejected. Ten of the sixteen separate confidence intervals in Columns 5 and 6 of Table 2 include zero. This suggests no agreement between the replicate gels of most samples.
Assessing the quality of a pre-processing technique
In Tables 3 to 10 the spot identification numbers in bold represent proteins that were subsequently identified and found to be biologically relevant to the system being studied (10). In all, eleven proteins that had significantly different intensities at alpha = 0.05 were identified. The measure of the quality of a particular pre-processing technique in this study was the proportion of these eleven proteins identified as statistically significant in a two-sample t-test after the particular technique was used.
The effect of log-transformation and minimum values substitution on the distribution of intensities
Log – Transformation
Figures 3a and 3b are the QQ plots for the raw spot intensity and normalized spot intensities for the 201 protein spots on a representative gel from the control group. These plots demonstrate that the normalization technique used does not alter the basic distribution of the raw data. They also demonstrate the highly non-gaussian distribution of the spot intensities. Figure 3c demonstrates that the log transformation converts the distribution of the intensities from a very non-normal distribution to a normal distribution. The points lie very close to the straight line that represents a normal distribution. We have found that the log transformation reduces the skew in the distribution of the spot intensities if the image analysis is done in PDQUEST® and most data sets produced by the software PROGENESIS®. Only one out of seven 2D gel data sets analysed by us so far did not respond well to this transformation. A closer examination of this data showed it had a large number of saturated spots, and thus needed to be rerun. The transformation of the distribution of the 201 spots also worked reasonably well at the level of individual spot intensities. This was important to confirm since the two sample tests were done at individual spot level. In cases where there was a considerable skew in the distribution, e.g., SSP 1733, the log transformation made the distribution of spot intensities normal. The Anderson-Darling test for normality for SSP 1733 in the control group, has a p-value = 0.03 before the transformation, and p-value = 0.456 after the transformation. In general, this was true of most spots we examined.
Table 3 compares the t-test results of non-log-transformed normalized spot intensities where the missing spot intensities were replaced with a zero; to the results using normalized spot intensities that were log-transformed. In the non-log transformed data without normalization, six of the eleven spots known to be significantly different were picked up. Six of the eleven proteins were picked after log transformation of the non-normalized data as well. It is important to note that the two lists of six proteins were not identical. After normalization, in the log-transformed data the t-test picks up all the eleven proteins, whereas, in the non-log-transformed data seven proteins are picked up (Table 4).
Effect of normalization
Table 5 compares the proteins that were found to be significant (p = 0.05) in a t-test when the spot intensities were not normalized, not log transformed, and the missing intensities were replaced with a zero (Column 1); proteins that were found to be significant in a t-test (p = 0.05) when the spot intensities were normalized using normalization 1 (see methods), not log transformed, and the missing intensities were replaced with a zero (Column 2); proteins that were found to be significant in a t-test (p = 0.05) when the spot intensities were normalized using normalization 2 (see Methods), not log transformed, and the missing intensities were replaced with a zero (Column 3); and the proteins that were found to have significantly different intensities by the image analysis software PDQUEST (Column 3), which normalizes (each intensity divided by the total intensity of the gel) the data but does not use a log transformation. As is evident from comparisons of Column 1 with Columns 2 and 3 in Table 1, normalization has an effect on the number of proteins that are detected. The difference in Columns 2, 3 and 4 suggests that the protocol used for normalizations also has an impact on the proteins that are picked up as significant. In non-log transformed data, data sets with no normalization, normalization 1 and normalization 2 picked up six of the eleven proteins, and PDQUEST method picked up seven of the eleven proteins. Over fifty percent of the proteins picked up by PDQUEST occurred in very small numbers of gels, and hence did not meet our selection criteria for inclusion into the analysis data set. A number of the other proteins picked up by PDQUEST had skewed distribution. After log-transformations these proteins were no longer statistically significantly different. Tables 6, 7 and 8 again demonstrate that normalization 2 has a significant impact on the number of proteins identified as significantly different in intensity. In all of these tables, it is important to note once again that the highlighted proteins in each column vary with the pre-processing technique.
Effect of imputation of missing spots
In Table 9, Columns 1, 2, and 3 offer a comparison of the spots identified as significantly different (alpha = 0.05) when the three different kinds of imputation of missing spots were used. The three different kinds of imputation did not make much difference to the spots identified as significantly different in intensities at alpha = 0.05. In fact in this case we have identical lists in all three columns of table 9. The purpose of using multiple imputation instead of single value imputation, however, has more to do with getting a better estimate of the variance of a quantity than with the correct estimation of the mean. Since the t-statistic is a function of both the difference in means as well as the variance of a variable, given a constant mean, underestimating variance would lead to false positives, and overestimating variance would lead to false negatives. This data set shows a good example of the degree of variability on the intensities. Table 10 demonstrates that single value imputations tend to either underestimate (var = 0 for SSP 6452), or over-inflate the estimates of variance (e.g. SSP 1509). The variance of SSP 1733, which has no missing spot intensities, gives us a rough idea of the degree of variability expected in spot intensities when there are no intensities missing. The estimates of variance for the proteins with missing intensities are much closer to the values seen in SSP 1733 when one uses a multiple imputation technique.
Averaging across replicates versus keeping replicates separate
Given the lack of association between technical replicates, we used replicate information in two ways: 1) Spot intensities were averaged across the replicates so that the t-tests compared average spot intensities in the five treatment samples versus five control samples, and 2) The replicate gels of each sample were treated as independent gels, and the t-tests compared spot intensities in the fifteen treatment gels to the intensities in the fifteen control gels. Columns 1 and 2 in Table 11 compare Method 1 above to Method 2. Six out of eleven proteins are picked up using Method 1, whereas all ten are identified as significantly different if the replicates are kept separate.
Discussion
Normalization
Differential sample loading and stain absorption and other process variables can contribute to variability in measured protein intensity. In order to ensure that the detected differences in protein intensity are not due to a "technical" variability introduced by the process of gel creation, spot intensities are "normalized." Dividing the intensity of each protein on a gel by the total protein intensity of that gel is a widely used technique to reduce the "individual gel effect" on protein intensities [14]. Normalizing the data is an important step in many datasets, but it becomes especially important in proteomics experiments, which in general have many more variables than samples. In this case a systemic error in processing samples or gels that affects only one or two gels can have a huge impact on the results. This study has demonstrated that the results of statistical tests are not independent of the normalization technique.
Testing for normality and transforming data
To the best of our knowledge, none of the image analysis software packages available to date provide the tools necessary test for the distribution of the data. All of them provide t-tests or the non-parametric Wilcoxon rank sum test or ANOVA to test for differences in individual spot intensities. The probability values (p-values) for differences between groups are based on the assumptions of the normality of the distribution of spot intensities and equal variances. In order to make an informed judgement about the validity of the p-values of the tests above, it is important to know if these assumptions are met [15,16]. If one uses the averaged spot intensities across gels, the argument could be made that the central limit theorem obviates the need for a log transformation. Given the highly skewed nature of the raw spot intensities, the dependence of the mean and the variance of intensities, and the fact that even the non-parametric Wilcoxon test assumes symmetry in the outcome variable [17], log transformation of the data is still advisable.
Missing spot intensities
Despite the fact that 2D-gel technology offers many advantages, one of the pitfalls associated with this technology is the need for several replicates for proper validation of results. There are instances where one does not observe reproducible spot patterns or individual proteins even in replicate gels of the same sample. Missing spot intensities are commonly observed in 2D gel datasets. Multivariate techniques such as Principal Component Analysis and Discriminant Analysis (DA) are ideal tools to use on databases that have multiple outcome variables (protein intensities). However, SAS or any other statistical software that is used to analyse the data using multivariate techniques such as PCA and DA requires data sets with non-missing values. Gels with missing spot information will thus be dropped from the analysis. Since all gels will have some spot information missing, this will result in no gels being available for data analysis. To the best of our knowledge, all image analysis software packages substitute zeroes for missing intensity values. Missing intensities may be caused by the fact that a protein spot truly does not exist in one group compared to the other or because the spot intensity is so low that it is not detected by the image analysis software. In this study we treated all missing spots as undetectable spots. The question we were trying to answer was "If a spot exists but is 'undetectable', what is the best 'detectable' value to substitute as its intensity?" The most intuitive value was the smallest "detectable" intensity in the experiment hence we used the lowest intensity value in the experiment for the single value imputation. However, substituting a single value for all missing spot intensities would skew the distribution of spot intensities considerably. Thus the second option was to use a random process to substitute missing spot intensities with a plausible set of "detectable" intensities. We created a set of lowest 'detectable' intensities from the lowest intensity on each of the thirty gels. These values were used to impute missing intensity values as described in the methods section. Although we have used the terms "imputation" and "multiple imputation", these terms are not to be equated with multiple imputation advocated by Rubin [18]. Rubin's techniques assume the missing intensities to be Missing at Random (MAR). By our assumption, the spots are undetectable because of the intensity level, or the probability of missing is a function of the intensity. This by Rubin's definition would make the missing spot intensities Non-Ignorable Non-Response. His multiple imputation techniques thus would not be valid in this context.
The issue of missing protein intensities is one that has not been addressed at all in the literature describing 2D gel studies. In this study we treated all missing intensities as the same. However, all missing intensities in 2D gels are not equal. Some missing intensities are missing because they truly do not exist in one group versus the other, whereas others are missing because of the inherent variability in the process of creating 2D gels. This suggests that one needs to approach the filling of missing values differently based on the probability of a spot being a truly missing protein or one that is missing due to the process of gel creation. One way to do this would be to assume that spots that occur in two out of three replicates (or three out of four replicates) of a sample are true spots. These missing intensities would then be replaced by the mean value of the remaining two (three) spots. On the other hand, the assumption that those proteins that are missing in all control gels or all treatment gels are proteins that are turned on or off is justifiable. This in turn suggests that for this set of proteins the random imputation of missing values with a set of plausible minimum intensity values, acting as placeholders so that the non-missing data can be used in analyses, can also be justified.
Which method should one use?
In the last few years, published reports of 2D gel analysis have concluded that heart failure was associated with protein modifications in three cellular systems [19], identified proteins expressed in six different regions of brains of Alzheimer's disease patients [7], had been used to establish genetic relationships in the Brasscacae family [20], to classify human ovarian tumours as malignant and benign [8], in the detection of polypeptides associated with the histo-pathological differentiation of primary lung cancer [21], and to identify eight protein feature changes that differentiated breast cancer cell lines that did or did not form tumours in nude mice [9]. These are all important studies that will be used as springboards to launch ever more expensive and sophisticated experiments. We have demonstrated that protein changes that are large (e.g. SSP 6452 present in controls and absent in all treatment gels) are independent of the statistical protocol used. The identification of more subtle changes can vary widely depending on the statistical algorithm used to pre-process and analyze the data. Our experience with the GSE data and a couple of subsequent experiments we have been involved in suggests that the algorithm we have developed is more sensitive with respect to identifying biologically relevant proteins that image analysis software might miss. However, it is a fact that pre-processing could also give rise to false positive results. It is important to establish the best statistical protocol for analysing the data from these studies. One way to get around the issue of the effect of pre-processing is to restrict a study to only those proteins that are picked up as significant by image analysis software. As we have mentioned above, in this study a large number of the spots selected by PDQUEST were poor quality spots either in terms of protein quality or consistency. Another option is to consider only those spots that appear in two sets of analyses (e.g. image analyses, and the protocol described here) as true changes. One is thus restricting oneself to gross changes. As the use of proteomic techniques moves forward, however, we think it will be important to identify more subtle changes in proteins. A number of studies have suggested that change in protein expression that starts the cascade of changes that leads to a diseased tissue need not always be gross or dramatic. Subtle changes in expression early in a pathway can cause significant changes downstream. Shapiro et al [22] suggest that subtle changes in the spatial or temporal expression of the patterning molecule Sonic Hedgehog (SHH) is linked to the proliferation and patterning of developing limbs. Similarly, a disease condition could be caused by small changes in expression in a number of proteins. Reneiri et al [23] suggest that the phenotypic expression of Retts in some but not all girls with the MECP2 mutation suggests that MECP2 causes deregulation of a very small subset of genes that have not yet been detected or that very subtle changes in many genes (by extension proteins) may cause the neuronal phenotype. The importance of picking up subtle changes in protein expression suggested by studies cited above point to a need to establish a way to identify the optimal statistical pre-processing techniques for 2D gel datasets.
An intuitively appealing way to do this is to create 2D gels with serially diluted quantities of commercially available proteins, establishing the relationship of protein quantity to spot intensity and then proceeding to compare different statistical pre-processing techniques to the datasets acquired from these gels. Since it is a known fact that commercially bought proteins may not necessarily be pure and may be present in multiple modified forms due to the process of isolation, the experiment described above may be enhanced by using controlled biological samples from a cell culture with at least a hundred resolved spots. Once again, the sample could be loaded in on gels in known concentrations and the process described above would be repeated. Clearly, the set of pre-processing techniques that picks up differences that come closest to the true differences would be chosen as the optimal techniques. Some recent publications have used similar techniques to establish the validity of emerging proteomic technology. Alban et al [24] used an Escherichia coli lysate "spiked" with varying amounts of four different known proteins to test a novel experimental design that exploits the sample multiplexing capabilities of DIGE by including a standard sample in each gel. Rabilloud et al [25] compared the staining sensitivity of RuBPS and Sypro Ruby of serial dilutions of molecular weight markers. However, there are no designed experimental studies that have looked at the impact of statistical pre-processing or the effectiveness of various statistical techniques on the conclusions drawn from 2D gel experiments. Given the proliferation and promise of 2D gel experiments, we suggest that the need to conduct these validation experiments is urgent.
In this study we have used the proteins that were subsequently identified by MALDI-TOF spectroscopy as a measure of how well particular statistical protocols perform. We concede that there is an inherent bias in that the spots that were identified by MALDI-TOF were selected on the basis of the protocol described in this paper. The described protocol therefore will seem to perform much better than others in this comparison. This however does not diminish the main thrust of this paper, which is that statistical protocols affect the conclusions drawn from a 2D gel experiment.
Conclusions
This study has demonstrated that the pre-processing of the data from 2D gel experiments can have a significant impact on the results of statistical tests. The purpose of the study was not to identify the particular statistical protocol used in this study as the optimal protocol, but rather to demonstrate that the results and conclusions from a biological experiment are not independent of the statistical protocol. The study has in effect looked at three different statistical protocols. The one described in Figure 1, the one described in Figure 2, in which the averaging of intensities across replicates allows one to proceed directly on to the t-tests, without the steps of testing distributions, or imputation of missing spots, and the protocol used by PDQUEST. Allowing for the inherent bias we have described above, this study shows that the protocol described in Figure 1, with normalization technique 2, and multiple imputations, is superior to the method used by PDQUEST, or the one in Figure 2. Given the possible bias in this study, the larger conclusion from the study is that there is a great need for research into developing optimal statistical methodology to analyze data from 2D gel experiments.
Methods
We used data from an experiment that compared the protein expression in the whole brain homogenate of rats that were fed a diet with 5% of grape seed extract to that of a normal rat diet. This experiment will be described in a separate article. The data from this experiment was exported to a database, which was saved as a text file. The database was imported into SAS V-9.0 (Statistical Analysis Software, 2003 Cary NC, USA), which was then used to do all the statistical analysis.
Normalization 1
The intensity of each protein spot was divided by the total protein content (total intensity) in the experiment.
Normalization 2
The intensity of each spot in each group was then normalized to the median intensity of its group, i.e. the intensity of each spot was converted into the intensity it would have had if the gel it was in had a total intensity equal to the median total intensity of the group. This is described by the following formula:
This normalization reduces intra group variability, but maintains the inter group variability.
Fold change is a common metric used in articles describing gene array and proteomic experiments. Fold change measures the degree of change in protein intensity in the treatment group, compared to the control group. This is measured by dividing the average spot intensity in the treatment group by the average spot intensity in the control group. In order for this ratio to be a true comparison of the intensities in the two groups, the intensities need to be expressed as proportions of the same quantity, i.e. they need to be divided by the same quantity. Normalization 1 has this property built into the formula. In normalization technique 2, the normalized intensities were divided by the total protein content in the experiment in order to make a fold change comparison meaningful.
Subset of spots included in analysis
A spot was considered present only if it was present in both replicates of a sample if there were only two replicates, in two out of three replicates if there were three replicates of a sample, and three out of four replicates if there were four replicates of a sample. We also included spots that were present in all controls and absent from all treatments, or present in all treatment gels but absent from all controls. Using these criteria, we had 201 spots that were available for statistical analysis from the GSE database.
Missing spot intensities
Given the criteria used to subset spots for the final analysis, there was a large number of missing spot intensities. We examined the effect of filling in the missing spots in three different ways.
1. Missing spot intensities were first replaced with the lowest value of log-transformed intensities. In this case the value was -17.28. The effect of the replacement of this single value for all missing spot intensities is the same as the effect of replacing non-log transformed spot intensities with zero.
2. We created 1000 separate data sets that randomly selected one of the 15 lowest spot intensity values from the 15 gels in the control group to replace each of the missing spot intensities in the controls, and repeated the process for the 15 treatment gels.
3. In the third imputation method, each missing spot intensity was replaced with a randomly selected value from the 30 lowest values from each gel in the experiment without regard to whether the value chosen was from a control or treatment gel. The two methods of random imputation of spot intensities were replicated 1000 times. two sample t-tests were repeated with each replication.
Authors' contributions
HK is the PI of the project that provided the data for this experiment. JD, HK's Research Associate, created the 2D gels, conducted the image analysis and created the database. SM designed this study and performed the statistical analyses.
Figures and Tables
Figure 1 Statistical protocol for 2D gels. The first protocol that we followed in the statistical analysis of data from 2D gel experiments is demonstrated in the flowchart in Figure 1. This consists of: 1) testing for differences between the groups with respect to total protein expression; 2) normalizing protein intensities on a gel to the mean total intensity of its group (e.g. treatment or control); 3) expressing each normalized intensity as a fraction of the total protein intensity in the experiment in order to make fold change comparisons meaningful; 4) testing the distribution of normalized intensities and using appropriate transformations if necessary to convert distribution to a normal distribution; 5) selecting the subset of protein spots to analyse; 6) imputing values for missing spot intensities; 7) using 2-sample t-tests and f-tests to identify protein spots that can be used to build classifiers; 8) building a linear (or quadratic) discriminant classifier; and 9) using Principal Components Analysis plots to demonstrate the separation between groups visually.
Figure 2 Statistical protocol for 2D gels. The second protocol that we followed in the statistical analysis of data from 2D gel experiments is demonstrated in the flowchart in Figure 2. This consists of: 1) testing for differences between the groups with respect to total protein expression; 2) normalizing protein intensities on a gel to the mean total intensity of its group (e.g. treatment or control); expressing each normalized intensity as a fraction of the total protein intensity in the experiment in order to make fold change comparisons meaningful; 3) selecting the subset of protein spots to analyse; 4) Average spot intensities across gels from the same sample; 5) imputing values for missing spot intensities; 6) using 2-sample t-tests and f-tests to identify protein spots that can be used to build classifiers; 7) building a linear (or quadratic) discriminant classifier; and 8) using Principal Components Analysis plots to demonstrate the separation between groups visually.
Figure 3 Intensity plots. (a) Plot of raw intensities before log-transformation and normalization. Probability plot of raw intensities of 201 spots in the final data set; a normally distributed variable is expected to plot a line close to the straight line; the intensities are very skewed. (b) Plot of Normalized spot intensities. Probability plot of normalized spot intensities of 201 spots in the final data set. Comparison of 3a and 3b demonstrates that the normalization does not alter the distribution of the spot intensities. (c) QQ plot of log transformed intensities. Figure 3c demonstrates that the log transformation successfully transforms the highly skewed distribution of spots into a normal distribution.
Table 1 The number of protein spots resolved in replicate gels of the same biological sample.
Sample No (Treatment group) No. of Replicates Total Proteins resolved (occurring in at least one replicate) (%) Number resolved in all replicates (%) Number resolved in all but one replicate (%) Number resolved in all but two (%) Number resolved in just one (%)
6 (GSE) 3 4064 (100) 309 (8) 563 (14) 3192 (78) -
7 (GSE) 4 546 (100) 169 (31) 130 (26) 97 (18) 150 (27)
8 (GSE) 4 954 (100) 186 (19) 120 (12) 105 (11) 543 (57)
9 (GSE) 2 904 (100) 342 (39) 562 (62) - -
10 (GSE) 2 396 (100) 229 (58) 167 (42) - -
22 (CONT) 4 924 (100) 234 (25) 89 (10) 109 (12) 492 (53)
23 (CONT) 4 950 (100) 161 (17) 151 (16) 102 (11) 536 (59)
24 (CONT) 2 879 (100) 312 (35) 567 (65) - -
25 (CONT) 3 957 (100) 272 (28) 117 (12) 568 (59)
26 (CONT) 2 432 (100) 183 (42) 249 (58) - -
The variability in the resolution of protein spots in technical replicates in a 2D gel experiments is one of the causes of the large number of missing spot intensities. The variability in the table above demonstrates the need for technical replicates as a quality control measure to identify spots that are most 'reliable' and common and therefore most useful to generalize to a larger population.
Table 2 Highest and lowest correlation among spot intensities between technical replicates in a 2D gel experiment; highest and lowest Kappa coefficients between technical replicates
Sample No of Replicates Highest Correlation Coefficient (R-sq%) Lowest Correlation Coefficient (R-sq%) Highest Kappa 95% CI Lowest Kappa 95% CI
6 (GSE) 3 0.774 (60%) 0.547 (30%) 0.4962 (-0.1039,1.000) 0.1476 (-0.1374,0.4326)
7 (GSE) 4 0.907 (82%) 0.589 (35%) 0.4437 (0.1004,0.7870) -0.0767 (-0.114,-0.0350)
8 (GSE) 4 0.932 (87%) 0.617 (38%) 0.4445 (0.1027,0.7864) 0.0099 (-0.0855,0.1052)
9 (GSE) 2 0.747 (56%) 0.747 (56%) 0.1299 (-0.1384,0.3982) 0.1299 (-0.1384,0.3982)
10 (GSE) 2 0.837 (70%) 0.837 (70%) 0.1231 (-0.0486,0.2948) 0.1231 (-0.0486,0.2948)
22 (CONT) 4 0.805 (65%) 0.467 (22%) 0.6538 (0.3705, 0.9372) 0.2599 (-0.0566,0.5765)
23 (CONT) 4 0.845 (71%) 0.632 (40%) 0.3269 (0.0304,0.6235) 0.0322 (-0.0296,0.0941)
24 (CONT) 2 0.711 (50%) 0.711 (50%) 0.0743 (-0.1284,0.2770) 0.0743 (-0.1284,0.2770)
25 (CONT) 3 0.837 (70%) 0.524 (27%) 0.2843 (0.0073,0.5613) 0.2384 (-0.0629,0.5397)
26 (CONT) 2 0.578 (33%) 0.578 (33%) 0.1946 (0.0531,0.3361) 0.1946 (0.0531,0.3361)
The Pearson correlation coefficient is a measure of the linear relationship between two variables. R square, the square of Pearson's correlation is a measure of how much variability in one variable is explained by the variability in the other. Since technical replicates are expected to be identical, the r-squares are expected to be very high, at least 0.95. The table demonstrates the degree of variability between technical replicates after normalization. The Kappa coefficients with the 95% confidence intervals confirm the same thing. Ten out of sixteen confidence intervals span zero, indicating no agreement between technical replicates of the same sample in those cases.
Table 3 The effect of log transformation using non-normalized data.
No Log Transform No Normalization, Missing replaced with zero (55% of identified spots picked up) Log Transformed No Normalization, Missing replaced with – 17.28 (55% of identified spots picked up)
SSP 1509 SSP 1509
SSP 1733
SSP 2307
SSP 3219
SSP 3806
SSP 4225
SSP 4435 SSP 4435
SSP 4438
SSP 4519
SSP 4724
SSP 5413
SSP 6314 SSP 6314
SSP 6452 SSP 6452
Column 1 has spots that have significantly different intensities (p = 0.05) before normalizing and log transforming data. Column 2 has spots that are significantly different in intensity before normalizing data, but after using a log transformation. Spots in bold were later identified by MALDI-TOF. These were all spots that were biologically relevant to the system being studied. The percentages in parenthesis measure in the header indicate how many of the ten proteins known to be different were identified before log transformation.
Table 4 The effect of log transformation using non-normalized data.
No Log Transform Normalized, Missing replaced with zero (63% of identified spots picked up) Log Transformed Normalized, Missing replaced with – 17.28 (100% of identified spots picked up)
SSP 1134
SSP 1509 SSP 1509
SSP 1733
SSP 2309 SSP 2309
SSP 3219
SSP 3806
SSP 4203
SSP 4225 SSP 4225
SSP 4435 SSP 4435
SSP 4519
SSP 4724
SSP 5309
SSP 5329
SSP 5413 SSP 5413
SSP 6205 SSP 6205
SSP 6228
SSP 6304 SSP 6304
SSP 6314 SSP 6314
SSP 6321
SSP 6349
SSP 6443
SSP 6452 SSP 6452
SSP 7223 SSP 7223
SSP 7334
SSP 7750 SSP 7750
SSP 8613
Column 1 has spots that have significantly different intensities (p = 0.05) after normalizing and log transforming data. Column 2 has spots that are significantly different in intensity after normalizing data (using normalization 2), but after log transformation. The percentages in parenthesis in the header measure how many of the ten proteins known to be different were identified after log transformation and normalization.
Table 5 The effect of log transformation using non-normalized data.
No Log Transform No Normalization Missing replaced with zero (54% of identified spots picked up) No Log Transform – Normalization 1 Missing replaced with zero (54% of identified spots picked up) No Log Transform Normalization 2, Missing replaced with zero (54% of identified spots picked up) No Log Transform Normalized-PDQUEST (64% of identified spots picked up)
SSP 03121
SSP 11121
SSP 1309
SSP 13211
SSP13311
SSP 1509 SSP 1509 SSP 1509
SSP 1733 SSP 1733
SSP 2307
SSP 2309 SSP 2309
SSP 32341
SSP 34371
SSP 35231
SSP 4225 SSP 4225 SSP 4225 SSP 4225
SSP 4435 SSP 4435 SSP 4435
SSP 4438 SSP 4438 SSP 44382
SSP 45171
SSP 4519 SSP 4519 SSP 4519 SSP 45192
SSP 46372
SSP 4724 SSP 4724 SSP 4724
SSP 47351
SSP 50111
SSP 5309
SSP 5329
SSP 5413 SSP 5413 SSP 5413 SSP 5413
SSP 6205
SSP 6304
SSP 6314 SSP 6314 SSP 6314
SSP 6321
SSP 6349
SSP 6443
SSP 6452 SSP 6452 SSP 6452 SSP 6452
SSP 7027
SSP 7231
SSP 7223
SSP 7334
SSP 74131
SSP 7750
SSP 8613
1 These are spots that were present in a very small number of gels, and therefore did not meet our criteria to be included.
2 These spots have highly skewed distributions or were very poor quality spots. Log transformation made out the distribution closer to normal and p-values were no longer significant.
Column 1 has spots that have significantly different intensities (p = 0.05) normalizing and log transforming data. Column 2 has spots that are significantly different in intensity after using normalization 1, but before using a log transformation. Column 3 has spots that are significantly different in intensity after using normalization 2, but before using a log transformation. Column 4 has the results from the image analysis software PDQUEST, which has an option for normalizing but no log transformation. Columns 1 and 2 are subsets of the 201 spots in the final data set that met our criteria for inclusion. Column 3 is a subset of all possible spots in the experiment. Spots in bold were later identified by MALDI-TOF. These were all spots that were biologically relevant to the system being studied. The percentages in parenthesis in the header measure how many of the ten proteins known to be different were identified after the different normalization techniques.
Table 6 T-Test results of log transformed intensities pre-and post normalization – Imputation Method 1
Normalization 1 Missing replaced by = -.17.28 Log transformed (36% of identified spots picked up) Normalization 2 Missing replaced by = -17.28 Log transformed (100% of identified spots picked up)
SSP 1134
SSP 1509 SSP 1509
SSP 1733
SSP 2309
SSP 3219
SSP 3806 SSP 3806
SSP 4203
SSP 4225
SSP 4435 SSP 4435
SSP 4724 SSP 4724
SSP 5413
SSP 6205
SSP 6304
SSP 6314 SSP 6314
SSP 6452 SSP 6452
SSP 7223
SSP 7750
The table shows the results of two sample t-tests on log-transformed intensities, pre and post normalization, when all missing intensities were replaced by the lowest intensity value in the experiment (-17.28). Spots in bold were later identified by MALDI-TOF. These were all spots that were biologically relevant to the system being studied.
Table 7 T-Test results of log transformed intensities pre-and post normalization – Imputation Method 2
Normalization 1 Missing replaced by Random sample of minimum from 15 gels in each group Log transformed (36% of identified spots picked up) Normalization 2 Missing replaced by Random sample of minimum from 15 gels in each group Log transformed (91% of identified spots picked up)
SSP1134
SSP 1509 SSP 1509
SSP 1733
SSP 2309
SSP 3806 SSP 3806
SSP 4203
SSP 4225
SSP 4435 SSP 4435
SSP 4724 SSP 4724
SSP 5413
SSP 6205
SSP 6228
SSP 6304
SSP 6314 SSP 6314
SSP 6452 SSP 6452
SSP 7223
SSP 7334
SSP 7750
The table shows the results of two sample t-tests on log-transformed intensities, pre and post normalization, when each missing intensities in GSE (control) gel were replaced by randomly selecting one of the 15 lowest spot intensity values from the 15 gels in the GSE (control) group. Spots in bold were later identified by MALDI-TOF. These were all spots that were biologically relevant to the system being studied.
Table 8 Test results of log transformed intensities pre-and post normalization – Imputation Method 2
Normalization 1 Minimum replaced by Random sample of minimum from all 30 gels Log transformed (36% of identified spots picked up) Normalization 2 Minimum replaced by Random sample of minimum from all 30 gels Log transformed (91% of identified spots picked up)
SSP 1134
SSP 1509 SSP 1509
SSP 1733
SSP 2309
SSP 3806 SSP 3806
SSP 4203
SSP 4225
SSP 4435 SSP 4435
SSP 4724 SSP 4724
SSP 5413
SSP 6205
SSP 6228
SSP 6304
SSP 6314 SSP 6314
SSP 6452 SSP 6452
SSP 7223
SSP 7334
SSP 7750
The table shows the results of two sample t-tests on log-transformed intensities, pre and post normalization, when each missing intensities in GSE or control gel were replaced by randomly selecting one of the 30 lowest spot intensity values from the 30 gels in the experiment. Spots in bold were later identified by MALDI-TOF. These were all spots that were biologically relevant to the system being studied.
Table 9 Comparing t-test results for the three different imputation methods
Normalization 2 Missing replaced by = -17.28 Log transformed (100% of identified spots picked up) Normalization 2 Missing replaced by Random sample of minimum from 15 gels in each group Log transformed (91% of identified spots picked up) Normalization 2 Minimum replaced by Random sample of minimum from all 30 gels Log transformed (91% of identified spots picked up)
SSP 1134 SSP1134 SSP 1134
SSP 1509 SSP 1509 SSP 1509
SSP 1733 SSP 1733 SSP 1733
SSP 2309 SSP 2309 SSP 2309
SSP 3219
SSP 3806 SSP 3806 SSP 3806
SSP 4203 SSP 4203 SSP 4203
SSP 4225 SSP 4225 SSP 4225
SSP 4435 SSP 4435 SSP 4435
SSP 4724 SSP 4724 SSP 4724
SSP 5413 SSP 5413
SSP 6205 SSP 6205 SSP 6205
SSP 6304 SSP 6228 SSP 6228
SSP 6314 SSP 6304 SSP 6304
SSP 6314 SSP 6314
SSP 6452 SSP 6452 SSP 6452
SSP 7223 SSP 7223 SSP 7223
SSP 7334 SSP 7334
SSP 7750 SSP 7750 SSP 7750
In column 1 missing values were replaced with the lowest intensity value in experiment; in column 2 values to replace missing intensities were randomly chosen from the 15 lowest intensity values within a treatment group; in values to replace missing intensities were randomly chosen from the 30 lowest intensity values without regard to treatment group.
Table 10 Comparing variances of spot intensities when using multiple values to impute versus a single value imputation.
Multiple Imputation Single Imputation
Treatment Control Treatment Control
SSP 1509 0.767 0.663 7.05 8.39 9 missing control
Range = (3.16) Range = (2.63) Range = (7.34) Range = (6.56) 3 missing treatment
SSP 1733 0.356 1.4249 0.356 1.4249 0 missing control
Range = (2.23) Range = (3.74) Range = (2.23) Range = (3.74) 0 missing treatment
SSP 6314 1.2055 1.178 3.41 1.17 0 missing control
Range = (3.94) Range = (3.51) Range = (7.15) Range = (3.51) 14 missing treatment
SSP 6452 0.547 0.564 0.547 0 15 missing control
Range = (2.79) Range = (2.14) Range = (2.79) Range = (0) 0 missing in treatment
The variances for spots with missing values are either under estimated or over estimated with single imputation values. The ranges and variance values of intensities are closer to that of SSP 1733 (spot with no missing intensities) in the case of multiple value imputation.
Table 11 Comparing averaging across replicates versus not.
Averaged across replicates (54% of identified spots picked up) Log Normalized Log Transformed Missing replaced with – 17.28 (100% of identified spots picked up)
SSP 1134 SSP 1134
SSP 1509 SSP 1509
SSP 1733 SSP 1733
SSP 2309
SSP 2864
SSP 3222
SSP 3219
SSP 3806
SSP 4203
SSP 4225 SSP 4225
SSP 4435
SSP 4724
SSP 5413 SSP 5413
SSP 6205
SSP 6236
SSP 6304
SSP 6314 SSP 6314
SSP 6349
SSP 6452 SSP 6452
SSP 7144
SSP 7223
SSP 7439
SSP 7750 SSP 7750
Column 1 has the results of two sample t-tests when the intensity values were averaged across replicates. Columns 2 represents results of two sample t-tests when replicates were treated as independent observations.
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| 15707480 | PMC553976 | CC BY | 2021-01-04 16:02:58 | no | BMC Biotechnol. 2005 Feb 11; 5:7 | utf-8 | BMC Biotechnol | 2,005 | 10.1186/1472-6750-5-7 | oa_comm |
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BMC BiotechnolBMC Biotechnology1472-6750BioMed Central London 1472-6750-5-81571322710.1186/1472-6750-5-8Methodology ArticleLow density DNA microarray for detection of most frequent TP53 missense point mutations Rangel-López Angélica [email protected]íguez Rogelio [email protected] Mauricio [email protected] Juana Mercedes [email protected]éndez-Tenorio Alfonso [email protected] Kenneth L [email protected] Escuela Nacional de Ciencias Biológicas IPN, México, 11340, D. F., México, 11340, D. F., México2 Unidad de Investigación Médica en Enfermedades Oncológicas. Hospital de Oncología. Centro Médico Nacional S XXI. IMSS, México, D. F., México3 Amerigenics, Inc., 1326 Open Range Rd., Crossville, TN 38555 USA2005 15 2 2005 5 8 8 1 7 2004 15 2 2005 Copyright © 2005 Rangel-López et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
We have developed an oligonucleotide microarray (genosensor) utilizing a double tandem hybridization technique to search for 9 point mutations located in the most frequently altered codons of the TP53 gene. Isolated and multiplexed PCR products, 108 and 92 bp long, from exons 7 and 8, respectively, were obtained from 24 different samples. Single-stranded target DNA was then prepared from isolated or multiplexed PCR products, through cyclic DNA synthesis. Independent ssDNA's were annealed with the corresponding pairs of labeled stacking oligonucleotides to create partially duplex DNA having a 7-nt gap, which contains the sequence that will be interrogated by the capture probes forming double tandem hybridization. In the case of multiplexed ssPCR products, only two stacking oligonucleotides were added per target, therefore the gap for the PCR products having two consecutive codons to be interrogated in exon 7 was 12 nt long, so only single tandem hybridization was produced with these respective probes.
Results
18 codon substitutions were found by DNA sequencing. In 13 of them a perfect correlation with the pattern of hybridization was seen (In 5 no signal was seen with the wt probe while a new signal was seen with the appropriate mutant probe, and in 8 more, as expected, no signal was seen with any probe due to the absence of the corresponding probe in the array). In 3 other cases a mutation was falsely suggested by the combination of the absence of the wild type signal along with a false signal in the other probe. In the other 2 cases the presence of the mutation was not detected due to the production of a false hybridization signal with the wild type probe. In both cases (false mutation or no mutation detected) relatively stable mismatched target/probe duplexes should be formed. These problems could be avoided by the addition of probes to improve the performance of the array.
Conclusion
Our results demonstrate that a simple TP53 microarray employing short (7-mer) probes, used in combination with single or double tandem hybridization approach and a simple or multiplex target preparation method, can identify common TP53 missense mutations from a variety of DNA sources with good specificity.
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Background
Cancer is a group of diseases characterized by uncontrolled cell growth. According to The World Cancer Report, cancer rates could further increase by 50% to 15 million new cases in the year 2020 [1]. The chances of surviving the onset of some common cancers depend largely on how early they are detected in addition to how well they are treated.
The progression of mammalian cells towards malignancy is an evolutionary process that involves an accumulation of mutations at the molecular and chromosomal level. A candidate for involvement in this process is the tumor suppressor gene TP53 (MIM # 191170) which encodes a transcription factor (p53 protein) with cancer inhibiting properties. This gene is the most frequently mutated gene in human cancer. Between 30% and 70% of cancers of almost every organ and histological subtype have a point mutation in one of the two TP53 gene copies together with loss of the other copy [2-4]. In many cancers the distribution of mutations along the TP53 gene is tumor-specific, clustered between exons 5, 7 and 8. This region is highly conserved throughout evolution [3]. In this domain, six mutational "hotspots" have been identified at codons 175, 245, 248, 249, 273, and 282. The mutational profile of the TP53 gene is different between cancers, for example, codons 157, 158, 248, 249, and 273 have been designated as TP53 mutational hotspots in lung cancer [5,6], while in breast cancer the mutational hotspots are in codons, 175, 245, 248, 249 and 273 [7]. On the contrary, in cervical cancer the frequency of the p53 mutations is very low. [8].
Because mutations in the TP53 gene are so common in human tumors and have been widely reported in the literature, extensive mutational databases exist, such as that maintained by IARC [9]. Walker et al. [10], have utilized the IARC TP53 mutation database to define 73 "hotspots" for mutation in TP53 related to changes in protein structure and function. Martin et al. [11] performed a systematic automated analysis of the effects of TP53 mutations on the structure of the core domain of the p53 protein to functionally classify the various types of p53 mutants according to predicted effects on protein folding or DNA-protein interactions. Most mutations are currently identified by conventional methods of polymorphism detection and DNA sequencing [12]. A growing number of nucleic acid hybridization techniques have recently been applied to biomedical problems, including development of DNA probe arrays for detection of TP53 mutations [13-15]. In many applications, DNA chips containing surface-bound oligonucleotides (probes) are used to interrogate sample (target) sequence information through complementary recognition (hybridization) in a highly parallel fashion [16]. Major applications of DNA microarrays include gene expression profiling [17,18] and gene mutation analysis [19,20]. These techniques allow parallel analysis of multiple DNA samples. Mutation studies with DNA microarrays are still at an early stage and are in continuous development [21,22]. Although the field of microarray assays is still in a relatively early stage, it is generally anticipated that microarray assays will offer decreased cost and faster results, compared with the traditional, more labor intensive dideoxy sequencing approach. We recently reported a novel tandem hybridization strategy and its application for identification of mutations. Reliable discrimination of point mutations has been achieved by double tandem hybridization with a set of seven-mer probes [23,24]. In the present work we designed a novel small format DNA chip (genosensor array) to search for nine point mutations in codons 248, 249 and 273 of the TP53 gene.
Results
PCR products and reference hybridization patterns
PCR products from exon 7 (codons 248 and 249) and exon 8 (codon 273) were obtained from each type of DNA sources listed in Materials and Methods. Figure 2A shows gel electrophoretic analysis of the fragments of 108 and 92 bp corresponding to exons 7 and 8, respectively. To prepare ssDNA targets PCR products were used as templates for cycle synthesis of a target strand, using the reverse PCR primer in single or multiplexed reactions cycled 25 times. The production of single-stranded target DNA's was assessed by its change in electrophoretic mobility in 4% agarose gels (Figure 2B).
The reference hybridization patterns produced using synthetic DNA targets are shown in Figures. 3A and 3B. As expected, all the synthetic targets, including those of the negative controls, yielded hybridization signals with their respective ("perfect-match") probes. Pairs of wild-type with each mutant synthetic target sequences (1:1 mixtures) were also hybridized to reproduce heterozygous conditions. All gave the expected signals, (Figure 3B).
Hybridization of DNA targets
Hybridization of individual or multiplexed ssDNA targets prepared from the 24 DNA samples (that correspond to 72 codons in 24 samples) was performed with similar results. Base substitutions were seen in 11 out of the 24 samples tested (Table 3). One, two or three codons were altered in 5 (P6, L1, L2, B3 and B6), 5 (P1, L4, L7, L9 and L10) and 1 (L8) samples, respectively. In the cases of the samples having two or three codons affected, at least one of the base substitutions was silent. In the other 13 samples only WT sequences were identified (data not shown). Representative hybridization patterns are shown in Figure. 4A and the overall results are summarized in Table 3. The DNA samples hybridized with one or two probes from each codon, corresponding to the homozygous or heterozygous condition, respectively. Among the 24 different samples multiplexed or individually tested in the microarray, six single base susbstitutions in codon 248 were found by sequencing the PCR product. All of them corresponded to base substitutions keeping the original aminoacid (Arginine). The same number of point mutations were also seen in codon 249 by DNA sequencing. However, no probes to search for these base substitutions were placed in the DNA microarray. Six point mutations in codon 273 were obtained. All of them, except the CGT→CCT change observed in plasmid P6, were searched with the set of probes contained in the DNA microarray. In this codon all the base substitutions are producers of aminoacid substitution.
Discussion
The TP53 tumor suppressor gene has proven to be one of the cellular genes most often mutated in human neoplasias. Analysis of the mutational events that target the TP53 gene has revealed evidence for both exogenous and endogenous mutational mechanisms. This gene mutational spectrum suggests evidence for a direct causal effect of tobacco smoke in lung cancer [26,27], aflatoxin B1 in liver cancer [28]. Therefore, identification of mutations in the TP53 gene may play an important role in the diagnosis, staging, and management of the cancer patients.
More than 70% of the molecular studies have focused on the central region of the TP53 gene, more specifically on exons 5 through 8 which encode the DNA binding domain [3]. Thus, missense mutations in this region can cause a modification that may alter DNA binding. It is therefore essential that clinical trials aim to accurately establish the effect of TP53 mutations on all clinical parameters. One approach is the p53 GeneChip of Affymetrix used to identify mutations in this gene [13,14]. However, this technology is expensive and poorly available to many clinicians, which makes it necessary to develop new mutation detection methods [29,30].
The updated IARC TP53 mutation database [38] shows that more than 1700 different point mutations, which are associated to cancers, are distributed along the TP53 gene. TP53 somatic mutations are found in most types of sporadic human cancers at various frequencies (from 20% to 60%) [13,14]. The database also shows that approximately 20% of all mutations are located in the five codons (hotspots) in the core domain: 175, 245, 248, 249 and 273. Therefore a primary screen of these codons would be attractive.
Most SNP diagnostic arrays use probes varying from 15 to 20 nt in length with similar Tm values [31]. However, under this circumstance some of the mutant sequences are difficult to discriminate, due to the relatively small destabilizing effect of certain single mismatches in the probe/target duplex [32].
The tandem hybridization method described here offers several advantages over the traditional oligonucleotide array configuration in which each interrogated target sequence is represented by a single surface-tethered probe. Because the long stacking probe targets the analysis to a single unique site within a nucleic acid, direct hybridization analysis of nucleic acid samples of high genetic complexity using short capture probes may be enabled [24].
Recently, we successfully used a double tandem hybridization strategy in the genesensor chip system to discriminate point mutations in codon 634 of RET oncogene in individuals affected by medullary thyroid carcinoma [23]. This DNA chip format involves a target sequence which is annealed with a pair of labeled oligonucleotides (stabilizing oligonucleotides) prior to hybridization to the array of capture probes, forming a single-stranded gap between the stabilizing oligonucleotides and corresponding to the length of the capture probes. The partially duplex product contains the sequences interrogated by the capture probes, and when the sequences between the target DNA and the capture probe (attached to slide) are perfectly complementary, the stability of the hybridization is enhanced due to coaxial stacking of bases between the contiguous ends of the probe and the stabilizing oligonucleotides. The energy involved varies with the identity of the base pairs and the coaxial stacking [22]. Tandem hybridization uses shorter capture probes than traditional oligonucleotide array hybridization, which may contribute to increased discrimination of point mutations because the single base mismatch acts on a less stable duplex formed between the target DNA and the shorter probe. Two additional attractive features inherent to tandem hybridization are that, i) the probability that formation of secondary structure within the single stranded DNA [25] will block accessibility of the target sequence is reduced, and ii) the label needed to reveal the hybridization can be introduced into the two stabilizing oligonucleotides, avoiding the extra step of labeling individual target strands [33,34].
The goal of the present work was to develop a simple format array able to perform a primary screening of the mutations occurring in the most frequently affected codons of the TP53 gene.
It has been recently reported that detection of mutations yields variable results depending on the type of DNA sample [35,36]. Is clear that, the sensitivity to detect mutations in TP53, may also differ depending on the method used. In the current study the double tandem hybridization assay was applied to genomic DNA samples extracted from a variety of sources, including blood cells, cell lines and plasmids. There were not any differences in the ability of our system to detect mutations with respect to the different DNA sources.
In the experiments employing synthetic DNA targets hybridized with arrays of mutant and WT probes the expected hybridization signals were seen both in homozygous and heterozygous conditions (Figure. 3). This result indicates that the probes are working properly.
The hybridization of natural targets was done both with isolated and multiplexed PCR products with similar results. Multiplexing was applied in all (PCR reaction ssDNA target preparation, annealing of target with stacking oligonucleotides and hybridization) the steps. So these results are suggesting that multiplexing can be confidently employed for this analysis.
Base changes were found in 11 DNA samples. Two were from plasmid, seven from cell lines and two from blood samples. In 5 of these samples a single codon was affected, in other 5 samples two codons were altered, and in the other sample three codons were affected. Therefore a total of 18 codon alterations were detected. In five of the 18 codon alterations (Samples L2 codon 273, L7 codon 273, L8 codon 273, B3 codon 273 and B6 codon 273) both the absence of hybridization signal with the wild type probe and the production of signal with the appropriated probe was seen. In eight cases (Samples P1 codon 248, P6 codon 273, L8 codons 248 and 249, L9 L8 codons 248 and 249 and L8 codons 248 and 249 and, L10 codons 248 and 249) no hybridization signal was seen even with the wild type sequence. This was due to the absence of the corresponding mutant probe in the array. In three cases (P1 codon 249, L4 codon 249 and L7 codon 249) having either homozygous (sample P1) or heterozygous (samples L4 and L7) genotypes (wtAGG→mutAGA) no hybridization signal with the wild type probe and, an additional false hybridization signal with the probe corresponding to the substitution of guanine for thymine in codon 249 (p-249 G2→T) was seen, which could be relatively stable under the conditions tested. Therefore an additional probe, searching by the AGA sequence, should be added in the future array to discriminate between the two alternative silent (AGA) and missense (AGT) genotypes. In the other two codons affected (L1 codon 248 and L4 codon 248) the presence of signal with the wild type probe was seen even when the sequencing was showing a base change (wtCGG→mutCGT in sample L1 or 248 wtCGG→mutCGK in sample L4). In both cases a G:N mismatch is formed which, according to SantaLucia [37], is relatively stable. So, as in the previous two cases the incorporation of additional probes in the array should help to improve performance of the DNA microarray to confidently identify the genotypes.
The base substitution observed in B3 and B6 blood samples is a mutation that substitutes Arginine by Leucine. The presence of this mutation in blood suggests that it could be from germinal origin.
In all the mutations (silent and missense) observed an Arginine was involved, which can be expected because it is coded by 6 codons and it frequently produces non conservative aminoacid substitutions.
The array is able to confidently detect most of the mutations searched, however, additional probes should be added to improve its performance.
Conclusions
Our results demonstrate that a simple TP53 microarray using short (7-mer) probes, used in combination with a double tandem hybridization approach and a simple multiplex fragment preparation method, can identify common TP53 point mutations from different DNA sources. All the previous data confirms that the tandem hybridization technology, with the appropriated set of probes, is highly specific and can be confidently employed for diagnostic purposes. This system could provide rapid, economical and accurate mutational screening in the TP53 gene. We are presently improving our system by adding probes for these and other TP53 mutational hot spots.
Methods
IARC TP53 gene database search
The distribution of nucleotidic changes in the TP53 gene associated with all cancers war retrieved from the IARC TP53 database [38]. Then, the single base substitutions occurring in the 3 exons of TP53 most frequently altered in cancers were obtained. Both wild type (accession # U94788) and mutated TP53 sequences were used to design the probes, stacking oligonucleotides, PCR primers and synthetic targets.
Oligonucleotides
Four new PCR primers of 20–25 nt length (Table 1) were used to amplify two DNA fragments of 108 bp (exon 7: codons 248 and 249) and 92 bp (exon 8: codon 273) within the TP53 gene. Six stacking oligonucleotides of 17–21 nt length were annealed with their corresponding target DNA's, in three pairs, to produce partially duplex DNA's having a 7-nt gap which is the sequence interrogated by the capture probes (Figure 1A). Fourteen capture probes (seven-mer length; Table 1) were designed, two of them to search the wild type sequences for codons 157 and 158, which were used as negative controls, three more for the wild type 248, 249 and 273 codon sequences and nine to look for mutant sequences. According to our previous experience, 7-mer oligonucleotides with base changes located at central positions were selected as probes in order to yield good discrimination of point mutations by tandem hybridization at room temperature. The probes, which had a 3'-aminolink (3'-aminopropanol) modification for covalent binding to the glass slide surface [24], were purchased from Integrated DNA Technologies Inc. (Coralville, IA, USA).
Biological samples (DNA targets)
Twenty-four DNA samples from different sources were probed, some having known wild type or mutant TP53 sequences and others with unknown TP53 genotype. These included eight blood samples (B), two obtained from healthy subjects and six obtained from lung cancer affected patients, and ten established cancer cell lines or recombinant DNA's acquired from the ATCC (L). Two of the cell lines were a generous gift from Dr Mary A Salazar (IIB, UNAM) and one was a gift from Dr José Sullivan (INER, SS, Mexico). The other eight cell lines were provided by the DNA bank from Genomic Oncology Laboratory (CMN S XXI). Finally, six recombinant plasmids (P) harboring TP53 (wild type or different point mutations), provided by MSc. Irene Correa (CMN S XXI) were employed to standardize the hybridization arrays. The names, identification numbers, source and known genotypes in the 24 samples studied are described in Table 2
Target preparation and PCR assay
Genomic DNA was isolated and purified using the Genomic DNA Extraction Kit (Life Technologies Inc., Gaithersburg, MD). DNA concentration was determined by absorbance at 260 nm in a spectrophotometer MBA 2000 (Perkin-Elmer) and by the PicoGreen® dsDNA Quantitation Kit (Molecular Probes).
Plasmid DNA was prepared from bacterial cultures using the alkaline lysis/SDS minipreparation according to the manufacturer's instructions (Life Technologies).
The quality of the DNA preparations was assessed by gel electrophoresis in 1% agarose. All the reagents for PCR reactions were purchased from Promega (Madison WI. USA). The PCR primers are described in Table 1. Oligonucleotides sequences and fragment sizes for each exon were as follows: PCREX7-F and PCREX7-R for a 108 bp product in exon 7, and PCREX8-F and PCREX8-R for a 92 bp fragment in exon 8. The final reaction mixture contained 0.2 mM each dNTP, 50 mM KCl, 10 mM Tris-HCl (pH 8.4), 1.5 mM MgCl2, 0.5 μM each primer, 2.5 U of Taq DNA pol (Promega) and purified DNA (50–100 ng) in a final volume of 100 μL. The PCR profile consisted of an initial heating at 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 30 s, with a final extension step at 72°C for 7 min. The PCR product was assessed by electrophoresis in 1 % agarose gel stained with ethidium bromide.
Single-stranded target DNA was prepared by cycle synthesis as follows. A 30-μL aliquot of PCR product was processed using Ultrafree (Millipore, Bedford, MA) spin filters (30,000 Mr cutoff), and resuspended in the same volume of HPLC-grade water (OMNI SOLV®, EM Science). A 5-μL aliquot of this solution was added to a 100-μL PCR reaction, along with the primer corresponding to the target strand, then incubated for 27 temperature cycles using the same temperature profiles described above. The formation of each amplicon was assessed by mobility shift in agarose gel electrophoresis.
Each one of the six stacking oligonucleotides was 5'-labeled by reaction with T4 polynucleotide kinase (Invitrogen, San Diego, CA) as follows. 5 pmol of each dephosphorylated stacking oligonucleotide; 5X Forward Reaction Buffer (5 μl); T4 Polynucleotide Kinase (10 unit); [γ32P]ATP (10 μCi/μl, 3000 Ci/mmol) 2.5 μl (NEN, Boston MA), specific activity 7000 Ci/mmol, diluted in sterile water to 7 μCi/μl, and autoclaved water HPLC-grade to 25 μl. The mix was incubated 10 min at 37°C and the reaction was stopped by adding 5 mM EDTA (2.5 μl).
To prepare labeled partially duplex (gapped) target DNA's, each pair of γ32P-labeled stacking oligonucleotides was preannealed with the respective synthetic DNA target, single-stranded PCR fragments or multiplexed, single-stranded PCR products to form a 7-nt gap in each target. The annealing mixture contained 50 μL 20X SSC, 10 μL 1 M Tris-HCl (pH 8.0), 3 μL 0.5 M EDTA, 0.2 pmol of each labeled stacking oligonucleotide, 10 μL of target DNA, and HPLC-grade H2O to a final volume of 90 μL. The mixture was incubated at 95°C for 5 min, 45°C for 5 min, then 6°C for 5 min. Excess [γ32P]ATP was removed by filtration through an Ultrafree spin filter (3,000 Mr cutoff) (Millipore. Bedford, MA), and the retained DNA was dissolved in 20 μL of 1X SSC.
In the case of multiplexed targets, only 4 stacking oligonucleotides (SO-248-R, SO-249-L, SO-273R and SO-273-L) were added, one pair for each exon in the study, forming gap sizes of 12 and 7 nt for exons 7 and 8, respectively. Thus, with exon 7 a single tandem hybridization was conducted with the glass-tethered 7-mer capture probes, whereas with exon 8 double tandem hybridization was conducted.
DNA probe arrays
Glass microscope slides were prepared by soaking in hexane 20 min, then 20 min in absolute ethanol followed by drying 5 h at 90°C. Oligonucleotide probes containing 3'-terminal aminopropanol modification were dissolved in sterile water to a final concentration of 20 μM, and 200 μl droplets of each probe were applied to the glass slides using a model 417 Affymetrix (Santa Clara, CA) arrayer to place submicroliter droplets onto glass slides. Each array contained 14 probes: three wild type sequences and nine mutant sequences corresponding to the TP53 targets under study, two negative controls consisting of 7-mers targeted to the wild type sequences from codons 157 and 158 of TP53 gene, plus one blank spot without probe. As depicted in Fig. 1B, each TP53 capture probe was arrayed in triplicate (to assess the reproducibility of the results) in an upward direction starting from position 1 and the printing was divided into three groups. Positions 1–14 were printed first, then the pattern was repeated twice more across the slide. Position 15 was left empty (nothing at all was applied) and served as a negative glass control. After spotting, each slide was washed with water to remove unbound oligonucleotides, air-dried, then stored in a dessicator at room temperature. This attachment procedure results in a surface density of 1010-1011 molecules mm2, which corresponds to intermolecular spacing of about 30–100 Å across the surface [25].
Hybridization experiments
Just prior to hybridization the slides were soaked for 1 h at room temperature with blocking agent (10 mM tripolyphosphate) and then rinsed twice with water and air-dried. The hybridization cocktail contained: 118 μL 5 M tetramethyl-ammonium chloride (TMAC) (Life Technologies), 9 μL 1 M Tris-HCl (pH 8.0) (Life Technologies), 0.72 μL 0.5 M EDTA, 1.8 μL 10% (w/v) sodium dodecyl sulfate, 14.4 μL 40% (w/v) polyethylene glycol, 20 μL labeled target DNA in 1X SSC, and 35.3 μL HPLC-grade water. A 40-μL aliquot of this cocktail was placed onto each array and a cover slip was applied. Slides were incubated in a humid chamber for 3 h at 25°C. After hybridization, the slides were washed by dipping several times in the same hybridization solution without polyethylene glycol and DNA, air-dried, and then wrapped in plastic film and placed against X-ray film (Kodak BioMax) for autoradiography. Detection and quantitation of 32P-labeled target molecules bound across the hybridization array was performed with a scanner (HP Scanjet 4400 c), and analysis of densitometric images was performed using ScanAnalyze 2 software (Stanford University).
To provide confidence in the interpretation of the signals produced by human samples, reference hybridization patterns were prepared using a full set of synthetic targets. For this purpose, thirteen synthetic targets (44–50 nt), representing the wild type and mutant sequences, were used independently and in combination, to represent homozygous and heterozygous conditions.
Sequencing of PCR products
The PCR products obtained were purified using the QIAEX II kit (QIAGEN Inc. USA). For each PCR product the presence or absence of a mutation in exons 7 and 8 was demonstrated via sequencing using the Big Dye Terminator kit (Perkin-Elmer) and a model 373 automated DNA sequencer (Instruments Core Center of Health Research Council- IMSS).
Authors' contributions
ARL performed all molecular assays, data collection and analysis, and drafted the manuscript. RMR conceived and designed the study, coordinated and managed the study, performed data analysis and participated in drafting the manuscript. MSV conceived and designed the study, coordinated and managed the study, performed data analysis and participated in drafting the manuscript. JMEL participated in the design and coordination of the study. AMT designed the oligonucleotide probes, performed bioinformatic analysis of targets, and participated in drafting the manuscript. KLB assisted in coordination of the study, data analysis, and drafting the manuscript. All authors read and approved the final manuscript.
Acknowledgements
During this work ARL was IMSS fellowship recipient. This work was partially supported by 34686-M and 7114 CONACYT (MSV), FOFOI-IMSS (MSV) and CGPI-20020638.
Figures and Tables
Figure 1 Layout of the TP53 low density DNA microarray. (A): Names and alignment of stacking oligonucleotides and probes to their respective synthetic wild type target sequence. Bold letters correspond to the nucleotide change in DNA sequence due to the point mutations. (B): Layout of the probe array on the glass slide. The probes were applied to the slide in triplicate as depicted at the top. The numbers correspond to the probes in Table 1.
Figure 2 Electrophoretic analysis of PCR products and single-stranded target DNA produced by "cycle sequencing." (A): Electrophoresis in 2% agarose gel from PCR reactions of regions from exons 7 and 8 of the TP53 gene: 100 bp ladder (Lane 1), 108 bp PCR product from exon 7 (lane 2), 92 bp PCR product from exon 8 (lane 3). (B): Electrophoresis in 4% agarose gel of two multiplex PCR reactions from exons 7 and 8 of the TP53 gene. Lanes 1 and 3 represent the products of two different samples; lane 2 is the 100 bp ladder.
Figure 3 Hybridization of synthetic targets. (A) Homozygous pattern. Hybridization pattern produced by synthetic target (ST) on probes (arrayed onto 14 slides). Synthetic targets were annealed with prelabeled stacking oligonucleotides and hybridized with the array of probes having sequence variations. (B) Heterozygous pattern. Hybridization patterns produced by heterozygous combination of synthetic targets (ST) on probes. Equivalent amounts of wild type and mutant synthetic targets were annealed with prelabeled 5'and 3'stacking oligonucleotides, then hybridized with the array of probes having sequence variations (see Table 1).
Figure 4 Hybridization of samples proposed in the multiplex format. (A). Sample with wild type genotype. Representative experiment of multiplex hybridization assay with sample P1. The red arrows indicates the wild type hybridization signals. The DNA obtained was individually annealed with the two pairs of stacking oligonucleotides and hybridized in one assay. Above Multiplex Assay with sample L7. The hybridized with their respective array of probes having sequence variations. The DNA obtained was annealed the two pairs of stacking oligonucleotides and hybridized in the multiplex format with. (B) The electropherogram showed the sequence wild type of the interest codon. The pink row pointed the signal of the probe in the microarray (point mutation).
Table 1 Primers, stacking oligonucleotides, synthetic targets and capture probes used.
Primers
Name Sequence (5'→3')
PCREX7-F GCTCTGACTGTACCACCATCCA
PCREX7-R ACCTGGAGTCTTCCAGTGTGA
PCREX8-F ACTGGGACGGAACAGCTTTGA
PCREX8-R GCTCCCCTTTCTTGCGGAGAT
Stacking oligonucleotides
Name Sequence (5'→3')
LSO108–248 CCTGCATGGGCGGCATG
RSO108–248 GGCCCATCCTCACCATCATC
LSO108–249 GCATGGGCGGCATGAACCG
RSO108–249 ATCCTCACCATCATCACTC
LSO92–273 GGGACGGAACAGCTTTGAGG
RSO92–273 TTGTGCCTGTCCTGGGAGA
Synthetic targets
Name Sequence (5'→3')
ST-157,158WT GGTGTGGGGGCGGGCCGTGGGCGCAGGCGCGGTACCGGTAGATGTTCGTC
ST-248,249WT GTGTGATGATGGTGAGGATGGGCCTCCGGTTCATGCCGCCCATGCAGG
ST-248C→T GATGATGGTGAGGATGGGCCTCCAGGTCATGCCGCCCATGCAGG
ST-248G1→T GATGATGGTGAGGATGGGCCTCAGGTTCATGCCGCCCATGCAGG
ST-248G1→A GATGATGGTGAGGATGGGCCTCTGGTTCATGCCGCCCATGCAGG
ST-249G1→T TGATGATGGTGAGGATGGGCATCCAGTTCATGCCGCCCATGCAGG
ST-249G2→T TGATGATGGTGAGGATGGGACTCAGGTTCATGCCGCCCATGCAGG
ST-249G2→C TGATGATGGTGAGGATGGGGCTCTGGTTCATGCCGCCCATGCAGG
ST-273WT CTCTCCCAGGACAGGCACAAACACGCACCTCAAAGCTGTTCCGTCCC
ST-273G→T CTCTCCCAGGACAGGCACAAACAAGCACCTCAAAGCTGTTCCGTCCC
ST-273G→A CTCTCCCAGGACAGGCACAAACATGCACCTCAAAGCTGTTCCGTCCC
ST-273C→T CTCTCCCAGGACAGGCACAAACACACACCTCAAAGCTGTTCCGTCCC
Capture probes
Name Sequence (5'→3')
p-157WT CGCGTCC@
p-158WT GTCCGCG@
p-248WT AACCGGA@
p-248C→T AACTGGA@
p-248G1→T AACCTGA@
p-248G1→A AACCAGA@
p-249WT GAGGCCC@
P-249G1→T GATGCCC@
P-249G2→T GAGTCCC@
P-249G2→C GAGCCCC@
P-273WT = TGCGTGT@
P-273-G→T TGCTTGT@
P-273G→A TGCATGT@
P-273C→T TGTGTGT@
@ = 3'-aminopropanol modification for glass attachment.
Base changes are represented in bold
Table 2 Biological samples tested.
ID Name Source The genotype was previously assumed as:
P1 pp53 Plasmid Unknown
P2 pSCX3 Plasmid Unknown
P3 pM47 Plasmid Unknown
P4 pC53 Plasmid Unknown
P5 XbaI Plasmid Unknown
P6 C1 Plasmid Unknown
L1 A-427 Human cell line Unknown
L2 C33 A Human cell line Point mutation in codon 273
L3 Hela Human cell line Unknown
L4 Jurkat Human cell line Heterozygous for p53 ***
L5 SiHa Human cell line Unknown
L6 T24 Human cell line Unknown
L7 SW480 Human cell line Unknown
L8 I-37 Human lung tumor sample Unknown
L9 I-51 Human lung tumor sample Unknown
L10 PCS-4 Human lung tumor sample Unknown
B1 Case 1 PBLCA Unknown
B2 Case 2 PBLCA Unknown
B3 Case 116 PBLCA Unknown
B4 Case 4 PBLCA Unknown
B5 Case 5 PBLCA Unknown
B6 Case 152 PBLCA Unknown
B7 Case 7 PBHS Unknown
B8 Case 8 PBHS Unknown
*PBLCA = Peripheral blood lung cancer affected.
**PBHS = Peripheral blood healthy subject.
***Personal communication.
Table 3 Detection of TP53 silent and missense base substitutions using DNA sequencing versus DNA microarray hybridization.
ID Genotype Mutation
DNA sequencing Results of hybridization
P1 Pp53 248 wtCGG→mutCGC No wild type signal: mutant probe absent Silent
249 wtAGG→mutAGA No wild type signal: p-249 G2→T Silent
273 wtCGT p-273 WT Silent
P6 cDNA 248 wtCGG p-248 WT Silent
249 wtAGG p-249 WT Silent
273 wtCGT→mutCCT No wild type signal: mutant probe absent Proline
L1 A-427 248 wtCGG→mutCGT p-248 WT: mutant probe absent Silent
249 wtAGG p-249 WT Silent
273 wtCGT p-273 WT Silent
L2 C33 A 248 wtCGG p-248 WT Silent
249 wtAGG p-249 WT Silent
273 wtCGT→mutTGT p-273 C→T Cysteine
L4 Jurkat 248 wtCGG→mutCGK* p-248 WT: mutant probe absent Silent
249 wtAGG→mutAGR** p-249 G2→T: mutant probe absent p-249 WT Silent
273 wtCGT p-273 WT Silent
L7 SW480 248 wtCGG p-248 WT Silent
249 wtAGG→mutAGR** p-249 G2→T: mutant probe absent p-249 WT Silent
273 wtCGT→mutCAT p-273 G→A Histidine
L8 I-37 248 wtCGG→mutCGC No wild type signal: mutant probe absent Silent
249 wtAGG→mutCGA No wild type signal: mutant probe absent Silent
273 wtCGT→mutTGT p-273 C→T Cysteine
L9 I-51 248 wtCGG→mutCGC No wild type signal: mutant probe absent Silent
249 wtAGG→mutCGA No wild type signal: mutant probe absent Silent
273 wtCGT p-273 WT Silent
L10 PCS-4 248 wtCGG→mutCGC No wild type signal: mutant probe absent Silent
249 wtAGG→mutCGA No wild type signal: mutant probe absent Silent
273 wtCGT p-273 WT Silent
B3 Case 116 248 wtCGG p-248 WT Silent
249 wtAGG p-249 WT Silent
273 wtCGT→mutCTT p-273 G→T Leucine
B6 Case 152 248 wtCGG p-248 WT Silent
249 wtAGG p-249 WT Silent
273 wtCGT→mutCTT p-273 G→T Leucine
*K = G or T (Heterozygous), **R = G or A (Heterozygous)
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| 15713227 | PMC553977 | CC BY | 2021-01-04 16:02:58 | no | BMC Biotechnol. 2005 Feb 15; 5:8 | utf-8 | BMC Biotechnol | 2,005 | 10.1186/1472-6750-5-8 | oa_comm |
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BMC EcolBMC Ecology1472-6785BioMed Central London 1472-6785-5-21570748110.1186/1472-6785-5-2Research ArticleLife history traits in selfing versus outcrossing annuals: exploring the 'time-limitation' hypothesis for the fitness benefit of self-pollination Snell Rebecca [email protected] Lonnie W [email protected] Dept of Biology, Queen's Univ., Kingston, ON, K7L 3N6, Canada2005 11 2 2005 5 2 2 22 6 2004 11 2 2005 Copyright © 2005 Snell and Aarssen; licensee BioMed Central Ltd.2005Snell and Aarssen; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Most self-pollinating plants are annuals. According to the 'time-limitation' hypothesis, this association between selfing and the annual life cycle has evolved as a consequence of strong r-selection, involving severe time-limitation for completing the life cycle. Under this model, selection from frequent density-independent mortality in ephemeral habitats minimizes time to flower maturation, with selfing as a trade-off, and / or selection minimizes the time between flower maturation and ovule fertilization, in which case selfing has a direct fitness benefit. Predictions arising from this hypothesis were evaluated using phylogenetically-independent contrasts of several life history traits in predominantly selfing versus outcrossing annuals from a data base of 118 species distributed across 14 families. Data for life history traits specifically related to maturation and pollination times were obtained by monitoring the start and completion of different stages of reproductive development in a greenhouse study of selfing and outcrossing annuals from an unbiased sample of 25 species involving five pair-wise family comparisons and four pair-wise genus comparisons.
Results
Selfing annuals in general had significantly shorter plant heights, smaller flowers, shorter bud development times, shorter flower longevity and smaller seed sizes compared with their outcrossing annual relatives. Age at first flower did not differ significantly between selfing and outcrossing annuals.
Conclusions
This is the first multi-species study to report these general life-history differences between selfers and outcrossers among annuals exclusively. The results are all explained more parsimoniously by selection associated with time-limitation than by selection associated with pollinator/mate limitation. The shorter bud development time reported here for selfing annuals is predicted explicitly by the time-limitation hypothesis for the fitness benefit of selfing (and not by the alternative 'reproductive assurance' hypothesis associated with pollinator/mate limitation). Support for the time-limitation hypothesis is also evident from published surveys: whereas selfers and outcrossers are about equally represented among annual species as a whole, selfers occur in much higher frequencies among the annual species found in two of the most severely time-limited habitats where flowering plants grow – deserts and cultivated habitats.
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Background
Most flowering plants that are predominantly self-pollinating have an annual life history [1-3]. Interpretations of this association usually involve one of two main hypotheses. (i) Compared with perennials, annuals may generally accrue greater fitness benefits from selfing through 'reproductive assurance', i.e., because ovules may be generally more outcross-pollen-limited and/or pollen grains may be more outcross-ovule-limited [2,4-8]. (ii) Perennials may incur a higher fitness cost of selfing through seed discounting and inbreeding depression; hence, possibly most selfers are annuals simply because relatively few perennials can be selfers [9,10].
A recent third hypothesis, the 'time-limitation' hypothesis, predicts that both selfing and the annual life cycle are concurrent products of strong 'r-selection' associated with high density-independent mortality risk in ephemeral habitats with a severely limited period of time available to complete the life cycle [11]. Both the traditional reproductive assurance hypothesis and the time-limitation hypothesis involve a fitness advantage for selfing through ensuring that at least some reproduction occurs, but they involve very different selection mechanisms – pollinator/mate-limitation (where outcross pollen is not available at all due to a lack of pollinators or mates), versus time-limitation (where outcross pollen is available but arrives too late to allow sufficient time for development of viable seeds). Accordingly, these two hypotheses for selfing involve very different assumptions and predictions.
The time-limitation hypothesis has direct and indirect components. The indirect component predicts higher selfing rates in annuals as a trade-off of selection for earlier reproductive maturity in annuals [12,13] (Figure 1a). More rapid floral maturation is expected to result in smaller flowers with increased overlap of anther dehiscence and stigma receptivity in both space (reduced herkogamy) and time (reduced dichogamy) thus, increasing the frequency of selfing as an incidental consequence [12] (Figure 1a). If selfing also shortens the time between flower maturation and ovule fertilization, then higher selfing rates for annuals in time-limited habitats may also be predicted as a direct fitness benefit; abbreviating the time between anthesis and ovule fertilization may ensure that there is enough remaining time in the growing season (after ovule fertilization) to allow complete seed and fruit maturation [11] (Fig 1b). Selection favors selfing here by favoring increased overlap in anther dehiscence and stigma receptivity in both space and time, which are in turn facilitated by smaller flower size and shorter flower development time, respectively (Figure 1b).
Figure 1 Two components of the 'time-limitation' hypothesis for the evolution selfing in annuals. In (a), selfing is a trade-off of selection favoring a shorter time to reproductive maturity (fully developed flowers) under strong r-selection. As a tradeoff (dashed arrows), flowers become smaller with greater overlap in location and timing of anther dehiscence and stigma receptivity, thus increasing the rate of selfing as an incidental consequence. In (b) strong r-selection favors a shorter pollination time directly; i.e., selfing is selected for directly because it shortens the amount of time between flower maturation and ovule fertilization, thus leaving sufficient remaining time for seed and fruit maturation before the inevitable early mortality of the maternal plant under strong r-selection. In this case, smaller flower size and shorter flower development time are favored by selection because they facilitate selfing (see text).
However, the two components of time-limitation cannot be separated clearly, as they operate simultaneously; i.e., earlier onset of flowering, shorter flower development time, smaller flowers and selfing can all be interpreted to have direct fitness benefits because they may all contribute directly to accelerating the life cycle [11]. Indeed, time-limitation associated with strong r-selection would be expected also to favor an acceleration of the final stage in the life cycle – seed/fruit development time (Figure 1) – resulting, as a trade-off, in smaller seeds and/or fruits [11].
The time-limitation hypothesis remains untested. Some recent studies have explored the rapid growth and maturation time of annuals in terms of bud development rates and ontogeny [13-15]. However, these studies have compared growth and development rates between selfing and outcrossing populations of only a single species. Since their effective sample size is only one, this makes it difficult to extrapolate the predominant selection pressures that may have promoted the general association of selfing with the annual life cycle.
The objective of the present study was to compare, for annuals exclusively, life history traits associated with selfing versus outcrossing using several species from a wide range of plant families. Phylogenetically-independent contrasts (PIC) were used to control for confounding effects due to common ancestry among species [16]. Using a database of 118 species involving 14 families, plant size, flower size, and seed size were compared between selfing and outcrossing annuals. The time-limitation hypothesis predicts that all of these traits should be smaller in selfing annuals because the severely time-limited growing season that promotes selfing also imposes an upper limit on the maximum sizes that can be attained for plant traits [11] (Figure 1). The trend for outcrossers to be taller, and have larger flowers and larger seeds has often been noted [1,17-19]. We used a multi-species, across-family comparison, however, to investigate whether this trend also holds true within annuals exclusively.
Data on the timing of life history stages (i.e. age at first flower, bud development time, and flower longevity) were also obtained from a greenhouse study of 25 annual species involving 5 families. The time-limitation hypothesis predicts that selfers should produce mature flowers more quickly and should have shorter flowering times.
Results
Data base analyses
Based on phylogenetically-independent contrasts, selfing annuals had significantly shorter plant heights (Wilcoxon test for matched pairs, n = 12, T = 15.5, one-tailed P = 0.032, Figure 2a), significantly smaller flowers (Wilcoxon test for matched pairs, n = 14, T = 13, one-tailed P = 0.0054, Figure 2b), and significantly smaller seeds (Wilcoxon test for matched pairs, n = 13, T = 13, one-tailed P < 0.01, Figure 2c).
Figure 2 (a) Plant height contrasts for 13 selfing and outcrossing pairs (some points overlap), where each pair consists of the median value of the selfing and outcrossing species within one family. (b) Flower size contrasts for 14 selfing and outcrossing pairs. (c) Seed size constrasts between 14 selfing and outcrossing pairs. (See Appendix A for list of families and species).
Greenhouse study
Bud development time (Figure 3) and flower longevity (Figure 4) were significantly (P < 0.05) shorter in selfing annuals in all of the families except the Fabaceae (P = 0.123 and P = 0.056 respectively). Selfing annuals also had significantly shorter bud development times (Figure 5), and floral longevities (Figure 6) in three of the four genus pairs. Selfing and outcrossing annuals of the genus Ipomoea did not differ significantly in either bud development time (P = 0.402) or flower longevity (P = 0.328). Age at first flower was not significantly related to mating system for any of the family or genus comparisons (P > 0.05; data not shown).
Figure 3 Mean (SE) bud development time for selfing and outcrossing species within each of 5 families. For each species, n = 4 or 5. P – values are from ANOVA. (See Appendix B for species list).
Figure 4 Mean (SE) flower longevity for selfing and outcrossing species within each of 5 families. For each species, n = 4 or 5. P – values are from ANOVA. (See Appendix B for species list).
Figure 5 Mean (SE) bud development time for selfing and outcrossing species within each of 4 genus pairs. For each species, n = 4 or 5. P – values are from ANOVA. (See Appendix B for species list).
Figure 6 Mean (SE) flower longevity for selfing and outcrossing species within each of 4 genus pairs. For each species, n = 4 or 5. P – values are from ANOVA. (See Appendix B for species list).
Discussion
There is a rich body of theory and empirical work on the evolution of selfing in flowering plants [e.g. [1,2,4-7,9,10]], but practically none of it involves an explicit role of selection involving time-limitation. The present paper is only the second to explore the implications of the time-limitation hypothesis and contribute to the maturation of this idea. According to the time-limitation hypothesis, selfing in annuals has evolved as a consequence of strong r-selection in ephemeral habitats, resulting either as an indirect consequence (trade-off) of selection for shorter time to reproductive maturity (Figure 1a), or as a direct consequence of selection for shorter pollination time, i.e., the time between flower maturation and ovule fertilization (Figure 1b), or both [11]. Consistent with the predictions of this hypothesis, we found, using phylogenetically-independent contrasts, that (compared with outcrossing annuals) selfing annuals in general had significantly shorter plant heights, smaller flowers, shorter bud development time, shorter flower longevity and smaller seed sizes.
At the same time, these results are not inconsistent with the predictions of selection resulting from pollinator/mate-limitation associated with the traditional reproductive assurance hypothesis. Just as with many situations where two different mechanisms can potentially produce the same outcome/pattern, it is not easy here to clearly distinguish between the roles of "pollinator/mate-limitation" and "time-limitation". Nevertheless there are two important contributions from our study: First, in reporting significant life history differences between selfers and outcrossers, our multi-species study is unique in its comparison of monocarpic annual species exclusively. All previous multi-species studies of trait comparisons between selfers and outcrossers have involved variable mixes of monocarpic and longer-lived polycarpic species. Second, by comparing annuals exclusively, our results provide indirect support for the time-limitation hypothesis, not by rejecting the role of pollinator/mate-limitation, but rather by representing a system in which it is more plausible to argue for the role of time-limitation; i.e., compared with pollinator/mate-limitation, time-limitation as a selection factor favoring selfing is likely to have been much stronger, more persistent and more widespread. The strength of this argument lies in the fact that the annual life history is unequivocally a product of some type of time-limitation favoring an abbreviated life cycle, which is promoted by (among other things) selfing (as opposed to outcrossing) (Fig. 1). It is much less plausible to suspect that selection associated with pollinator/mate-limitation has been sufficiently strong and persistent to favor selfing in such a wide range of annual taxa across the many genera and families considered here. We emphasize, therefore, that for annuals the time-limitation hypothesis provides a more parsimonious explanation for the differences in traits between selfers and outcrossers. We consider each of these traits in turn below.
Plant height and time to anthesis
Taller plants may attract more pollinators and, hence, experience greater outcrossing rates [20,21]. The pollination benefit of being relatively tall, therefore, is presumably experienced only by outcrossers. If, however, selfers have evolved from outcrossers [3], then why should selfers be shorter than their outcrossing ancestors? The relatively small size, including short height of selfers can be predicted as an indirect consequence of selection, from time-limitation, favoring precocious maturation time [22,23] (Figure 1a). In the present study, however, selfers and outcrossers did not differ significantly in age at first flower. Andersson [18] found similar results between selfing and outcrossing populations of Crepis tectorum. Arroyo [24], however, reported that selfing individuals of Limnanthes floccosa flowered earlier than the outcrossing L. alba, as predicted by the time-limitation hypothesis. The results for flowering times in the present study may be confounded by the controlled greenhouse environment of constant day-length, temperature and moisture regime. In the field, flowering times may be triggered by environmental cues. L. floccosa, for example, uses soil moisture to trigger the early onset of flowering, thus escaping the detrimental effects of soil desiccation during seed development [24]. Note also that age at first flower is only a crude estimate of time to reproductive maturity. Future studies may employ more detailed measures such as rate of mature flower production.
Flower size
One of the most well established trends of predominantly self-fertilizing species is their reduced flower sizes compared with outcrossing species [1,17]. The present results indicate that this trend is also evident even within annuals exclusively. In all but three of the 14 PICs, selfing annuals had smaller flowers than the outcrossing annuals (Figure 2b). Outcrossers and selfers had similar flower sizes in the Fabaceae and Plantaginaceae. In the Poaceae, outcrossing annuals had smaller flowers than selfers.
Under the time-limitation hypothesis, smaller flowers and selfing may be tradeoffs of selection for precocity (Figure 1a), or smaller flowers may be favored by selection because they promote selfing and hence, direct fitness benefits by abbreviating pollination time (Figure 1b). Also, if selfing evolves from outcrossing (by whatever mechanism), then selection may subsequently favour a reduction in flower size since relatively large flowers are no longer needed to attract pollinators. Hence, higher fitness may result if the resources required to construct and support these larger flowers are invested instead in other functions (e.g. seed and fruit development) [17].
Bud development time
Selfers had significantly shorter bud development times in all but one of the independent family contrasts (Figure 3) and all but one of the genus comparisons (Figure 5). Results from previous studies, however, are inconsistent. Shorter bud development times were found in selfing populations of Mimulus guttatus [25] and in Clarkia xantiana [14]. However, no significant differences in bud growth rates were found between the selfing and outcrossing populations of C. tembloriensis [15]. Hill, Lord and Shaw [13] reported that flowers from selfing populations of Arenaria uniflora develop over a longer period of time than observed in outcrossing populations. In the field, selfing populations of A. uniflora were also observed to flower at the same time or even later than outcrossing populations [13], suggesting that time-limitation is not currently a strong selection pressure. Self-fertilization in A. uniflora may have arisen through reproductive assurance in response to competition for pollinators [7]. The evolution of self-fertilizing species from outcrossing progenitors has occurred repeatedly and independently in several lineages [1,3,14], each of which may have been associated with different contexts of natural selection vis-à-vis the fitness benefits of selfing.
Flower longevity
The families and genera in which selfers had shorter bud development times also had significantly shorter flower longevities (Figure 4). In fact, all of the selfers had flowers that remained open for less than four days (except in Trifolium hirtum; Fabaceae), with a large proportion of flowers open for only one day, which is common amongst self-fertilizing species [17]. The present data again indicate that this generalization apparently holds true even within annuals exclusively. By having flowers that remain open longer, outcrossers increase the probability of visitation by pollinators and successful cross-pollination [17]. This fitness benefit is realized, however, only if there is sufficient time remaining after cross-pollination to complete seed and fruit development before the maternal plant succumbs to density-independent mortality in strongly r-selecting habitats [11]. If time is limiting in this context, selection should favor selfing (Figure 1b) with no advantage in having long-lived flowers.
It is important to note that our data measure maximum flower longevity, since there were no pollinators in the greenhouse, nor was hand pollination conducted. Pollination has been shown to induce floral senescence in numerous species [26]. This effect was not tested on any of the study species, which means that our observed flower longevities in outcrossing species may be longer than would normally be seen in the wild. Nevertheless, since selfing may have evolved as a method of shortening pollination time, and flower longevity was used as a measure of pollination time, the maximum floral longevity gives an indication of how long outcrossers can delay flower abscission or self-pollination (i.e. through delayed selfing).
Seed size
Strong r-selection associated with the annual life form presumably favors wide dispersal mechanisms (for colonizing new and distant sites) which may be conferred by small seed sizes [19]. The reproductive assurance hypothesis would predict, therefore, that most selfers are annuals because annuals are more likely than perennials to disperse further, or colonize new habitats where conditions are unsuitable for successful outcrossing (because of a shortage of mates or pollinators) and where selfing, therefore, provides reproductive assurance. The present study indicates that even among annuals only, selfers have smaller seeds than outcrossers (Figure 2c). Future studies are required to test whether smaller-seeded selfing annuals are more likely than their outcrossing annual relatives to disperse further or colonize new habitats and thereby incur potential reproductive assurance benefits of selfing.
An alternative explanation, however, is offered by an extension of the time-limitation hypothesis: strong r-selection favors an acceleration of all stages of the life cycle (Figure 1), including not only earlier reproductive maturity (Figure 1a) and a shorter pollination time (facilitated through selfing) (Figure 1b), but also a shorter seed and fruit maturation time, which, on a per-seed basis, is facilitated in turn through the production of smaller seeds. Andersson [18] found that self-fertilizing individuals of Crepis tectorum took an average of 16 days for fruit maturation, whereas outcrossing individuals of the same species required 43.3 days. Small seed size may also be simply a trade-off of selection for high fecundity, also favored by strong r-selection [11].
Habitat selection and time-limitation
While most selfers are annuals, it is not the case that most annuals are selfers. An unbiased literature survey [27] suggests that roughly half of all annual species are selfers and half are outcrossers. If, however, selfing annuals evolved in habitats with a short window of time for completing the life cycle (Figure 1), then selfing annuals should be significantly more common than expected (i.e. comprising greater than 50% of resident annuals) within habitats associated with historically regular, early-season disturbances (e.g. cultivated fields, gardens), or in habitats where severe droughts follow quickly after a wet season (i.e. deserts, Mediterranean climates, vernal pools). Hence, we should expect to find more selfers than outcrossers among annual weeds of cultivated habitats and among desert annuals in particular. Similarly, for annuals with both selfing and outcrossing ecotypes or races, we should expect selfers (or a higher selfing rate) to be more commonly associated with these severely time-limited habitats [11].
While rigorous tests of these predictions have yet to be explored, some preliminary support is available from published surveys. From a representative sample of Mediterranean annuals [28], we find a much greater representation of selfers: i.e. 34 selfers versus 11 outcrossers. Selfing and outcrossing desert annuals have been shown to be distributed along a moisture gradient. Outcrossing annuals are found generally in the wetter areas and selfers in the more arid zones, as seen in Clarkia xantiana [29] and between outcrossing populations of Limnanthes alba and its selfing relative L. floccosa [24]. Since the length of the growing season is limited by the amount of moisture in the soil, selfers have a much narrower window of time to complete their life cycle before desiccation. During a severe drought, seed production in L. alba was reduced by one sixth, whereas the seed set of L. floccosa found in the same area was virtually unaffected by the identical drought [24].
The association between 'weediness' and self-fertilization has also been noted [2,30]. An extensive survey of colonizing herbaceous plants of Canada showed that agricultural weeds of row crops and grain fields are almost exclusively annuals, and most of these are self-compatible [31]. A published list of the world's worst weeds of agricultural crops [32] includes 76 species, 41 of which are annuals. Based on previous literature, we were able to identify the breeding system for 24 of these annuals, and, as predicted, the majority (20 out of 24) are selfers.
Conclusions
Botanists have long known that selfing is particularly associated with the annual life cycle in flowering plants [2]. The present study shows further that, among annuals exclusively, selfing is particularly associated with shorter plant heights, smaller flowers, shorter bud development time, shorter flower longevity and smaller seed sizes compared with annuals that are outcrossing. Also, in spite of the null prediction that selfing and outcrossing annuals should be equally represented if there is no bias associated with time-limitation, we found instead that two of the most time-limited habitats on earth that support flowering plants have a significantly higher percentage of selfers among the resident species that are annuals. Because we focused on annual species only, all of these results are explained more parsimoniously by selection associated with time-limitation than by selection associated with pollinator/mate limitation. The role of pollinator/mate-limitation (as traditionally associated with the reproductive assurance hypothesis for the evolution of selfing) is likely to be of greater importance in longer-lived polycarpic species (not considered here), simply because by comparison, there is no convincing basis to argue that selection associated with time-limitation is likely to have been important in species with longer life cycles. We suggest therefore, that most selfers, because most of them are annuals, are likely to have evolved not because of fitness benefits through reproductive assurance associated with selection from pollinator/mate limitation, but rather because of fitness benefits associated with selection from time limitation.
The effect of time-limitation under strong r-selection is to minimize the duration of the life cycle, with selfing favored directly (Figure 1b) and/or indirectly (Figure 1a). There is no basis for predicting that either mechanism is more probable than the other; both are likely to operate simultaneously and perhaps indistinguishably. Indeed, the predicted effects under direct and indirect selection involve the same phenotypic outcome for the same suite of traits (Figure 1). It is particularly significant that the shorter bud development time reported here for selfing annuals is predicted explicitly by the time-limitation hypothesis but not by selection associated with pollinator/mate limitation. Although, we cannot of course rule out the possibility that shorter bud development time may be a pleiotropic consequence of the evolution of autonomous selfing through other mechanisms.
Designing empirical studies that clearly distinguish between mechanisms involving time-limitation versus pollinator/mate limitation remain a challenge but we anticipate that our results and our discussion of these issues may help to inspire further research along these lines. Future studies may be designed to test more directly the role of limited pollination time (vis-à-vis Figure 1b) by comparing the time required for effective pollination under selfing versus outcrossing for closely related species or ecotypes within natural habitats, taking care of course to control for other aspects of the pollination environment (such as mate and pollinator availability) that might affect time-to-effective pollination.
Methods
Data base analyses
The literature was surveyed to obtain breeding information (i.e. selfing versus outcrossing) for as many annuals species as possible. For each species, data on plant height, flower size, and seed size were obtained where possible from standard floras and other published literature. A complete database was assembled for 118 species from both Europe and North America, involving 14 families (Table 1). For each species, the maximum published value for each trait was used. Plant height was the maximum recorded vertical extent of the plant. The measure used for flower size depended on the usual convention specific for each family, e.g. maximum petal length, corolla width, lemma length (in the Poaceae). Seed size was measured as the length of the longest axis. Within each family, for each trait, the median value across selfing species and the median value across outcrossing species was calculated and used in the phylogenetically-independent contrasts.
Table 1 Species list, with breeding system (O – outcrosser; S – selfer), for database from published literature.
Family Family
Species Species
Asteraceae Malvaceae
Anthemis cotula (O) Abutilon theophrasti (S)
Cosmos bipinnatus (O) Hibiscus trionum (O)
Centaurea cyanus (O) Malva neglecta (O)
Centaurea montana (O) Malva rotundiflora (S)
Crepis capillaris (O)
Crepis tectorum (O) Plantaginaceae
Helianthus annuus (O) Plantago arenaria (O)
Lapsana communis (O) Plantago virginica (S)
Matricaria maritima (O)
Matricaria matricarioides (S) Poaceae
Senecio viscosus (S) Aira praecox (O)
Senecio vulgaris (S) Avena fatua (S)
Silybium marianum (S) Avena sativa (S)
Sonchus oleraceus (S) Bromus hordeaceus (S)
Xanthium strumarium (S) Bromus secalinus (O)
Bromus sterilis (S)
Boraginaceae Bromus tectorum (S)
Anchusa arvensis (O) Desmazeria rigida (S)
Borago officinalis (O) Echinochloa crus-galli (S)
Lappula squarrosa (S) Hordeum vulgare (S)
Myosotis arvensis (S) Lolium temulentum (S)
Myosotis ramosissima (S) Panicum miliaceum (S)
Myosotis stricta (S) Phalaris canariensis (O)
Plagiobothrys calandrinioides (S) Poa annua (S)
Secale cereale (O)
Brassicaceae Setaria italica (S)
Arabidopsis thaliana (S) Setaria verticillata (S)
Berteroa incana (O) Setaria virdis (S)
Brassica juncea (O) Triticum aestivum (S)
Brassica nigra (O) Zea mays (O)
Brassica rapa (O)
Cakile edentula (S) Polemoniaceae
Capsella bursa-pastoris (S) Allophyllum gilioides (S)
Cardamine hirsute (S) Allophyllum integrifolium (S)
Descurainia pinnata (S) Collomia grandiflora (O)
Diplotaxis muralis (O) Collomia linearis (S)
Erucastrum gallicum (S) Gilia australis (S)
Erysimum cheiranthoides (O) Gilia capitata (O)
Erysimum repandum (S) Gilia caruifolia (O)
Lepidium sativum (O) Gilia clivorum (S)
Lepidium campestre (S) Gilia inconspicua (S)
Lepidum ruderale (S) Gilia millefoliata (S)
Rorippa palustris (S) Gilia sinuata (S)
Sinapis alba (O) Gilia tenuiflora (O)
Sinapis arvensis (O) Gilia transmontana (S)
Sisymbrium officinale (S) Gilia tricolor (O)
Thlaspi arvensis (S) Navarretia atrictyloides (O)
Thlaspi perfoliatum (S) Navarretia squarrosa (S)
Caryophyllaceae Polygonaceae
Agrostemma githago (O) Fagopyrum esculentum (O)
Arenaria serpyllifolia (S) Polygonum aviculare (S)
Cerastium nutans (O) Polygonum convolvulus (S)
Silene dichotoma (O) Polygonum hydropiper (S)
Silene noctiflora (S) Polygonum lapathifolium (S)
Spergula arvensis (S) Polygonum persicaria (S)
Stellaria media (S)
Ranunculaceae
Fabaceae Myosurus minimus (S)
Medicago lupulina (O) Ranunculus reptans (O)
Trifolium arvense (S) Ranunculus sceleratus (O)
Trifolium aureum (S)
Trifolium campestre (S) Scrophulariaceae
Vicia sativa (S) Chaenorrhinum minus (S)
Vicia tetrasperma (O) Veronica agrestis (O)
Veronica arvensis (S)
Lamiaceae Veronica peregrina (S)
Galeopsis tetrahit (O) Veronica persica (S)
Lamium amplesicaule (S)
Lamium purpureum (S) Apiaceae
Aethusa cynapium (S)
Anethum graveolens (O)
The contrasts were based on 14 phylogenetically-independent pairs, where each pair consisted of median values of the selfing and outcrossing species within one family, which by definition are species that are more closely related to each other than to any other species in the data set [19]. For plant height, only 13 pairs were used due to missing information. The data were analyzed using a Wilcoxon matched pairs test.
Greenhouse study
The species included in this study were chosen if there was a known breeding system, if germinable seeds were available, and if a complementary species (i.e. in the same family with the opposite breeding system) was known and could also be obtained as germinable seeds. Seeds were obtained from a variety of sources; Herbiseed, Rancho Santa Ana Botanic Gardens, Chiltern Seeds, S&S Seeds, and the National Plant Germplasm System. Our search lead to an unbiased sample of 25 candidate species, allowing five pair-wise family comparisons and four pair-wise genus comparisons (Table 2).
Table 2 List of species, with breeding system (O – outcrosser; S – selfer), used in the greenhouse study
.
Family Family
Species Species
Asteraceae Convolvulaceae
Crepis capillaris (O) Ipomoea hederacea (S)
Helianthus annuus (O) Ipomoea purpurea (O)
Matricaria maritime (O)
Matricaria matricarioides (S) Fabaceae
Senecio viscosus (S) Lupinus bicolor (O)
Senecio vulgaris (S) Lupinus nanus (S)
Lupinus succulentus (O)
Boraginaceae Trifolium hirtum (S)
Borago officinalis (O)
Myosotis arvensis (S) Lythraceae
Cuphea laminuligera (O)
Brassicaceae Cuphea lanceolata (O)
Brassica juncea (O) Cuphea lutea (S)
Brassica nigra (O)
Capsella bursa-pastoris (S) Polemoniaceae
Cardamine hirsuta (S) Navarretia squarrosa (S)
Sinapis alba (O) Phlox drummondii (O)
Sinapis arvensis (O)
Most species were germinated in 15 cm pots filled to 3 cm below the top with standard potting soil (Promix BX©). Pots were placed in a greenhouse and watered daily until the appearance of their first true leaves. Subsequently, they were watered uniformly every second day to ensure that the soil was kept moist. Some species were germinated in a petri-dish in a growth chamber (23°C, 12 hour cycles of light and dark), after which they were transplanted into pots and placed in the greenhouse. Each species was replicated five times, with one plant per pot. Pots were arranged randomly on benches at a density of 1 pot per 0.093 m2.
The plants were exposed to 16 hours of daylight each day, with maximal natural light levels at ca 1200 μE. Before sunrise and after sunset, artificial lights (250–300 μE) were used to supplement the light exposure to 16 hours of light per day. The greenhouse was kept at an average temperature of 23.1°C during the day and dropped to 20.0°C at night. The plants were fertilized every 2 weeks with 200 ml per pot of a 2g/L concentration of 20–20–20 N-P-K fertilizer.
For each plant, age at first flower, bud development time, and flower longevity were measured. Emergence of the first pair of true leaves, after the cotyledons, was considered day 1 of the plant's life. Age at first flower was measured in days from day 1 to when the first flower opened on each plant. Bud development time (n = 3 buds per plant) was calculated as the number of days from the first appearance of a new bud until the flower opened. The same three buds on each plant were then monitored every day after opening, and the number of days until the flower senesced (flower longevity) was recorded for each. A flower was considered to be senesced when the corolla wilted, fell apart, or became discolored, as designated by Primack [17]. Any flower that was open for only one day was considered a one-day flower, regardless of whether it was open for the whole day or only part. Flowers in the Asteraceae were considered withered when the whole inflorescence had senesced, rather than the individual florets [17].
For bud development time and flower longevity, the three replicate measurements for each plant were averaged, and then these values for the five replicate plants were averaged to obtain a mean value for the species. The data were analyzed with a standard least squares one-way analysis of variance (ANOVA) model, with a post-hoc contrast between selfers and outcrossers. These analyses were done for each family and genus separately in order to control for phylogeny at these levels. In cases where the data were non-normal, a log-transformation was applied which corrected the distribution.
Authors' contributions
RS collected the data, performed most of the analyses, participated in the design of the study, and wrote the first draft as a B.Sc.(Hons) thesis. LWA conceived of the study, participated in its design and coordination, and wrote the final draft for submission to BMC. Both authors read and approved the final manuscript.
Acknowledgements
This research was supported by the Natural Sciences and Engineering Research Council of Canada through a research grant to LWA and a USRA to RS. Thanks to Spencer Barrett for provided breeding information for several species, Dale Kristensen and Richard Gold for assistance in the greenhouse, and Christopher Eckert and two anonymous reviewers for comments on an earlier version of the text.
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| 15707481 | PMC553978 | CC BY | 2021-01-04 16:29:15 | no | BMC Ecol. 2005 Feb 11; 5:2 | utf-8 | BMC Ecol | 2,005 | 10.1186/1472-6785-5-2 | oa_comm |
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BMC Complement Altern MedBMC Complementary and Alternative Medicine1472-6882BioMed Central London 1472-6882-5-31571323210.1186/1472-6882-5-3Research ArticleMedicinal herb use among asthmatic patients attending a specialty care facility in Trinidad Clement Yuri N [email protected] Arlene F [email protected] Derick [email protected] Ronald [email protected] Nadya [email protected] Rochelle [email protected] Odia [email protected] Deneil [email protected] Faculty of Medical Sciences, The University of the West Indies, St. Augustine, Trinidad and Tobago2005 15 2 2005 5 3 3 27 8 2004 15 2 2005 Copyright © 2005 Clement et al; licensee BioMed Central Ltd.2005Clement et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
There is an increasing prevalence of asthma in the Caribbean and patients remain non-compliant to therapy despite the development of guidelines for management and prevention. Some patients may self-medicate with medicinal herbs for symptomatic relief, as there is a long tradition of use for a variety of ailments. The study assessed the prevalence of use and the factors affecting the decision to use herbs in asthmatic patients attending a public specialty care clinic in Trinidad.
Methods
A descriptive, cross-sectional study was conducted at the Chest Clinic in Trinidad using a de novo, pilot-tested, researcher-administered questionnaire between June and July 2003.
Results
Fifty-eight out of 191 patients (30.4%) reported using herbal remedies for symptomatic relief. Gender, age, ethnicity, and asthma severity did not influence the decision to use herbs; however, 62.5% of patients with tertiary level schooling used herbs, p = 0.025. Thirty-four of these 58 patients (58.6%) obtained herbs from their backyards or the supermarket; only 14 patients (24.1%) obtained herbs from an herbalist, herbal shop or pharmacy. Relatives and friends were the sole source of information for most patients (70.7%), and only 10.3% consulted an herbalist. Ginger, garlic, aloes, shandileer, wild onion, pepper and black sage were the most commonly used herbs.
Conclusions
Among patients attending the Chest Clinic in Trinidad the use of herbal remedies in asthma is relatively common on the advice of relatives and friends. It is therefore becoming imperative for healthcare providers to become more knowledgeable on this modality and to keep abreast with the latest developments.
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Background
Recent reports from the Caribbean suggest that the incidence of asthma is following the global trend of increasing prevalence. In Jamaica, a prevalence of 20.8% for exercise-induced asthma was estimated in a cross-sectional study in schoolchildren [1]. About one in ten patients attending an Accident and Emergency Department in Trinidad were treated for acute severe asthma [2] and over 15,000 patients attended four A&E departments throughout the island over a 12-month period [3].
Inhaled corticosteroids as prophylaxis and 'as required' bronchodilator for symptomatic relief are established modalities for asthma management and prevention and the Commonwealth Caribbean Medical Research Council/Global Initiative for Asthma guidelines were adopted in the Caribbean in 1997 [4]. It has been noted that inefficient management predisposes patients to frequent hospitalization and reduced quality of life. In Trinidad, non-compliance and inadequate inhaler technique negatively impact on effective disease management [5,6]. The frequent unavailability of medication at public health facilities and the prohibitive cost at private pharmacies are significantly associated with non-compliance and consequently poor disease control. In these studies, some patients indicated their use of herbal remedies as an alternative to conventional medicines.
Over the last few decades, a global resurgence in the use of herbal remedies has fuelled the growing multi-billion dollar international trade of botanical products. Many patients, dissatisfied with conventional medicines because they expect permanent cures, believe that herbal remedies are 'natural' and sometimes self-medicate without informing their attending physician.
Although there is a long history of traditional use of medicinal herbs throughout the Caribbean [7,8] few studies were done to assess the prevalence of use. Surveys in Jamaica reported an almost 100% use of herbal teas and remedies by respondents throughout the island [9] and 71% in paediatrics inpatients at the University Hospital [10]. These studies, however, assessed only the lifetime use of medicinal herbs and did not identify their use for any particular disease. In Trinidad and Tobago, the use of 'bush medicine' in diabetic patients attending primary healthcare facilities throughout the island was assessed and although 42% reportedly used herbs, only 24% used this healthcare modality for self-management of diabetes [11]. Another survey conducted at an outpatient surgical facility in Trinidad indicated a lifetime prevalence of 86% among patients [12] for any healthcare issue.
This study was undertaken to assess the extent of use of herbal remedies by asthmatic patients attending a specialty chest clinic in Trinidad for symptomatic relief and to determine the factors influencing the patient's decision to use herbs.
Methods
The study was approved by the Ethics Committee of the Faculty of Medical Sciences, University of the West Indies, St. Augustine campus and permission to interview patients was granted by the Director of the Chest Clinic of the Ministry of Health, Trinidad and Tobago. The study was conducted over the two-month period June to July 2003.
Sample and setting
The Chest Clinic was chosen as the source of subjects as this is the only national tertiary level health facility specializing in the management of respiratory diseases. Patients entering the study were physician-diagnosed asthmatics based on self-reporting symptoms of wheezing, chest tightness and nocturnal coughing in the previous year. Patients were recruited by consecutive sampling and the nature and purpose of the study were explained on an individual basis. Those confirming their willingness to participate signed their informed consent and were interviewed using a de novo, pilot-tested, researcher-administered questionnaire.
Interview instrument
The questionnaire assessed demographic data such as age, gender, ethnicity, residential district, education, employment and socioeconomic status. Subjects reported their disease severity as intermittent, moderate or severe as determined by the Global Initiative for Asthma (GINA) guidelines with respect to symptom frequency [4]. Patients also reported their use of herbal remedies, identified the herbs used, the frequency of use, source of herbal medicines and the reasons for the use of herbs.
Statistical analysis
The sample size was calculated as 185 patients assuming a prevalence of 86% [13] with a confidence level of 95%. Since all variables were categorical, χ2 tests were performed to determine whether there were statistically significant associations between the use of herbs and these variables. The p value was set at <0.05 for statistical significance. The data was analyzed using SPSS for Windows (Version 9.0, Chicago, IL).
Results
Demography
During the study period one hundred and ninety one patients consented to participate. The demographic details of the sample are given in Table 1. Patients between 35 and 64 years of age formed the largest portion of the sample (62.3%). There was a significant gender difference with females outnumbering males by a 2:1 ratio, p < 0.01. Most patients were of Asian Indian origin (58.1%) and resided in suburban areas (60.2%). There was a high level of unemployment (30.4%); this could be correlated to primary schooling (seven or less years of formal education) being the highest educational level attained in 52.9% and no formal schooling in 5.2% of the sample population. Income was low, with 42.9% of the sample population earning below US$4,000 per year.
Table 1 Demographic details of patient sample
Factors (n = 191) No. (% of sample) No. (%) using herbs
Gender
Male 61 (31.9) 19/61(31.2)
Female 130 (68.1)* 39/130 (30.0)
Age groups
16–34 34 (17.8) 11/34 (32.4)
35–50 52 (27.2) 21/52 (40.4)
51–64 67 (35.1) 18/67 (21.1)
≥ 65 38 (19.9) 8/38 (13.8)
Ethnicity
African 45 (23.6) 13/45 (28.9)
Asian Indian 110 (57.6)* 31/110 (28.2)
Mixed 35 (18.3) 14/35 (40.0)
Other 1 (0.5) 0/1 (0.0)
Asthma severity
Intermittent 90 (47.1) 29/90 (32.2)
Mild Persistent 29 (15.2) 9/29 (31.0)
Moderate Persistent 27 (14.1) 7/27 (25.9)
Severe Persistent 45 (23.6) 13/45 (28.9)
Antiasthmatic drug use
The GINA guidelines were recently adopted in the Caribbean and asthmatic patients are currently treated according to their symptom severity. In our sample population, particularly in patients with moderate and severe symptoms, corticosteroids (controllers) and β2-agonists (relievers) were prescribed at very high rates, Table 2. Almost 90% of all patients with moderate symptoms were prescribed drugs in these classes. Almost all patients with severe symptoms were prescribed β2-agonists. This high level of prescription and use of β2-agonists suggest a lack of symptomatic control in our sample population. Theophylline and anticholinergics were prescribed in both categories of patients, but to a lesser extent.
Table 2 Antiasthmatic drug use and self-reported compliance in patient sample
Drug Use & Compliance Asthma Severity
Moderate Persistent (n = 27) Severe Persistent (n = 45)
Corticosteroids 24 (88.9) 39 (86.7)
β2 agonists 24 (88.9) 44 (97.8)
Anticholinergics 5 (18.5) 13 (28.9)
Theophylline 9 (33.3) 19 (42.2)
Self-reported Compliance 27 (100) 43 (95.6)
Factors influencing the use of herbal remedies
Gender, age, ethnicity, residential district, employment status, income and asthma severity had no statistically significant effect on the use of herbal remedies within the sample population, Table 3. However, almost two-thirds (62.5%) of patients with tertiary education used herbal remedies for asthma, p = 0.025.
Table 3 Socioeconomic details of patient sample
Factors (n = 191) No. (% of sample) No. (%) using herbs
Residential district
Rural 27 (14.1) 6/27 (22.2)
Suburban 119 (62.3)* 36/119 (30.3)
Urban 42 (22.0) 15/42 (35.7)
No response 3 (1.6) 1/3 (33.3)
Highest educational level attained
No formal education 10 (5.2) 2/10 (20.0)
≤ 7 years of formal education 101 (52.9) 26/101 (25.7)
≤ 12 years of formal education 64 (33.5) 20/64 (31.3)
> 12 year of formal education 16 (8.4) 10/16 (62.5)*
Employment status
Unemployed 58 (30.4) 19/58 (32.8)
Technical 12 (6.3) 6/12 (50.0)
Professional 16 (8.4) 5/16 (31.3)
Clerical 13 (6.3) 6/13 (46.2)
Vocational 28 (12.6) 5/28 (17.9)
Student 8 (4.2) 4/8 (50.0)
Housewife 23 (12.0) 7/23 (30.4)
Pensioner 33 (17.3) 6/33 (18.2)
Annual income (US$)
≤ 3,999 82 (42.9) 22/82 (26.8)
4000 – 9,999 73 (38.2) 23/73 (31.5)
10,000 – 11,999 12 (6.3) 4/12 (33.3)
12,000 – 19,999 15 (7.9) 7/15 (46.7)
≥ 20,000 9 (4.7) 2/9 (22.2)
Characteristics of patients using herbal remedies
Most patients (70.7%) using herbs were advised by a relative or friend and only 10.3% sought the advice of an herbalist, Table 4. A cultural/traditional basis was the reason for herbal remedy usage in twenty-one (36.2%) patients and another twelve (20.7%) patients used herbs because they felt that were either 'natural' or 'healthy'. Twelve (20.7%) patients used herbs because they believed that their physician-prescribed allopathic medicines were not working.
Table 4 Characteristics of patients using medicinal herbs (n = 58)
Patient characteristics No. (%) (Out of 58)
Reason for using herbs
Traditional/Cultural 21 (36.2)
Natural/Healthy 12 (20.7)
Conventional medicine not working 12 (20.7)
Other 13 (22.4)
Source of herbs
At home/backyard garden 25 (43.1)*
Supermarket 9 (15.5)
Herbalist/Herbal Shop/ Pharmacy 14 (24.1)
Relative/friend 4 (6.9)
Other 6 (10.3)
Source of information
Relative/friend 41 (70.7)*
Herbalist 6 (10.3)
No consultation 9 (15.5)
Other 2 (3.5)
Time last used herbs
Within last week 17 (29.3)
Within last month 9 (15.5)
Within last 3 months 6 (10.3)
Within last 6 months 3 (5.2)
More than 6 months ago 23 (39.7)
Most patients (58.6%) obtained their herbs or medicinal plants from either their backyards or the supermarket. Only fourteen (24.1%) obtained their herbal supplies from an herbalist, herbal shop or pharmacy. Seventeen (29.3%) of these patients reported using herbs within the last week and most these patients (60.3%) used herbs within the last six months.
Many of these patients were using both physician-prescribed antiasthmatic drugs and herbal remedies, Table 5. No patient with either moderate or severe symptoms indicated that herbal remedies alone were sufficient to relieve symptomatic episodes. It is interesting to note that most patients with moderate symptoms (57.1%) believed that concurrent use of conventional medications and herbs gave better symptomatic relieve. One the other hand, most patients with severe symptoms (53.8%) believed that physician-prescribed medications worked better than herbal remedies, while 23.1% believed that neither relieved their symptoms.
Table 5 Antiasthmatic drug use and self-reported compliance in patients using herbal remedies
Drug Use & Compliance Asthma Severity
Moderate Persistent (n = 7) Severe Persistent (n = 13)
Corticosteroids 6 (85.7) 10 (76.9)
β2 agonists 6 (85.7) 13 (100)
Anticholinergics 1 (14.3) 5 (38.5)
Theophylline 2 (28.6) 7 (53.8)
Self-reported Compliance 5 (71.4) 8 (61.5)
Subjective Benefits of therapy
Herbal remedies alone better 0 (0) 0 (0)
Herbal remedies and drugs better 4 (57.1) 3 (23.1)
Drugs alone better 3 (42.9) 7 (53.8)
Neither herbs and/or drugs work 0 (0) 3 (23.1)
Herbs used in asthma
Most patients in the sample used more than one medicinal herb simultaneously, which were usually prepared and administered as mixtures in teas. Almost one in four patients using medicinal herbs (22.5%) used either garlic (Allium sativum) or ginger (Zingiber officinale) for symptomatic relief of asthma, Table 6. Aloes (Aloe vera) shandileer (Leonotis nepetifolia), wild onion (Hymenocallis tubiflora), pepper (Capsicum spp.) tulsi (Ocimum gratissimum), black sage (Cordia curassavica), shadon beni (Eryngium foetidium), lemongrass (Cymbopogon citratus) and nutmeg (Myristica fragrans) were the more popular traditional indigenous West Indian medicinal plants used. Two patients reported using marijuana (leaves and roots). Herbs of European and North American origin, identified as Echinacea (Echinacea purpurea), Golden Seal (Hydrastis canadensis) and Chamomile (Matricaria chamomilla) were less frequently used. Five patients reported using trade name imported tablets for asthma.
Table 6 Medicinal plants commonly used by respondents (n = 58) using herbal remedies, ranked by prevalence
Common name Botanical name n %
Garlic Allium sativum L. 13 22.4
Aloes Aloe vera (Aloe barbadensis Miller) 13 22.4
Ginger Zingiber officinale Roscoe 9 15.5
Shandileer Leonotis nepetifolia (L.) R.Br. 8 13.8
Wild Onion Hymenocalis tubiflora 8 13.8
Pepper Capsium spp. L. 6 10.4
Black Sage Cordidia cylindristachya R.S. 6 10.4
Tulsi Ocimum grastissimum L. 5 8.6
Echinacea Echinacea purpurea L. Moench 5 8.6
Shadon beni Eryngium foetidium L. 5 8.6
Nutmeg Myristica fragrans Houtt. 4 6.9
Lemongrass Cymbopogon citratus (DC.) Stapfl 4 6.9
Christmas bush Chromolaena odorata (L.) R.M. King & H. Rob. 4 6.9
Golden Seal Hydrastis canadenesis L. 3 5.2
Bayleaf Pimenta racemosa (P. Mill) J.W. Moore 3 5.2
Charmomile Matricaria chamomilla L. 3 5.2
Hibiscus Hibiscus rosa-sinensis Linn. 3 5.2
Noni Morinda citrifolia Linn. 3 5.2
Marijauna Cannabis sativum L. 2 3.5
Effect of income and education on the use of herbs
Patients using easily accessible herbs such as ginger (Zingiber officinale) and aloes (Aloe vera), and traditional indigenous medicinal herbs such as shandileer (Leonotis nepetifolia) and tulsi (Ocimum gratissimum) were more likely to be earning less than US$12,000, Table 7. Herbs of European or North American origin (Echinacea purpurea and Matricaria chamomilla) were more likely to be used by patients earning in excess of US$12,000 per annum. Income did not affect the use of either garlic or cocoa onion.
Table 7 Income and education effects on use of herbs
Medicinal herb used Percentage of patients with annual income Percentage of patients with formal education
≤ US$12,000 > US$12,000 ≤ 12 years > 12 years
Ginger (Zingiber officinale) 18.4* 0.0 14.0 20.0
Garlic (Allium sativum) 22.5 22.2 16.7 50.0*
Aloes (Aloe vera) 24.5* 11.1 25.0 10.0
Shandileer (Leonotis nepetifora) 16.3* 0.0 14.6 10.0
Cocoa Onion (Hymenocalis tubiflora) 10.2 11.1 10.4 10.0
Tulsi (Ocimum grastissimum) 10.2* 0.0 10.4 0.0
Golden Seal (Hydrastis canadenesis) 6.1 0.0 6.3 0.0
Echinacea (Echinacea purpurea) 4.1 33.3* 4.2 30.0*
Chamomile (Matricaria chamomilla) 2.0 11.1* 3.9 10.0
Aloes (Aloe vera), tulsi (Ocimum gratissimum) and golden seal were preferred in patients with at least twelve years of formal education, Table 7. Garlic and Echinacea were the preferred herbal medicines in patients with more than twelve years formal education. Educational level did not affect the patients' decision to use shandileer (Leonotis nepetifolia), wild onion (Hymenocallis tubiflora) or ginger (Zingibe officinale).
Discussion
This is the first study of its kind in the Caribbean to assess the use of medicinal herbs by asthmatic patients attending a specialty care clinic. The findings of this study are instructive as the use of medicinal herbs for self-medication in disease management has far reaching implications on the quality of healthcare delivery [14]. We report a prevalence of 30.4% in our patient sample, which is significantly higher than that in the UK, Denmark, Singapore and in the US [15-18].
Most patients using medicinal herbs relied on the advice of relatives and friends as their sole source of information, as were caregivers of children in a US study [19]. We suggest that this information on the use of medicinal plants could have come from traditional/cultural knowledge, anecdotal evidence or from the greater public awareness through information networks such as the internet on the potential medicinal benefits of herbs. Asthma is an emerging chronic disease in the Caribbean and we suggest that the traditional knowledge in this area may be relatively 'new' and exist in relation to other diseases affecting the respiratory tract, such as cough, the common cold and the flu. This may be one of the reasons for the low prevalence of use of herbs in elderly asthmatic patients, as a strong traditional knowledge may not have existed.
We expected a higher prevalence of herbal use in individuals living in rural areas as these districts are depots for traditional knowledge as was reported in Jamaica where rural respondents used a larger variety of herbs than those living in urban areas [10]. As suggested earlier, we suspect that due to the recent emergence of asthma as a chronic disease in the Caribbean it is reasonable to expect that traditional knowledge in the management of this disease is not strong and our results are indicative of this.
We suspected that employment status could have predicted the use of herbs, however, this was not the case in our study sample. Unemployed patients did not improvise more in their use of herbal remedies than those in other income groups, even though most of the herbs used were relatively common, readily available and cheap. The low socioeconomic status of the majority of the sample may have prohibited both consultation with qualified herbalists and the purchase of imported, processed herbs that would have incurred additional out-of-pocket expense to the patient. What we noted was that there was no difference in the use of herbs across the income ranges and that in fact, patients earning relatively modest annual incomes between $US12,000 and $US19,999 were most likely to use herbs, although this did not reach statistical significance.
Attaining a higher education positively influence the decision to use herbs. We suggest that in the absence of traditional knowledge regarding the medicinal use of herbs for asthma, a higher educational level may predispose an individual to greater access to general knowledge, especially with greater exposure to the internet and other sources of information, and this could be a factor in positively influencing the individual's decision to use medicinal herbs. The availability of scientific evidence-based information on the efficacy of herbs for diverse healthcare problems may be particularly significant in patients with the resources to avail themselves to such information, particularly those with higher educational and income levels. This is particularly true for garlic and Echinacea, which have been extensively researched and furthermore patients with higher educational and income levels would be more likely be at an advantage to access information via literature or on the world wide web regarding the use of these medicinal plants.
Patients using imported, processed, and obviously more expensive herbal medications were on the higher end of the socioeconomic scale and were more likely to afford these medications. It was also observed that garlic and Echinacea were the herbs of choice in patients with higher educational levels. These herbs have a long tradition of use and are widely researched in Europe and North America. The traditional use and strong scientific evidence to support their therapeutic efficacy could be important factors influencing the patient's decision. It has been suggested elsewhere that patients with higher educational levels also tend be more involved in the management of their health; they tend to self-medicate or even suggest to their physicians the course of therapy.
Although one in five patients using medicinal herbs stated that "conventional medicines were not working" as the reason for using this alternative healthcare modality, we noted that asthma severity does not affect the decision to use herbs. In previous studies, poor management was associated with non-compliance with prescribed pharmacotherapy and poor inhaler technique [5,6].
The backyard and home garden were major sources of readily available herbs such as aloes, shadon beni and lemongrass. Wild growing 'weeds' such as shandileer, tulsi, cocoa onion and black sage were also identified. The supermarket was a major source of inexpensive common medicinal herbs such as garlic, ginger and nutmeg. The identification of these medicinal herbs provides an opportunity to investigate West Indian plants used to treat asthma to determine whether they possess pharmacological properties. Scientific investigations have shown that some of these herbs possess pharmacological and anti-inflammatory properties, and these may be useful in suppressing the characteristic exaggerated immune response in asthma [20-24]. Pepper and bayleaf have also been shown to exhibit anti-inflammatory properties [[25,26]27]. There is an imperative to commence scientific investigations on traditional West Indian medicinal plants to determine their therapeutic efficacy and safety.
The survey instrument specifically asked questions on the use of medicinal herbs in asthma and did not inquire about the use of herbs as customary teas or tonics. We therefore did not determine lifetime prevalence for the use of herbs in our patient sample, but we suppose that had this been included that there might have been a prevalence similar to those reported in the Jamaica [10,11] and Trinidad [13] surveys. The survey was also limited in that by electing to conduct the study at a public health facility we obviously had a bias towards patients at the lower rung of the socioeconomic ladder, with lower income and educational status. As a consequence, the results reflected patients from this demographic background. We may have expected a different outcome in asthmatic patients attending private institutions, where their characteristics would have been slightly different, as we noted that even in our sample the small number of persons with higher income and educational status tended to use more medicinal herbs for symptomatic relief.
We did not assess whether patients informed their attending physician at the clinic about their use of herbs or determined whether the knowledge or attitudes of these physicians regarding the use of herbs influenced the patients' decision to use herbs. The study was also limited in that we did not ascertain the out-of-pocket expense for herbal remedies by patients, although most stated that herbal medicines (which we supposed were processed, imported products) were more expensive than conventional medicines. We assumed that an additional expense would have only been incurred by those patients purchasing processed, imported herbs obtained from a herbalist, herbal shop or pharmacy (24.1%) and who actually consulted a herbalist (10.3%). We also reasoned that since all the other herbs used were inexpensive and available from either the backyard garden or supermarket (58.6%) that the cost to patients selecting these remedies was minimal.
Conclusions
The findings of this study are important in that local medicinal plants in Trinidad have been identified in the self-management of asthma in a significant number of patients attending the specialty clinic. These identified herbs can now be targeted for scientific investigation to determine whether their pharmacological efficacy will assist in the development of viable healthcare alternatives in a developing country. These findings are also important for policymakers in the health sector who are given the mandate to regulate issues pertaining to the public's health. We are also becoming more aware of the potential for critical interplay between herbs and drugs when taken concomitantly to produce life-threatening interactions. Since herbs are here to stay and patients will continue to self-medicate with increasing frequency, it is imperative that healthcare providers become more knowledgeable on this modality and keep abreast with the latest developments in herbal therapy.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
YNC was the P.I. in this study. He was responsible for the study concept, development of methodology, coordinating the research activities, analyzing the data, and writing the manuscript. AFW was responsible for data input and analysis. DA was involved in methodological development, data collection, data input and analysis and presentation at regional conference. RC was involved in methodological development, data collection, data input and analysis. NW was involved in methodological development, data collection and input. RM was involved in methodological development, data collection and input. OS was involved in methodological development, data collection and input. DW was involved in methodological development, data collection and input. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgement
Dr. Dottin Ramoutar, Director of the Chest Clinic and his staff are to be thanked for their kind indulgence in allowing us the facilities to interview patients. We wish to also thank Dr. Celia Poon King for her assistance in assessing the study's epidemiological soundness and Mr. Rudy Singh for his advice on matters relating to statistical analysis. Professor Peter Knott was kind enough to review and make critical comments on the manuscript.
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| 15713232 | PMC553979 | CC BY | 2021-01-04 16:31:46 | no | BMC Complement Altern Med. 2005 Feb 15; 5:3 | utf-8 | BMC Complement Altern Med | 2,005 | 10.1186/1472-6882-5-3 | oa_comm |
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BMC Plant BiolBMC Plant Biology1471-2229BioMed Central London 1471-2229-5-21572072910.1186/1471-2229-5-2Research ArticleThe Arabidopsis Mei2 homologue AML1 binds AtRaptor1B, the plant homologue of a major regulator of eukaryotic cell growth Anderson Garrett H [email protected] Maureen R [email protected] Molecular Biology and Genetics, Cornell University, Ithaca, NY 14850, USA2005 21 2 2005 5 2 2 4 10 2004 21 2 2005 Copyright © 2005 Anderson and Hanson; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
TOR, the target of the antibiotic rapamycin in both yeast and mammalian cells, is a potent cell growth regulator in all eukaryotes. It acts through the phosphorylation of downstream effectors that are recruited to it by the binding partner Raptor. In Arabidopsis, Raptor activity is essential for postembryonic growth. Though comparative studies suggest potential downstream effectors, no Raptor binding partners have been described in plants.
Results
AtRaptor1B, a plant Raptor homologue, binds the AML1 (Arabidopsis Mei2-like 1) protein in a yeast two-hybrid assay. This interaction is mediated by the N-terminal 219 residues of AML1, and marks AML1 as a candidate AtTOR kinase substrate in plants. The AML1 N-terminus additionally carries transcriptional activation domain activity. Plants homozygous for insertion alleles at the AML1 locus, as well as plants homozygous for insertion alleles at all five loci in the AML gene family, bolt earlier than wild-type plants.
Conclusion
AML1 interacts with AtRaptor1B, homologue of a protein that recruits substrates for phosphorylation by the major cell-growth regulator TOR. Identification of AML1 as a putative downstream effector of TOR gives valuable insights into the plant-specific mode of action of this critical growth regulator.
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Background
TOR, the target of the antibiotic rapamycin in both yeast and mammalian cells, is a major regulator of cell growth and translation [1]. TOR is a large (over 2,400 residues) protein kinase [2] present in all eukaryotes analyzed. It is thought to act in a nutrient-sensitive complex TORC1 with Raptor (regulatory associated protein of TOR) and another protein to regulate cell growth [3-7] – though there is some debate on the nutrient sensitivity of the complex [8]. Raptor, a protein with HEAT and WD-40 protein interaction domains, recruits substrates for phosphorylation by TOR in yeast and mammals [3,7]. TOR also acts in a second, nutrient-insensitive complex without Raptor to regulate the cytoskeleton [3,8,10].
Disruption of the Arabidopsis TOR homologue AtTOR is lethal early in plant embryonic development [11]. Disruption of AtRaptor (encoded by two paralogous loci in Arabidopsis) causes seedling developmental arrest but allows normal embryonic development [[12], manuscript in preparation], suggesting that AtTOR embryonic activity is independent of AtRaptor and that the TOR-Raptor complex has been adapted in the ancestor of the angiosperms to regulate post-embryonic growth. In support of this, AtTOR has been show to be expressed in dividing and expanding cells [11]. Thus, identifying downstream activators of TOR signaling may provide insights into the activity of AtTOR in post-embryonic growth.
Mei2 is a putative TOR substrate and potent meiosis-signaling molecule identified in the fission yeast Schizosaccharomyces pombe [13,14]. Mei2 is bound by the Raptor homologue Mip1 [15], and is an inactive phosphoprotein under high nutrient conditions [16,17] – conditions which promote TOR kinase activity. The kinase governing two of the three Mei2 phosphorylation sites is known [16]; TOR is a strong candidate for the kinase governing the third. In diploids under low nutrient conditions, unphosphorylated Mei2 accumulates and localizes to the nucleus, where it binds to a noncoding, mRNA-like RNA molecule meiRNA in an interaction mediated by the third of its three RNA Recognition Motifs (RRMs)[14,16,18]. The Mei2-meiRNA interaction occurs as meiRNA is being transcribed, tethering Mei2 to the meiRNA locus [19]. Accumulation of Mei2 at this focused point immediately precedes meiosis.
Mei2-like proteins are predicted in a wide range of organisms [20,21] including some fungi, alveolates, a diatom, and all land plants, but they are absent from metazoans and budding yeast. In land plants, predicted Mei2-like proteins form a small conserved gene family, many of whose members' transcripts accumulate in the shoot apical meristem specifically or in the shoot apical meristem in addition to a range of mature tissues [20].
Arabidopsis Mei2-like 1 (AML1), the first member of this family to be described, was isolated in a screen for plant cDNAs whose expression could complement defects in the fission yeast meiosis signaling pathway [22]. Like Mei2, AML1 has three RRMs. Expression of a protein fragment containing only the third AML1 RRM was sufficient for restoration of meiosis signaling in fission yeast lines with defects upstream of Mei2, but not in lesions of the mei2 locus itself. Terminal Ear 1 (te1), a more divergent member of the mei2-like family of genes, regulates leaf initiation in maize; the tassel of mutant plants is encased a whorl of leaves superficially resembling a maize ear [23].
Given the potential role of downstream AtTOR effectors in post-embryonic growth, the intriguing signaling activity of Mei2 in fission yeast, and the known interaction between Mei2 and the fission yeast Raptor protein, we asked if the Mei2-Raptor interaction was conserved between its Arabidopsis orthologues AML1 and AtRaptor1B.
Results
AML1 and AtRaptor1B interact in a yeast two-hybrid assay
To test for an interaction between the Arabidopsis proteins AML1 and AtRaptor1B, the open reading frames (ORFs) of each of the transcripts predicted to encode these proteins were amplified via PCR with primers carrying suitable restriction sites for cloning into the yeast two hybrid vectors pGADT7 and pGBKT7. pGADT7 encodes a transcriptional activation domain (AD) which can recruit the yeast transcription machinery. pGBKT7 encodes a DNA binding-domain (BD) which binds to the promoters of ADE2 and HIS3. pGADT7 and pGBKT7 additionally carry the genes LEU2 and TRP1. When transformed into the yeast line AH109, which is leu2-trp1-ade2-his3-, co-transformed cells will grow on yeast medium lacking leucine and tryptophan. Cells co-transformed with two-hybrid constructs encoding proteins that interact will grow on media additionally lacking histidine and adenine (selective media).
AH109 yeast cells co-transformed with clones encoding AML1 and AtRaptor1B in complementary two-hybrid vectors grew on selective media. Co-transformations of control pGBKT7 and pGADT7 empty vectors, or either control vector co-transformed with its complement harboring the AML1 or AtRaptor1B ORF, yielded cells able to grow on media lacking leucine and tryptophan but not on selective media.
Evidence from other systems indicates that Raptor protein fragments lose the ability to bind substrates [4]. Therefore, only full length AtRaptor1B was tested in this assay. Mei2, the fission yeast AML1 homologue, is highly modular. It is divided into distinct N-terminal and C-terminal domains. The N-terminal half of the protein appears to play a regulatory role [24]. The C-terminal half of Mei2 is sufficient to complement lesions in the mei2 locus [16]. Additionally, the AML1 C-terminal half, expressed in fission yeast meiosis signaling mutants, is able to complement meiosis signaling defects upstream of Mei2 [22]. Therefore we generated clones encoding fragments of AML1 and assayed them for interactions with full length AtRaptor1B.
AML1 fragments N412 and N219, comprised of the first 412 or the first 219 residues of the 915 residue AML1 protein, restored growth on media lacking leucine, tryptophan, histidine and adenine when cloned into pGADT7 and co-transformed with pGBK-Raptor (Fig. 1). Neither N163, nor 155–219, (the fragments which together comprise N219), nor 155–412 (which with N163 comprises N412), could restore growth when cloned into pGADT7 and co-transformed with pGBK-Raptor. AML1 fragment 402C, comprised of residues 402 to the C-terminus of the protein, and all C-terminal fragments tested (695C, 402–704) failed to restore growth on selective medium. None of the AML1 fragments cloned into pGADT7, and cotransformed with empty pGBKT7 control vector, yielded transformants able to grow on selective medium.
AML1 fragments N412 and N163 harbor activation domain activity in a yeast one-hybrid assay
AML1 N412, cloned into pGBKT7, restored growth on selective medium to cells co-transformed with either pGAD:Raptor or pGADT7. To investigate this result further, we tested all AML1 fragments for native transcriptional activation domain activity in a yeast one-hybrid assay. AML1 fragments were cloned into pGBKT7, singly transformed into AH109 yeast cells, and assayed for growth on media lacking tryptophan (to confirm transformation) and media additionally lacking adenine. The DNA binding-domain of the pGBKT7 tethers any C-terminally fused fragments to the ADE2 promoter. In the absence of a binding partner, BD-fusion chimeric proteins trigger transcription of ADE2 only if the protein fused to the BD contains native transcriptional activation activity.
AML1 N412 and AML1 N163, but not full length AML1, AML1 N219 or any C-terminal AML1 fragments, were able to restore growth on media lacking tryptophan and adenine (Fig. 2). AML1 N122 was similarly unable to restore growth. AML1 fragments 155–219 and 155–412, in pGBKT7, could not be stably transformed into yeast. These results were observed in multiple independent transformant lines for a given construct. Cotransformation of any of the pGBKT7-derived constructs with pGADT7 did not affect the growth of any of the transformed lines on media lacking tryptophan and adenine.
Plants homozygous for insertion alleles of AML1 and of all five AML family members show early flowering
An interaction with AtRaptor1B points to AML1 as a downstream effector of TOR signaling in plants. Additionally, the dramatic phenotype mei2 disruption and the intriguing mode of Mei2 action led us to ask what the consequences would be of disruption of the AML1 locus and of all five AML gene family members. To obtain insertion alleles in AML1 and other AML gene family members, we screened the insertion allele populations at the University of Wisconsin Arabidopsis Knockout facility [25], obtaining alleles harboring insertions in AML1, AML3, AML4 and AML5 (Fig. 3). An AML2 insertion allele was obtained from the SIGnAL collection at the Salk Institute [26].
By RT-PCR using primers which anneal to the cDNA at sites spanning the insertion site of each insertion allele, we established that no wild-type transcripts accumulate in homozygous mutants (Fig. 4). By a series of crosses, we then generated higher-order insertion homozygotes, culminating in the quintuple insertion homozygotes Q6 and Q17. All lines were viable and fertile.
AML insertion homozygotes bolted earlier than wild-type lines (Fig. 5A and 5B). This effect was independent of the number of insertion alleles carried by the mutants; AML 'quint' lines Q6 and Q17 were not qualitatively different than lower order insertion allele homozygotes.
Additionally, AML insertion homozygote seedlings were assayed for a differential response to a range of signaling molecules. Seedlings were germinated on culture medium supplemented with the gibberellic acid GA3, paclobutrazol, the auxin 2,4-D, 1-amino-cyclopropane-1-carboxylic acid, 1% sucrose, 6% sucrose and kinetin, and in the dark. Quintuple insertion mutants responded slightly more than wild-type seedlings to GA3 as measured by the change in seedling length in the presence vs. in the absence of the hormone. This effect was repeatable but weak, and no other differential hormonal response was observed (data not shown).
Sequence downstream of the mutant allele insertion sites is transcribed
Given the mild phenotype of the AML insertion homozygotes, we further investigated the extent of the effect of the insertions at each locus. RT-PCR, as previously stated, showed that no wild-type transcripts accumulate in lines Q6 and Q17. In fission yeast, however, the C-terminal half of the protein is sufficient to complement lesions of the mei2 locus [16]. The insertion alleles of all but AML4 are disrupted at or near the 5' ends of their predicted coding regions. We therefore designed primers that anneal to the region downstream of the insertion site in each allele and performed polymerase chain reactions to assay for accumulation of fragments capable of encoding the C-terminal half of any of the AML proteins (Fig. 4). To determine whether the amplified fragments corresponded to AML cDNAs, we performed restriction digests on the PCR products, which confirmed that the cDNAs originated from AML gene transcripts. Weak amplification of cDNA representing transcripts originating downstream of the insertion site was observed for AML1, AML2 and AML3; amplification of the AML5 3' region was indistinguishable from the amplification seen from of wild-type cDNA template.
Transgenic lines overexpressing AML1:GFP or GFP:AML1 fusion proteins could not be recovered
The AML1 ORF was separately cloned into the pCambia1302 35S::GFP plant transformation vector both 5' and 3' of the GFP ORF, and the construct was transformed into Arabidopsis via Agrobacterium-mediated floral dip [27]. Transformants, identified by resistance to the antibiotic hygromycin and confirmed through PCR, were recovered at a very low rate of less than .01%. No fluorescence was observed in any tissues of any transformants assayed, and AML1:GFP transcripts could not be detected via RT-PCR performed on cDNA transcribed from RNA extracted from bulk shoot tissue (data not shown).
Discussion
Raptor proteins in yeast and mammals function by recruiting substrates for TOR, a central regulator of cell growth in response to nutrients [4,7,28]. An interaction with Raptor therefore strongly suggests that a given protein is a TOR substrate and downstream effector of TOR signaling. Plants homozygous for lesions at both AtRaptor loci show normal embryonic development but are unable to maintain shoot meristem activity [12]. TOR substrates, then, may play a role in regulating meristem-driven post-embryonic growth. The interaction between AML1 and AtRaptor1B implicates the AML family of proteins in TOR signaling. It points specifically to a role for the AML proteins in regulation of shoot meristem activity.
An interaction between Mei2 and the fission yeast Raptor homologue Mip1 has been reported previously; indeed, Mip1 (Mei2 interacting protein 1) was the first Raptor homologue characterized in any eukaryote [15]. The conservation of this interaction from fission yeast to plants suggests that the well-characterized Mei2 signaling pathway may provide insight into the function of the AMLs.
Mei2 is a potent meiosis-signaling molecule. It triggers pre-meiotic cell differentiation and meiosis in response to nutrient stress [13,14]. Meiosis signaling in fission yeast is a model for cell differentiation in response to external nutrient cues. Thus the AMLs may also play a role in cellular differentiation or in meiosis signaling.
Aside from the effect of Mei2 in development, there is the intriguing issue of its mode of action. Mei2 sub-cellular localization is mediated by an interaction with a noncoding, mRNA-like molecule [14,19]. There is a fairly large population of mRNA-like transcripts conserved among land plants despite lacking large conserved open reading frames [29]. Of these, the conserved alfalfa transcript ENOD40 has been shown to mediate the sub-cellular localization of an RNA-binding protein [30] and to mediate phytohormone responses [31]. AML1 may be a binding partner of one or more of these mRNA-like noncoding molecules in plants.
The transcriptional activation activity of the AML1 N-terminus observed in the yeast one hybrid assay has not been ascribed to Mei2 and may represent a novel activity of plant Mei2-like proteins. This activation activity localizes to the N-terminal 163 residues, but is strongly influenced by the adjacent residues. Activity is lost in N219, regained on N412 and lost again in full length AML1. This suggests that the AML1 N-terminal half has multiple configurations, and that the accessibility of the activation domain varies among configurations. We should emphasize, however, that the AML1 N-terminal fragment transcriptional activation activity has yet to be shown in planta, and that the activity may result from fusion to the DNA binding domain rather than being present in native AML1.
AtRaptor1B binding to AML1 is also localized to the N-terminus, and appears to be mediated by multiple sites in this region. This suggests that the N-terminus may contain a TOR phosphorylation site, and that this site may influence the configuration of the N-terminus.
The repeated failure to recover transgenic lines expressing AML1 suggests that its unregulated overexpression is lethal. Future efforts to characterize the AML proteins in transgenic plants may benefit from the use of inducible promoters driving transgene expression to circumvent the putative lethality of unregulated AML expression.
Disruption of any of the AML loci causes early bolting in plants grown under long days. However, lines homozygous for insertions in all five AML loci did not differ dramatically from lower-order insertion homozygotes, despite the fact that RT-PCR performed with primers spanning the insertion sites show that the wild-type transcript does not accumulate.
Transcripts originating downstream of the insertion sites but still capable of encoding the C-terminal half of the wild-type protein accumulate from all loci but AML4. This raises the possibility that the AML quintuple insertion homozygote lines do not represent total disruption of AML activity. Four of the five AML open reading frames in the insertion mutant are apparently truncated and all are divorced from their native promoters, but some promoter activity (perhaps from the 35S viral promoters harbored within the inserted DNA) remains and may be sufficient to cause transcription of AML coding region DNA downstream of the insertion site.
Viewed in this light, the early flowering phenotype of AML insertion homozygotes may arise not from the total disruption of AML activity but from the accumulation of AML proteins which, due to the truncations in their N-termini caused by the insertions, are no longer bound by AtRaptor1B, no longer phosphorylated by AtTOR, or no longer able to activate transcription of floral repressors.
Finally, these results provide a cautionary tale. RT-PCR performed using primers which span an insertion site may not be sufficient to conclude that all activity of a protein of interest is abolished.
Conclusion
TOR is a major regulator of cell growth in eukaryotes, but little is known about its downstream effectors in plants. This work shows that AML1 binds AtRaptor1B, and suggests that the AML protein family may be phosphorylated by AtTOR in an AtRaptor1B-mediated interaction. The interaction with AtRaptor1B implicates AML1 as a downstream effector of AtTOR kinase signaling, and provides insight into the mode of action of this critical growth regulator.
Methods
Generating the two-hybrid constructs
AML1 was cloned via RT-PCR. The cDNA template was reverse-transcribed using Omniscript (Qiagen) from RNA extracted from bulk shoot tissue using Trizol Reagent (Invitrogen). Restriction sites NcoI and XmaI/SmaI were added to the 5' and 3' ends of the ORF and of all smaller AML1 fragments via PCR using ExTaq high-fidelity polymerase (Takara). An EST clone (RZL03b06) tagging AtRaptor1B was obtained from Kazusa DNA institute and sequenced. Restriction sites NcoI and EcoRI were added at the 5' and 3' ends of the ORF via PCR. pGBKT7 and pGADT7 are distributed by BDBiosciences.
Yeast two-hybrid assay
AH109 cells (leu2-trp1-ade2-his3-) were grown in YEPDA liquid plates or on YEPDA plates with 17 g/L Agar-Y (Bio101 Systems). Cells were transformed using the Yeastmaker Yeast Transformation System2 (BD Biosciences) and plated on medium lacking the appropriate macronutrients (Bio101 Systems). Colonies were observed 3–7 days after transformation.
Genotyping of insertion alleles
DNA from lines harboring insertion alleles was extracted using the alkaline boiling method [32]. Provisional homozygotes were confirmed via a second extraction using the C-TAB DNA extraction protocol. PCR to assay for wild-type and insertion alleles was performed in 20 uL volumes using ExTaq polymerase and buffers and the following cycling parameters: 94°C, 15 seconds; 61°C, 30 seconds; 72°C, 2 minutes; 35 cycles. Genotyping primers were as follows: AML1-5sm 5'atagaaggaaacaaaaaggaaaggaggaa3'; AML1-3sm 5'tagcatatcacttccctgtagccgcactg3'; AML2-5sm 5'attgctctgtctctgatgatgttttgtcg3'; AML2-3sm 5'gcagcaatatttctaaagcatcgggttca3'; AML3-5sm 5'ctttagttccctctttcctctgctgtgat3'; AML3-3sm 5'ctgccaagaacgggaaaacaaacataaa3'; AML4-5sm 5'ttgcaagcggtagtccatataaatcctc3'; AML4-3sm 5'atgctaccgggagaacctaagtgaaatc3'; AML5-5sm 5'tctttagccacatcaatcattctcatcct3'; AML5-3sm 5'atcagcgtcaagttccattcctcctccac3'; JL-202 5'cattttataataacgctgcggacatctac3'; JL-270 5'tttctccatattgaccatcatactcattg3'; pROC-737 5'gggaattcactggccgtcgttttacaa3'. The wild-type loci were assayed with the above pairs. The insert was assayed using the following pairs: AML1-5sm or AML1-3sm with JL-270 or JL-202, AML2-5sm with pROC-737, AML3-5sm with JL-270 or JL-202, AML4-3sm with JL-270 or JL-202, AML5-5sm with JL-202.
Insertions in the AML1 and AML5 loci were obtained from the University of Wisconsin alpha collection using their described protocol and are in the Wassiljewskia (Ws) ecotype background. The AML1 insertion was found in pool CSJ8-46-H35. The AML5 insertion was found in pool CSJ1091-H45. Insertions in the AML3 and AML4 loci were obtained from the University of Wisconsin Basta collection and are in the Ws background. The AML3 insertion was found in pool 67-6-F. The AML4 insertion was found in pool 18-2-H. The insertion in AML2 was obtained from the line 029713 from the Salk collection and is in the Columbia (Col-0) ecotype background. Aside from regions genetically linked to any of the insertion loci, the AML quintuple insertion allele homozygote lines are in the genomic background of a Col-0 × WS F3 individual once backcrossed to the WS background.
RNA extraction, RT-PCR
RNA extraction was performed using TRIzol™ Reagent (Invitrogen) essentially according to manufacturer's instructions. Total RNA was treated with DNA-free™ DNase (Ambion) and reverse transcribed using Omniscript reverse transcriptase (Qiagen) with an oligo-dT primer. Primer pairs spanning the insertion site for RT-PCR on all five lines are as follows: AML1 117–138 5'gtgatggatgcgattggataga3', AML1 556-534rc 5'attgtggcttcagctggtaactt3'; AML2 86–109 5'tttgcttctccgattctcttcctt3', AML2 456-435rc 5'agcatcgggttcaacatcttcc3'; AML3 548–568 5'gtagcggaggaggtcttgaat3', AML3 1060-1039rc 5'tctccttgatctcgccataaac3'; AML4 1932–1955 5'aagcggtagtccatataaatcctc3', AML4 2944-2927rc 5'tcccctgaatccgaccat3'; AML5 104–124 5'cgtgatcatcgtcggtgttgg3', AML5 1047-1024rc 5'ctcgacgaatttgtgatgcctctta3'. Reactions were performed using Takara ExTaq and 35 cycles of 94°C for 30 sec, 58°C annealing (AML1,2,4) or 60°C annealing (AML3,5) for 30 sec, and 72°C for one minute.
Primer pairs amplifying a region 3' of the insertion site for RT-PCR on AML1, AML2, AML3 and AML5 insertion homozygotes are as follows: AML1 +1635 5'aggctctcgccgccctatta3', AML1 -2466 5'cgttgccaccttctcgctatt3'; AML2 +1779 5'accggggaacagtagtgaac3', AML2 -2107 5'ctgtcggcaagcatagaaag3'; AML3 +1756 5'tctggcctgctgctacaatgg3', AML3 -2326 5'cgccgacaagaagatgagaaaac3'; AML5 +1268 5'gcaacggcttccaacagtca3', AML5 -1869 5'acgaggcctaccattttcatacaa3'. All reactions were performed with a 59°C annealing temperature.
Authors' contributions
GHA conceived of the study and performed all experimental manipulations. MRH provided guidance and arranged funding for the project. GHA and MRH drafted the manuscript, and both authors approve the final manuscript.
Acknowledgements
This work was funded by a fellowship for the NSF/DOE/USDA Training Group in Molecular Mechanisms of Plant Processes to G.H.A. and USDA Hatch Program and DOE Energy Biosciences (DE-F602-89ER14030) grants to M.R.H. Insertion alleles were generated by Michael Sussman and the University of Wisconsin Arabidopsis Knockout Facility, and by Joseph Ecker and the SIGnAL lab at the Salk Institute. Lines were distributed by the Arabidopsis Biological Resource Center.
Figures and Tables
Figure 1 AML1 interacts with AtRaptor1B in a yeast two-hybrid assay. A. Schematic diagram of AML1 fragments cloned into pGADT7. The AML1 RNA Recognition Motifs (RRMs) are indicated. Fragments interacting with AtRaptor1B are labeled in red. B. Yeast two-hybrid results. Numbers on each plate refer to the pGAD construct in A. For each plate, the set of six cultures on the top half were co-transformed with pGBK:Raptor; the set of six cultures on the bottom half were co-transformed with pGBKT7 vector. The plate at left lacks leucine, tryptophan, histidine and adenine and selects for a protein-protein interaction. The control plate at right lacks leucine and tryptophan, and indicates co-transformation of the yeast cells with both a pGAD and a pGBK plasmid.
Figure 2 The AML1 N-terminus harbors transcriptional activation domain activity. One-hybrid assay on AML1 fragments cloned into pGBK. The plate at left lacks tryptophan and adenine, and growth indicates transcriptional activation domain activity in the tested fragments. The control plate at right lacks adenine, and indicates transformation of the cells with the desired pGBK construct.
Figure 3 AML loci and insertion alleles. The position of the insertion in each locus is indicated by a triangle below the locus picture. The wild-type loci were described previously (17). The thin central line indicates genomic DNA. Solid blue blocks spanning the central line indicate coding exons. Solid blue blocks below the line indicate untranslated regions. T-DNA insertion left borders are indicated with a triangle below the locus. Binding sites for primers used to genotype the wild-type allele of each locus are indicated with arrows. Binding sites for primers used to ascertain the effect of homozygosity at the mutant allele are indicated with triangles tagged by a single hash line if the PCR product spans the insertion site, or a doublet hash line if the PCR product is from a region downstream of the insertion site.
Figure 4 AML mutant allele characterization. i) PCR product to identify the wild-type allele of each AML locus. ii) PCR product to identify the mutant allele of each AML locus. iii) PCR product to assay for accumulation of cDNA from transcripts transcribed across the insertion site. iv) PCR product to assay for accumulation of cDNA from transcripts transcribed from the AML locus downstream of the insertion site. AML4 was excluded from this assay because the mutant allele is disrupted in the center of its coding region. Q6 refers to genomic DNA or cDNA template extracted from quintuple insertion allele homozygotes; Ws refers to genomic DNA or cDNA template extracted from wild-type (Wassilijewskia ecotype) plants.
Figure 5 AML insertion mutants bolt early. A. Wassilijewskia (Ws) and AML1 insertion homozygotes (AML1-/-) grown under long days. B. Bolting time and number of rosette leaves at time of bolting for AML single insertion and higher order insertion mutants. Plants were grown under 16 hour days. Ws and Columbia (Col 0) ecotypes are the two points in the top left of the graph; the cluster of points at the lower right are all AML-/- mutants.
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| 15720729 | PMC553980 | CC BY | 2021-01-04 16:03:52 | no | BMC Plant Biol. 2005 Feb 21; 5:2 | utf-8 | BMC Plant Biol | 2,005 | 10.1186/1471-2229-5-2 | oa_comm |
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BMC Med Res MethodolBMC Medical Research Methodology1471-2288BioMed Central London 1471-2288-5-81571591610.1186/1471-2288-5-8Research ArticleEvaluating the role of quality assessment of primary studies in systematic reviews of cancer practice guidelines Brouwers Melissa C [email protected] Mary E [email protected] Manya L [email protected] Steve E [email protected] Alejandro R [email protected] George P [email protected] Department of Clinical Epidemiology and Biostatistics, McMaster University, Hamilton, Canada2 Program in Evidence-based Care, Cancer Care Ontario, Hamilton, Canada3 University of Toronto and University Health Network, Toronto, Canada4 Hamilton Regional Cancer Centre, Hamilton, Canada2005 16 2 2005 5 8 8 30 8 2004 16 2 2005 Copyright © 2005 Brouwers et al; licensee BioMed Central Ltd.2005Brouwers et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The purpose of this study was to evaluate the role of study quality assessment of primary studies in cancer practice guidelines.
Methods
Reliable and valid study quality assessment scales were sought and applied to published reports of trials included in systematic reviews of cancer guidelines. Sensitivity analyses were performed to evaluate the relationship between quality scores and pooled odds ratios (OR) for mortality and need for blood transfusion.
Results
Results found that that whether trials were classified as high or low quality depended on the scale used to assess them. Although the results of the sensitivity analyses found some variation in the ORs observed, the confidence intervals (CIs) of the pooled effects from each of the analyses of high quality trials overlapped with the CI of the pooled odds of all trials. Quality score was not predictive of pooled ORs studied here.
Conclusions
Had sensitivity analyses based on study quality been conducted prospectively, it is highly unlikely that different conclusions would have been found or that different clinical recommendations would have emerged in the guidelines.
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Background
Quality assessment of trials included in systematic reviews of evidence is a resource intensive and scientifically controversial endeavour. On the one hand, the routine use of quality assessment in the development of systematic reviews is encouraged by the Evidence-based Practice Center Program of the Agency for Healthcare Research & Quality (AHRQ) and the Cochrane Collaboration, two well respected groups that coordinate a substantial number of systematic reviews [1-3]. Indeed, West et al released a 2002 evidence report sponsored by the AHRQ comparing and contrasting various systems to rate the strength and quality of research evidence to assist in these activities [4]. Furthermore, many journal editors consider it important to include an assessment of study quality in reports featuring meta-analyses [3].
The concept of incorporating study quality assessment into systematic review methodology has also found empirical support. There is evidence that studies of lower methodological quality tend to report larger treatment effects than high quality studies [5-7]. For example, Moher and his colleagues found a 34% greater estimate of treatment effect for low quality versus high quality trials and a 37% greater estimate of treatment effect for inadequately concealed versus adequately concealed trials associated with reviews addressing a variety of clinical conditions [5]. Similar bias was found by Schultz and his colleagues [6] in their analysis of trials included in the Cochrane Collaboration's Childbirth and Pregnancy reviews. In addition, Colditz and colleagues found that nonrandomized and open studies were more likely to produce positive treatment effects than randomized and double-blinded studies [7].
Although this seminal work yields compelling results, these findings are not universal and the issue is not without detractors [8-14]. Some studies have found no reliable relationship between quality score and effect size [10-12] and another has found that low study quality was associated with diminished effect sizes [13]. Further, Juni et al [14] found the relationship between study quality and effect size depended on the scale used in the assessment.
Together these results suggest the study quality issue is controversial and that the merits of this methodological step in systematic review requires thoughtful analysis. Indeed, West et al conclude with recommendations advocating for research dedicated to comparing quality rating systems and the role of quality assessment within individual clinical contexts and for studies targeted at determining specific quality factors that make a difference in final quality scores [4].
The Practice Guidelines Initiative of the Cancer Care Ontario's Program in Evidence-based Care (PEBC) uses the Guidelines Development Cycle to create cancer practice guidelines comprised of a systematic review of the research literature, an interpretation and consensus of the evidence by members of the guideline development team, clinical recommendations informed by the evidence, and an external review process by Ontario clinicians [15-19]. We face the challenge of balancing scientific rigour and the timely production of guideline documents in an environment defined by limited financial and human resources. Hence, we try to approach our methodological decisions with a critical scientific and practical eye. We took note of the growing controversy in the study quality assessment literature and conducted an evaluation, reported below, to evaluate the benefits of assessing the quality of each study included in our systematic reviews. Our overall objective was to decide whether to augment our current practice of simply describing study characteristics to also incorporate study quality assessment as a routine formal component of the guideline development methodology. The evaluation was conducted in three steps, each of which was designed to address three specific issues:
1. What valid and reliable quality assessment instrument would be most appropriate for our context?
2. How is study quality currently being used in published systematic reviews of cancer trials and what is the relationship between effect size and study quality in this disease area?
3. What impact would study quality assessment have on the clinical recommendations made in evidence-based practice guidelines developed by the PEBC?
Methods
Search for a valid and reliable quality assessment tool for the PEBC context
For a comprehensive review of the strengths and weaknesses of quality assessment instruments, readers are referred to the 2002 West et al. evidence report commissioned by the AHRQ [4]. For our study, which began before the release of this report, we used components of Moher et al's definition of study quality that are related to internal validity (i.e., design, conduct and analysis) [20] and updated his 1992 published reports of check lists and scales used to measure the phenomenon [21]. We searched the Medline database using the following search strategy: (quality adj rat:).tw OR (quality adj assess:).tw. OR (quality adj scale:).tw. OR (quality adj checklist:).tw. AND randomized controlled trials.sh. OR clinical trial:.tw. OR random:.tw. Reference lists of reviews were scanned for additional citations.
Systematic review of the oncology literature on study quality
To locate systematic reviews on oncology topics, the strategy suggested by Moher et al for finding systematic reviews [3] was combined with the terms "neoplasms.sh. OR cancer.tw. OR carcinoma.tw" to search the Medline, CINAHL, and Cancerlit databases. To ascertain if the authors assessed the quality of the studies included in the systematic reviews, the search was narrowed to include the terms [(quality adj rat:).tw OR (quality adj assess:).tw. OR (quality adj scale:).tw. OR (quality adj checklist:).tw. OR (study adj quality).tw.]. Textwords were used to search the Cochrane Library for systematic reviews on oncology topics. Systematic reviews that included analyses exploring the relationship between study quality (using any assessment instrument, not just validated tools that met our criteria as described above) and effect size were examined. Because survival following cancer treatment is commonly used as the primary outcome variable in our practice guidelines, this variable was selected as the primary outcome measure of interest.
Impact of study quality on PEBC practice guidelines
The validated scales were applied by two methodologists (MJ and MC) to studies reported in any our practice guidelines that included a pooled analysis based on at least ten randomized trials related to the main guideline question. Intraclass correlation coefficients with 95% confidence intervals (CI) were calculated using a random sample of the RCTs to assess inter-rater reliability, one coefficient calculated for each of the scales used. Because of budgetary limitations for staff time, the analysis was conducted on 18 randomly selected studies rather on the whole group of articles. This is a methodological limitation as fewer studies result in larger confidence intervals and less precise estimates. This may account for the difference in reliability ratings we found for the Sindu scale compared to published norms (see Table 1).
Table 1 Quality assessment tools with published validity and reliability data – information reported by the scale developers
Instrument Description Item Generation Validity Inter-Rater Reliability Application in Study
Jadad et al (22) number of items: 3 or 6
scoring for 3 item: 0, 1, or 2 for two items 0 or 1 for 1 item range 0 to 5
scoring for 6 item: 0, 1, or 2 for two items 0 or 1 for 4 items range 0 to 8
cut offs not specified for either version consensus among 6 judges (pain management &/or instrument development)
pretested with 3 raters on 13 clinical trial reports 3 groups of pain studies were identified a priori:
• studies rated as excellent by experts (n = 7); overall score = 3.4
• studies rated as poor by experts (n = 6); overall score = 0.7
• a random selection of RCTs from a MEDLINE search (n = 23); overall score = 2.7 ICC = 0.66 (95% CI: 0.53, 0.79)
14 raters 3-item and 6-item versions in their entirety
Sindhu et al (24) number of items: 53
scoring: items weighted from 1 to 10 range 0 – 100
cut offs not specified 8-member Delphi panel (clinicians and methodologists) Compared with the Chalmers scale for five studies of non-pharmacologic nursing interventions for pain management; r = 0.94 (Pearson) 11 studies considered for a published meta-analysis on hypertension were evaluated:
• 9 included studies scored 73–93%;
• 2 rejected studies scored below 70% ICC = 0.93
2 raters 12 of 53 items eliminated because of difficulty applying to cancer studies
41 items related to 12 domains range 0 – 79.5
Downs & Black (25) number of items: 27
scoring 0 or 1 for 25 items
1 or 2 for 1 item
0 to 5 for 1 item
cut offs not specified based on epidemiological principles, reviews of study designs and existing checklists
pilot tested by 2 epidemiologists rating 20 studies Compared with the Standards of Reporting Trials Group checklist scale for 10 RCTs on surgery for stress incontinence; r = 0.90 (Spearman) Spearman r = 0.75
2 raters used 13 internal validity items only
items not related to our definition of quality eliminated (10 reporting items, 3 external validity items, 1 power item) range 1 to 13
RCT, randomized controlled trial; ICC, intraclass correlation coefficient; CI, confidence interval
To assess the impact of study quality on effect sizes, sensitivity analyses were conducted for the meta-analysis from each guideline report. For each scale, studies were divided into two groups (low quality and high quality) based on total quality score. Where the scale developer suggested a cut-off point for low versus high quality, this was used. Where no cut point was specified, the observed median study quality score was used as the dividing point between low and high quality. Meta-analyses were repeated with the high quality studies. Because there would never be a situation in which guideline developers would consider low quality studies only, a meta-analysis using this sample of the studies was not conducted.
Results
Valid and reliable quality assessment tools for our context
Four scales meeting our criteria were found; two instruments, Jadad et al [22] and Cho & Bero [23], were originally uncovered in the Moher review [21] and two instruments, Sindhu et al [24] and Downs & Black [25], were uncovered in our update of this review. While none of these scales were developed in the oncology setting, they all purport to be generic assessment tools that measure the quality of specific study designs regardless of clinical condition reflected in the design. The procedures undertaken to create the instruments followed appropriate methodological processes for questionnaire design. In addition, while a number of additional scales and checklists emerged from our search, validity and reliability data were not reported. Because our practice guidelines are based primarily on evidence from randomized trials, we decided to reserve to employ the scales that focused specifically on RCTs. As such, the Cho & Bero scale, which is applicable to a range of study designs, was not employed here but will be considered at a later date when we have a portfolio of diverse study designs. The characteristics of the instruments included in our study are summarized in Table 1 and 2 and detailed descriptions and comparisons can be found in West et al. [4].
Table 2 Quality assessment tools – comparison of key quality constructs
Jadad Sindhu Downs & Black
Randomization Max.= 2 points out of total score of 5
1. Was the study described as randomized? (1 point for yes) Give an additional point if the method to generate the sequence of randomization was described and it was appropriate. Deduct a point if it was inappropriate. Max. = 10 points out of total score of 100
2. Have the patients been randomly allocated to treatment groups? (1 point for yes)
If yes: i) Is the method of randomization explicitly detailed? (1.5 points)
ii) Is it valid? i.e. Are there any threats to internal validity re: designation of subjects to groups? (2.5 points)
iii) Is patient consent sought prior to randomization? (2.5 points)
iv) Is it secure and 'blind' to the assessors? (2.5 points) Max. = 2 points out of total score of 13
23. Were the study subjects randomised to intervention groups? Studies which state that subjects were randomised should be answered yes except where method of randomisation would not ensure random allocation. For example alternate allocation would score no... (1 point for yes)
24. Was the randomised intervention assignment concealed from both patients and health care staff until recruitment was complete and irrevocable? (1 point)
Blinding Max. = 2 points out of total score of 5
2. Was the study described as double blind? (1 point)
Give an additional point if the method of double blinding was described and it was appropriate. Deduct a point if it was inappropriate. Max. = 5 points out of total score of 100
9. Is the assessment blind?
a) If yes, who is blinded:
i) patients? (2 points)
ii) therapist/carer? (2 points)
iii)assessor/data collector? (1 point) b) If no, i) are reasons given as to why assessment is not blind? (2 points)
ii) Is there discussion of bias resulting from non-blind assessment? (3 points) Max. = 2 points out of total score of 13
14. Was an attempt made to blind study subjects to the intervention they have received? (1 point)
15. Was an attempt made to blind those measuring the main outcomes of the intervention? (1 point)
Withdrawals and dropouts Intention to treat analyasis Max.= 1 point out of total score of 5
3. Was there a description of withdrawals and dropouts? 1 point) Max. = 12 points out of total score of 100
6. Has an 'intention-to-treat analysis been performed? i.e. everyone randomized is retained in the study; everyone randomized is included in the final analysis; and no selective dropouts. (8 points)
b) if not, is it clear what was done, its justification and impact on bias? (8 points)
11. Loss to follow-up
a) (<) 20% loss to follow up (2 points)
b) <10% loss to follow-up (2 points) Max. = 2 points out of total score of 13
25. Was there adequate adjustment for confounding in the analyses from which the main findings were drawn? The questions should be answered no for trials if: the main conclusions of the study were based on analyses of treatment rather than intention to treat;.... (1 point)
26. Were losses of patients to follow-up taken into account? If the number of patients lost to follow-up are not reported, the question should be answered unable to determine. If the proportion lost to follow-up was too small to affect the main findings, the question should be answered yes? (1 point)
Appropriate statistical analysis no items Max. = 6 points out of total score of 100
7. Statistical analysis
a) Is the analysis appropriate/specific to the hypothesis and to the data? (1 point)
b) Is the analysis adequately described? (1 point)
c) Do the statistical assumptions hold? (1 point)
d) Are adequate summary statistics provided at:
i) baseline? (0.5 point)
ii) outcome? (05. point)
e) Is the overall significance level reported protected against inflation due to multiple testing? (1 point)
f) If confounders exist, are they adjusted for via multivariate techniques even if differences between groups are not significant? (1 point) Max. = 4 points out of total score of 13
16. If an of the results of the study were based in 'data dredging', was this made clear? (1 point)
17. Do the analyses adjust for different lengths of follow-up of patients? (1 point)
18. Were the statistical tests used to assess the main outcomes appropriate? (1 point)
25. Was there adequate adjustment for confounding in the analyses from which the main findings were drawn? (1 point)
Compliance with treatment no items Max. = 4 points out of total score of 100
14. Has patient compliance been assessed? (4 points) Max. = 1 point out of total score of 13
19. Was compliance with the interventions reliable? (1 point)
Outcome Measures no items Max. = 14 points out of total score of 100
3. Measurement of outcomes
a) Is the form of measurement stated? (3 points)
b) Has an attempt been made to validate the measures? (3 points)
c) Has an attempt been made to test the reliability of the measures? (2 points)
d) Is the outcome objective as compared to subjective? (2 points)
12. Outcomes
a) How many outcomes are used (1/2 point for each, to a max. of 2)
b) Are they relevant? (1 point)
c) Are they independent? (1 point) Max. = 1 point out of total score of 13
20. Were the main outcome measures used accurate (valid and reliable)? (1 point)
The relationship between study quality and effect size in the oncology literature
The literature review located 32 published systematic reviews on oncology-related topics that included some measure of study quality. Five of the reviews examined changes in pooled estimates of effect size of mortality rates when meta-analysis was restricted to high-quality randomized trials [26-30]. As shown in Table 3, four of the five reviews found somewhat larger effects (i.e., larger differences between experimental and control groups) with high-quality trials compared to all trials [26-29]. With one exception, the statistical relevance of the differences between the groups (i.e., significant differences or no significant differences) remained the same regardless of the number of trials included. Specifically, two of the reviews did not detect a statistically significant difference in survival between groups when all studies were included or when the meta-analysis was restricted to high-quality studies [26,29]. For one data set, the meta-analysis was repeated with study quality ratings used as weights [29]; there was still no significant difference between experimental and control groups. Two analyses detected significant differences between experimental and control treatments with analysis of all trials and when the analysis was restricted to high-quality trials [27,28]. In the fifth review, a significant difference between experimental and control interventions was detected when all trials were synthesized that became only marginally significant (p < .07) when the meta-analysis was adjusted for study quality [30].
Table 3 Systematic reviews of randomized oncology trials with sensitivity analysis exploring the relationship between study quality scores and effect sizes for mortality
Systematic Review Interventions Outcome Quality Scale Definition of High Quality Effect Size (95% confidence interval)
All Studies (# studies) High Quality (# studies)
McAlister et al, 1998 (26) allogenic blood transfusion versus autologous or leucocyte-depleted allogenic blood during cancer surgery relative risk of death* Jadad (22) score ≥3 out of 5 RR, 0.94 (0.76 to 1.16) (n = 5) RR, 0.84 (0.47 to 1.52) (n = 2)
Caubet et al, 1997 (27) nonsteroidal anti-androgens (plus LHRH or orchiectomy) versus LHRH or orchiectomy alone for advanced prostate cancer relative risk of death* Chalmers (31) score ≥50 % of total possible score RR, 0.81 (0.70 to 0.94) (n = 13) RR, 0.78 (0.66 to 0.92) (n = 4)
Dube et al, 1997 (28) adjuvant chemotherapy versus control for colorectal cancer odds ratio for death* Chalmers (31) score >50 % of total possible score OR, 0.82 (0.77 to 0.89) (n = 29) OR, 0.77 (0.71 to 0.85) (n = 14)
Detsky et al, 1992 (29) total parenteral nutrition versus control in cancer patients undergoing chemotherapy odds ratio for survival** Chalmers (31) score >42 % of total possible score; quality score also used as a weighting factor in meta analysis OR, 0.74 (0.42 to 1.3) (n = 8) OR, 0.69 (0.38 to 1.3) (n = 2) weighted OR, 0.61 (0.23 to 1.6)
Klein et al, 1986 (30) total parenteral nutrition versus control in cancer patients undergoing surgery odds ratio for operative death* Developed specifically for the systematic review quality score used as a weighting factor in meta analysis OR, 0.44 (0.21 to 0.90, p = 0.02) (n = 10) weighted OR not reported but p = 0.07 after weighting for study quality
LHRH, luteinizing hormone-releasing hormone; RR, relative risk; OR, odds ratio
*RR or OR <1.0 indicates fewer deaths in the experimental group than in the control group
** OR <1.0 indicates more deaths in the experimental group than in the control group
Impact of study quality on PEBC practice guidelines
Three of the PEBC practice guidelines included at least 10 RCTs in their systematic reviews of the evidence and were eligible for inclusion in this evaluation [31-33]: concomitant chemotherapy and radiotherapy in squamous cell head and neck cancer (18 trials) [31]; adjuvant therapy for stage II colon cancer following complete resection (11 trials) [32]; and neoadjuvant chemotherapy in locally advanced squamous cell carcinoma of the head and neck (23 trials) [33]. For the latter guideline [33], data could not be reliably reconstructed and is not discussed further.
At the conclusion of our study, we identified a fourth practice guideline which originally did not meet our 10 RCT inclusion criteria, but later did so after it was updated. The guideline focused on the role of erythropoietin (EPO) in the management of cancer patients with non-hematologic malignancies [34]. Unlike the chemotherapy trials included in the practice guidelines described above, which were not placebo-controlled and where the primary outcome was death, one-third of the EPO trials were double blind and all used the need for blood transfusion as the primary outcome. Although by the time this practice guideline emerged as eligible we had identified a preferred scale (see below), we chose to include it here and apply only the preferred scale as a demonstration of its use on a report that had differing characteristics than the chemotherapy topics covered.
Inter-rater reliability
Intraclass correlation coefficients used to established inter-rater reliability were 0.71 for the 3-item Jadad scale (95% CI, 0.38 to 0.88), 0.80 for the 6-item Jadad scale (95% CI, 0.54 to 0.92), 0.62 for the Sindhu scale (95% CI, 0.24 to 0.84), and 0.63 for the Downs & Black Scale (95% CI, 0.25 to 0.84). Disagreements were resolved by consensus. Where consensus could not be reached, a third rater (MB) assessed the items and provided the tiebreaker score.
Application of quality scales to primary studies informing practice guidelines
While the total quality scores emerging from each of the different scales did all significantly correlate with one another (range r = .35 to r = .73), there was considerable variation in the classification of studies as high quality or low quality as a function of the scale that was applied (Table 4). For example, of the 11 comparisons from 11 trials comprising the stage II colon cancer review, the application of the Jadad 3-item, the Jadad 6-item, the Sindhu, and Downs & Black scales yielded 0, 8, 9, and 6 of these as high quality, respectively. The 6 studies categorized as high quality using the Downs & Black tool were also categorized as high quality when the Jadad 6-item and Sindhu scales were applied. Similarly, the 8 studies categorized as high by the Jadad 6-item were also categorized as high quality by the Sindhu scale.
Table 4 Meta-analysis of all trials and high-quality trials from evidence-based practice guidelines
Practice Guideline
Colon Cancer Adjuvant chemotherapy Outcome: Mortality (32) Head & Neck Concomitant therapy Outcome: Mortality (31) Systemic Therapy erythropoietin Outcome: need for blood transfusion (34)
All Studies # studies (# comparisons) 11 (11) 18 (20) 15 (15)
OR (CI) 0.82 (0.62 – 1.09) 0.62 (0.54 – 0.72) 0.57 (0.47–0.70)
High Quality Studies By Assessment Scale Criteria Jadad (3-item) range = 0–5 criteria >2 # studies (# comparisons) 0 2 (2) not applied
OR (CI) - 0.54 (0.34 – 0.86)
Jadad (6-item) range = 0–8 criteria >3 # studies (# comparisons) 8 (8) 13 (14) 9 (9)
OR (CI) 0.88 (0.69–1.13) 0.53 (0.44 – 0.64) 0.57 (0.44–0.72)
Sindhu (41-item) range = 0–79.5 criteria > 44.8 # studies (# comparisons) 9 (9) 11 (12) not applied
OR (CI) 0.86 (0.68–1.09) 0.62 (0.49 – 0.79)
Downs & Black (13-item) range = 0–13 criteria > 7 # studies (# comparisons) 6 (6) 12 (14) not applied
OR (CI) 0.86 (0.59–1.25) 0.56 (0.46 – 0.68)
OR, odds ratio; CI, 95% confidence interval
The 20 comparisons from the 18 trials included in the head and neck concomitant therapy systematic review yielded 2, 14, 12 and 14 high quality studies, respectively, when the Jadad 3-item, the Jadad 6-item, the Sindhu and the Downs & Black scales was used. Although both Jadad 6-item and Downs & Black scales both assessed 14 comparisons to be from high quality studies, only 11 of these 14 studies were the same. For the 12 comparisons from studies categorized as high quality with the Sindhu scale, 10 of these were also rated high quality by both the Jadad 6-item and the Downs & Black scales, the other 2 were rated as high quality by the Jadad 6-item scale only. There was 1 comparison from a study rated as high quality by the Jadad 6-item scale only and two from studies rated as high quality by the Downs & Black scale only.
Impact on pooled estimates of outcome measures
Mortality data (i.e., numbers of deaths and number of patients randomized for each allocation group, abstracted from published trial reports) used for the meta-analysis included in the guideline reports were available for two guidelines and need for blood transfusion data were available for the third [31,32,34]. For each guideline, the pooled odds ratio based on only the high-quality trials was compared with the odds ratio from meta-analysis of all trials that had been included originally in the review (Table 4). For the first guideline [31], there was a significant survival benefit for concomitant chemotherapy and radiotherapy compared with radiotherapy alone for squamous cell head and neck cancer in the meta-analyses that included all studies and the meta-analyses restricted to high quality studies, regardless of quality appraisal tool used. Although the effect size was larger for meta-analysis of high-quality RCTs than for all RCTs (irrespective of quality scale used), the confidence intervals between the two calculations overlapped and the overall conclusions and the recommendations informed by the meta-analysis would have been the same. For the second guideline [32], no survival benefit was detected for adjuvant chemotherapy compared to standard therapy for stage II colon cancer in the meta-analysis of all the studies or the high quality studies, again, regardless of quality appraisal tool used. Although the meta-analysis of the high study quality studies was associated with smaller effect sizes than the calculation including all of the studies, the confidence intervals overlapped and the conclusions and the recommendations would have remained the same.
Only the 6-item Jadad scale was applied to the studies of the EPO guideline and the data were pooled to calculate an overall risk ratio for blood transfusion [34]. The risk ratio for all 15 trials was 0.57 (95% CI, 0.47 to 0.70); for nine trials that scored more than three out of eight on the 6-item Jadad scale, the risk ratio was also 0.57 (95% CI, 0.44 to 0.72) (see Table 4).
Conclusions
Several conclusions can be drawn from this study and review of the literature. First, there are established methods for assessing the quality of randomized controlled trials in which data on adequate reliability and validity were available. West et al uncovered 32 scales, check lists and component systems concerned with evaluating RCTs [4]; more than the four strategies we applied here. Although most (87%) of the instruments found by West included quality domains for which there is an empirical basis, most failed to report the use rigorous methods in their development and most failed to report data regarding reliability and validity, criteria we set for our study. Interestingly, West et al did not include the Jadad 6-item in their analysis [4], although the Jadad 3-item, Downs & Black, and Sindhu tools were reported.
Although all of the scales we used have established reliability and validity estimates, we found that the number of trials categorized as high quality or low quality depended specifically on the scale that was applied. For the head and neck cancer systematic review, the number comparisons from high quality studies ranged from 2 (when the Jadad 3-item scale was applied) to 14 (when the Jadad 6-item or Downs & Black scales were applied). The range for the colon cancer review was 0 to 9. There was also considerable variability regarding the specific quality category in which each trial was placed. These finding are consistent with those of Juni et al [14] and suggest caution should be applied if the intent of quality rating scales is to restrict the number of studies considered in the systematic review; clearly the choice of scale will have a significant impact regarding what studies are eligible. The problem of identifying to which quality category, high or low, studies should be placed is exacerbated by the lack of clear cut-off criteria identified by the instrument developers. This poses a significant methodological limitation to the utility of these instruments. In our study, we chose the median score as the cut-off criteria in situations where none was reported. However, it would be useful for researchers of these tools to continue the development work to create the evidence-base from which valid criteria can be established.
The lack of consistency of study classification from one scale to the next and the lack of clear cut-off criteria for users to employ when measuring quality of studies, presents a challenge to guideline developers when they need to make choice about which instrument they ought to adopt if the choose to adopt an instrument at all. Rather than clear evidence driving our decisions, we considered other features of the instrument in our decision making. Of the rating scales we examined, our preferred choice would be the Jadad 6-item instrument. In contrast to the others considered in this report, this instrument is relatively easy to implement and interpret and good inter-rater reliability was established. Further, although the 3-item version of the Jadad scale is most commonly used, we found the original 6-item version to be more relevant in our clinical context as it provides greater variation in scores. In the cancer discipline, few trials are placebo-controlled and treatment allocation tends to be poorly reported. In contrast to the pain trials which were profiled in the development phase of the Jadad instrument, the majority of the items in the 3-item version (randomization and blinding items) yield no variation in scores in our context and are, therefore, not useful to discriminate among cancer trials. The 6-item version of the scale more aptly differentiates quality across studies and includes more quality domains for which an empirical basis has been established [4].
Another conclusion that can be drawn from this study is that effect size can be related to study quality but that the nature of the relationship in one clinical area may not generalize to another clinical area. Some of the original work examining the role of study quality reinforces the need to be mindful of the variation among studies included in systematic review [5-7]. However, when we examined five published reviews that had conducted sensitivity analyses on pooled mortality data from RCTs, four of these found that larger effect sizes were associated with high-quality studies, not lower quality trials as has been convention, and the absence or presence of statistical differences between the two allocation groups remained constant. One of challenges in examining this work is that the number of high quality studies is limited; there is a reduction in power that subjects the point estimates to bias. Nonetheless, the potential bias of study design and quality requires thoughtful consideration within a given clinical field.
We conducted sensitivity analysis on the systematic reviews comprising the guidelines developed by the PEBC. Only four systematic reviews among 36 eligible practice guidelines included more than 10 trials with data appropriate for pooling; three from which we could extract data. Although there was some variation in the odds ratios observed, the confidence intervals of the pooled effects from each of the analyses of high quality trials overlapped with the confidence intervals of the pooled odds with all of the trials. In no case would the conclusions based on these results be affected by restricting the meta-analysis to only high quality studies; the recommendations remained the same. Had sensitivity analysis based on study quality been conducted prospectively, it is highly unlikely that different conclusions would have been drawn from the systematic review or that different clinical practice guidelines would have been formulated.
Together, these findings lead to our final conclusion that measuring study quality did not translate into altered conclusions from a systematic review in the oncology domain for the outcomes we used here. Thus, at this time we have decided that measuring study quality using a numerical assessment scale for the purposes of sensitivity analysis will not be a routine part of our guideline development program. We will, however, encourage guideline developers to describe the variation among studies and to point out methodologic flaws. In addition, it will be important for us to repeat this study looking at other outcome measures, such as quality of life and adverse effects, as they become more routinely reported in primary cancer research and incorporated into our practice guidelines. Outcomes other than those studied here may be more sensitive to the issues of study quality.
This study highlights a strategy that may be useful for guideline programs to utilize in making decisions regarding the methods employed in their guideline development process. It is important that scientific inquiry be maintained in studying the value and role of study quality assessment rather than accepting its role as convention. By exploring it within a specific clinical context one can identify it's most appropriate application.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
MB, MJ and GB conceived of the project idea and developed the protocol. MB, MJ, and MC conducted by study. SH provided statistical advice and AJ provided conceptual advice. The manuscript was drafted initially by MB and MJ. All authors contributed to the final version submitted for publication.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgement
We would like to thank Cancer Care Ontario and the Ontario Ministry of Health and Long-Term Care for their financial support.
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| 15715916 | PMC553981 | CC BY | 2021-01-04 16:32:51 | no | BMC Med Res Methodol. 2005 Feb 16; 5:8 | utf-8 | BMC Med Res Methodol | 2,005 | 10.1186/1471-2288-5-8 | oa_comm |
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BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-5-211572370910.1186/1471-2407-5-21Research ArticleDocetaxel plus cisplatin is effective for patients with metastatic breast cancer resistant to previous anthracycline treatment: a phase II clinical trial Park Se Hoon [email protected] Eun Kyung [email protected] Soo-Mee [email protected] Dong Bok [email protected] Jae Hoon [email protected] Young Don [email protected] Division of Hematology and Oncology, Internal Medicine, Gachon Medical School Gil Medical Center, Incheon 405–760, Korea2 Department of Breast Surgery, Gachon Medical School Gil Medical Center, Incheon 405–760, Korea2005 22 2 2005 5 21 21 30 6 2004 22 2 2005 Copyright © 2005 Park et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Patients with metastatic breast cancer (MBC) are frequently exposed to high cumulative doses of anthracyclines and are at risk of resistance and cardiotoxicity. This phase II trial evaluated the efficacy and toxicity of docetaxel plus cisplatin, as salvage chemotherapy in patients with MBC resistant to prior anthracyclines.
Methods
Patients with MBC that had progressed after at least one prior chemotherapy regimen containing anthracyclines received docetaxel 75 mg/m2 followed by cisplatin 60 mg/m2 every 3 weeks for a maximum of 6 cycles or until disease progression.
Results
Between Jan 2000 and May 2002, 24 patients with tumors primary resistant and 15 with secondary resistant disease were accrued. All 39 patients were evaluable for safety and 36 for efficacy. The objective response rate was 31% (95% CI, 16–45%) with 3 complete responses. The median time to disease progression was 7 months, and the median overall survival was 23 months (median follow-up of 41 months). Neutropenia was the most frequently observed severe hematologic toxicity (39% of patients), whereas asthenia and nausea were the most common non-hematologic toxicities. No treatment-related death was observed.
Conclusion
In conclusion, we found docetaxel plus cisplatin to be an active and safe chemotherapy regimen for patients with MBC resistant to anthracyclines.
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Background
In the management of breast cancer, anthracycline-based chemotherapy regimens remain standard adjuvant or first-line palliative treatment. Furthermore, some patients cannot be treated with anthracyclines due to impaired cardiac function. It is thus important to identify active, well-tolerated, not anthracycline cross-resistant, salvage regimens [1].
Taxanes (docetaxel and paclitaxel) are currently the most extensively studied new chemotherapeutic agents for metastatic breast cancer (MBC). Single-agent docetaxel has demonstrated significant survival advantages over other recognized regimens in 2 large randomized trials in patients with anthracycline-pretreated MBC [2,3]. Phase II data suggest that docetaxel is the most active agent yet available for the treatment of MBC [4]. Docetaxel also has some activity in paclitaxel-resistant MBC [5].
Cisplatin monotherapy has shown response rate of 9% in salvage settings, and 50% as first-line therapy [6]. Because docetaxel and cisplatin are both active and have different mechanisms of action, this combination may provide additive effect against MBC. Although docetaxel and cisplatin were not actively synergistic in preclinical studies [7,8], this combination chemotherapy has been widely used for treatment of a variety of tumor types [9-13]. Prior phase I studies showed the feasibility of this combination and its activity on different tumors [14]. Considering the single-agent activity of both drugs, their different mechanism of action and distinct toxicity profile, we designed this phase II study, in which the combination was evaluated as a salvage therapy in patients with anthracycline-resistant MBC.
Methods
Eligibility
Eligible patients had measurable or assessable histologically confirmed breast cancer that had progressed after at least one prior chemotherapy regimen containing anthracyclines. All patients had Eastern Cooperative Oncology Group performance status of 2 or lower and adequate bone marrow, hepatic, and renal function, defined as white blood cells ≥4000/μL, absolute neutrophil count ≥1500/μL, platelets ≥100,000/μL, total bilirubin ≤2.0 mg/dL, transaminases ≤3 times the upper normal limit, and serum creatinine ≤1.5 mg/dL. Concurrent radiation or hormonal therapy was not allowed; however, patients with clinically stable metastases of the brain or other sites who had completed radiation therapy were permitted. Patients were eligible regardless of the nature of prior therapy, including high-dose therapy with stem cell support and prior exposure to paclitaxel. Any prior antitumor treatment had been completed at least 1 month before entering this study. Patients were excluded if they had severe comorbid illness, symptomatic peripheral neuropathy of any origin, or a history of anaphylaxis of any type. All patients were provided a thorough explanation of the study, and they signed informed consent prior to enrollment into the study.
Definition of anthracycline-resistance
Patients were classified as primary and secondary anthracycline-resistant. Primary anthracycline-resistance was defined as relapse during or within 12 months after anthracycline-based adjuvant therapy, or disease progression with no intervening response during anthracycline-containing chemotherapy for MBC. Secondary resistance was defined as relapse more than 12 months after receiving adjuvant anthracycline chemotherapy, or disease progression at some time after a documented clinical response to anthracycline-based chemotherapy for MBC.
Pretreatment and follow-up evaluation
Pretreatment evaluation included a complete medical history and physical examination, a computed tomographic (CT) scan of the chest and abdomen, magnetic resonance imaging (MRI) of the brain, and a bone scintigraphy to assess the extent of disease. Follow-up consisted of physical examination, monitoring of toxic effects, a complete blood count, liver function tests, chest radiography, and CT scan as clinically indicated.
Tumor response and toxicity were recorded in accordance with the World Health Organization (WHO) criteria. Responses were assessed every 2 cycles of chemotherapy, and patients were evaluated before each new treatment cycle for toxicities.
Treatment plan
The treatment consisted of docetaxel (Taxotere®, Aventis, Bridgewater, NJ) 75 mg/m2 given by a 1-hour intravenous infusion immediately followed by cisplatin 60 mg/m2 in a 2-hour infusion. Cycles were repeated every 3 weeks if the patient's blood count had returned to normal and non-hematologic toxicities had resolved. Dosage of subsequent cycles was adjusted according to the observed toxicities that developed during the preceding cycle. The treatment continued for a maximum of 6 cycles or until disease progression. All patients received standard supportive care regimen consisting of adequate hydration, dexamethasone, and antiemetic therapy according to the guidelines of the American Society of Clinical Oncology [15].
Statistics
The primary end points of the trial were the efficacy of the therapy, which were measured as objective response rate and time to progression, with a secondary end point of toxicity. All patients who received at least 2 cycles of treatment or who progressed after the first cycle were considered assessable for response. The study was conducted using a single-stage, phase II trial design [16]. Based on the results from previous phase II trials reporting response rates of about 30% in patients with anthracycline-resistant MBC, we selected a 50% target response probability and a 30% null response probability, with type I and type II error set at 10%. The number of patients required was 39.
Statistical calculations were performed using SPSS software, version 11.5 (SPSS, Inc, Chicago, IL). Comparisons were performed using chi-square test and survival curves were generated using Kaplan-Meier method. Results were considered significant at the P = .05.
Results
Patients characteristics
Between Jan 2000 and May 2002, 24 patients with tumors primary resistant and 15 with secondary resistant disease were accrued. Among 39 patients assessable for safety, 3 were not evaluable for response due to early discontinuation of treatment.
Baseline characteristics of the eligible patients are listed in Table 1. Median age was 51 years (range, 41–64) and median performance status was 1. Thirty-three patients (85%) had visceral metastases as the dominant site of disease. All patients had received prior anthracycline-based chemotherapy with a median number of prior chemotherapeutic regimens of 2 (range, 1–3). Three patients (8%) had exposed to paclitaxel in their previous chemotherapy regimen.
Efficacy
According to the "intent-to-treat" analysis, the objective response rate was 31% (95% confidence interval [CI], 16–45%). There were 3 complete responses, 12 partial responses and 15 patients had stable disease. The three patients who obtained a complete response had non-visceral metastatic pattern (neck lymph nodes and/or soft tissue lesions). Most patients who achieved a response did so at the end of the third cycle (median time to response, 2.2 months; 95% CI, 2.1–2.4 months).
At a median follow-up of 40.5 months (95% CI, 36.4–44.6 months), the median time to progression was 6.6 months (95% CI, 3.9–9.4 months) and the median overall survival was 22.9 months (95% CI, 17.2–28.5 months). The time to progression in responders were 16.7 months (P < 0.0001). The Kaplan-Meier method was used to estimate overall survival and time to progression, as shown in Figure 1. At the time of present analyses, 21 patients (54%) died.
We observed no significant differences in overall response rate or survival between primary and secondary anthracycline-resistant groups. The overall survival and time to progression were higher in patients with objective response (P < 0.0001 and P = 0.05, respectively).
Toxicity
Patients received a median of 6 cycles (range, 1–6) of docetaxel plus cisplatin. Relative dose intensities were 84% (95% CI, 73–96%) and 86% (95% CI, 75–97%) for docetaxel and cisplatin, respectively. Twenty-four patients (62%) completed 6 cycles of chemotherapy as planned; Nine patients had progression of disease while receiving treatment and 6 patients discontinued treatment because of toxicity. Treatment delay was occurred in 54 cycles (29%).
Toxicity was evaluable in all 39 patients and 189 chemotherapy cycles. Even if the most frequent hematological toxicity was neutropenia (grade 3/4 in 22% of treatment cycles), only 12 episodes of non-fatal febrile neutropenia were observed. The most commonly encountered non-hematologic toxicities were asthenia and nausea. Other toxicities are reported per patient and per cycle in Table 2. Six patients had severe toxicity precluding further treatment. No treatment-related deaths were observed.
Discussion
Single-agent docetaxel has been considered a standard treatment for patients with anthracycline-pretreated MBC for several years. Docetaxel monotherapy often demonstrated response rates of 50% or more in this setting [17,18]. Ahn et al. also reported, in a similar patient population, 36% response rate and 69 weeks median survival with docetaxel 75 mg/m2 every 3 weeks[19].
Platinum-based combinations also have significant activity in previously treated as well as previously untreated patients. In several small clinical trials testing the cisplatin-based regimens, response rates of 50%-83% were reported [20]. The North Central Cancer Treatment Group (NCCTG) evaluated carboplatin combined with paclitaxel as first-line chemotherapy for MBC [21]. The overall response rate was 62% and the median time to progression was 7.3 months. Clearly, platinum-based combinations are very active against MBC, but the relative degree of toxicity compared with other agents limited their use in routine clinical practice. Recently, synergism between platinum and trastuzumab, a novel monoclonal antibody directed against the protein product of the HER2/neu oncogene, awaked interest in the use of cisplatin for breast cancer [6,22].
There are relatively few completed clinical studies involving platinum-based combination chemotherapy for anthracycline-resistant MBC. Spielmann et al. evaluated docetaxel 75 mg/m2 plus cisplatin 80 mg/m2 every 3 weeks in 38 patients with anthracycline-resistant MBC, giving an objective response rate of 36% [23]. Japanese investigators performed a phase II study of docetaxel 60 mg/m2 and cisplatin 80 mg/m2 in patients with anthracycline-pretreated MBC [24]. They reported an overall response rate of 64%. In a phase II study of docetaxel and carboplatin, overall response rate of 61% was achieved in patients with chemotherapy-pretreated MBC [25]. Gelmon et al. combined biweekly paclitaxel with cisplatin and achieved a response rate of 85% with few septic events [26].
We classified "anthracycline-resistant" into primary and secondary. Ando et al. suggested that the status of anthracycline-resistance is important for the prediction of response to second-line treatment with docetaxel [27]. However, we could not observe significant difference in the efficacy of docetaxel plus cisplatin between primary and secondary resistant groups. The different outcomes might be due to the current situation that there have been no standard criteria defining anthracycline-resistance and often intermingled with "anthracycline-pretreated" or "anthracycline-refractory."
This study demonstrated that a combination of docetaxel plus cisplatin in a 3-week cycle was an effective and well-tolerated regimen for patients with anthracycline-resistant MBC. In this study, the use of docetaxel plus cisplatin resulted in an overall response rate of 31% and the median time to progression was 7 months. Significant improvements in the actuarial survival rate and time to progression were observed in the group of patients who achieved objective responses. The main toxicities, gastrointestinal, hematological and asthenia, were manageable with dose adjustment and supportive care. Dose intensities of more than 80% were delivered and 61% of patients completed 6 cycles of chemotherapy, which is considered acceptable and expected. We used the planned dose of cisplatin 60 mg/m2 every 3 weeks, rather than 75 mg/m2 cited in a phase I study [14], because we believed that tolerability of treatment is indispensable in the salvage setting in the management of solid tumors.
With the increasing use of anthracycline-based chemotherapy as adjuvant treatment, as well as first-line chemotherapy against MBC, patients are frequently exposed to high cumulative doses of anthracyclines and are therefore at risk of resistance and cardiotoxicity [28]. This combination of docetaxel with cisplatin may be particularly useful in patients previously treated with anthracyclines (but naïve to either docetaxel or cisplatin). In addition, for patients who have already had cardiac failure and have not received chemotherapy with taxanes as an adjuvant or as first-line treatment, use of docetaxel plus cisplatin is considered a better option. More recently, reports that trastuzumab has a powerful synergistic interaction with docetaxel and with cisplatin [29], have prompted evaluation of the combination of trastuzumab with docetaxel and/or platinum in the treatment of MBC [30,31].
Conclusion
In summary, the combination of docetaxel plus cisplatin is active and safe in patients with anthracycline-resistant MBC. The activity observed in anthracycline-resistant and heavily-pretreated patients suggests relative non-cross-resistance with other drug combinations. Therefore, we hope that this study could result in a prospective trial to determine whether this activity translates into actual improvement in survival and quality of life in patients with anthracycline-resistant MBC.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
SHP collected the data, performed the statistical analysis and drafted the manuscript. EKC, SB, JHL, YDL followed the patients. DBS designed the study, followed the patients and helped with the manuscript. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Figures and Tables
Figure 1 Estimated curves of overall survival and time to progression. At a median follow-up of 40.5 months (95% CI, 36.4–44.6 months), the median time to progression was 6.6 months (95% CI, 3.9–9.4 months) and the median overall survival was 22.9 months (95% CI, 17.2–28.5 months).
Table 1 Patient characteristics
No. %
Patients
Treated 39 100.0
Evaluable for response 36 92.3
Age
Median (years) 51.3
Range 41.2 – 64.1
ECOG performance status
0 3 7.7
1 27 69.2
2 9 23.1
Site(s) of metastatic lesion(s)*
Lung 27 69.2
Liver 9 23.1
Lymph node 15 38.5
Soft tissue 3 7.7
Bone 24 61.5
Brain 3 7.7
Response to prior anthracyclines
Primary resistant 24 61.5
Secondary resistant 15 38.5
Interval from the cessation of last chemotherapy
Median (months) 4.1
Range 1.0 – 11.2
No. of prior chemotherapy regimens
Hormonal therapy (total) 20 51.3
1 9 23.1
2 21 53.8
3 or more 9 23.1
Characteristics of prior chemotherapy†
CMF 24
CAF 15
FEC 9
VE 21
High-dose therapy with stem cell rescue 6
AT 3
* Because patients could have metastases at multiple sites, the total numbers of metastases are greater than the number of patients.
† CMF, cyclophosphamide 600 mg/m2, methotrexate 40 mg/m2, 5-fluorouracil 600 mg/m2, days 1 & 8, every 4 weeks; CAF, cyclophosphamide 500 mg/m2, doxorubicin 50 mg/m2, 5-fluorouracil 500 mg/m2, every 3 weeks; FEC, 5-fluorouracil 600 mg/m2, epirubicin 60 mg/m2, cyclophosphamide 600 mg/m2, every 4 weeks; VE, vinorelbine 25 mg/m2, days 1 & 8, epirubicin 60 mg/m2 day 1, every 3 weeks; AT, doxorubicin 50 mg/m2, paclitaxel 175 mg/m2, every 3 weeks.
Table 2 Toxicity of chemotherapy*
Grade 1,2 Grade 3,4
/ 39 pts / 189 cycles / 39 pts / 189 cycles
Febrile neutropenia 12 (31%) 12 (22%)
Neutropenia 18 (46%) 28 (15%) 15 (39%) 42 (22%)
Thrombocytopenia 18 (46%) 24 (13%) 6 (15%) 9 (5%)
Nausea/vomiting 27 (69%) 42 (22%) 24 (62%) 42 (22%)
Oral mucositis 4 (10%) 5 (3%) 3 (8%) 3 (2%)
Asthenia 12 (31%) 12 (22%) 15 (39%) 24 (13%)
Peripheral neuropathy 6 (15%) 7 (4%) 6 (15%) 9 (5%)
Hearing impairment 3 (8%) 3 (2%)
Renal insufficiency 1 (3%) 1 (1%)
* Others: abdominal pain(1), skin rash(1), fatigue(6)
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| 15723709 | PMC553982 | CC BY | 2021-01-04 16:03:07 | no | BMC Cancer. 2005 Feb 22; 5:21 | utf-8 | BMC Cancer | 2,005 | 10.1186/1471-2407-5-21 | oa_comm |
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BMC Med EthicsBMC Medical Ethics1472-6939BioMed Central London 1472-6939-6-210.1186/1472-6939-6-2Research ArticleEthical challenges in surgery as narrated by practicing surgeons Torjuul Kirsti [email protected] Ann [email protected]ørlie Venke [email protected] Sør-Trøndelag University College, Faculty of Nursing, Trondheim, Norway2 Centre for Medical Ethics, University of Oslo, Norway3 Institute of Nursing and Health Sciences, Faculty of Medicine, University of Oslo, Norway2005 28 2 2005 6 2 2 13 12 2004 28 2 2005 Copyright © 2005 Torjuul et al; licensee BioMed Central Ltd.2005Torjuul et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The aim of this study was to explore the ethical challenges in surgery from the surgeons' point of view and their experience of being in ethically difficult situations.
Methods
Five male and five female surgeons at a university hospital in Norway were interviewed as part of a comprehensive investigation into the narratives of nurses and physicians about being in such situations. The transcribed interview texts were subjected to a phenomenological-hermeneutic interpretation.
Results
No differences in ethical reasoning between male and female surgeons were found. They reasoned in both action and relational ethical perspectives. Surgeons focused on their relationships with patients and colleagues and their moral self in descriptions of the ethical challenges in their work. Dialogue and personal involvement were important in their relationships with patients. The surgeons emphasized the importance of open dialogue, professional recognition, and an inclusive and accepting environment between colleagues.
Conclusion
The surgeons are personally challenged by the existential realities of human life in their relationships with patients. They realized that ethical challenges are an inherent part of performing surgery and of life itself, and say that they have to learn to "live with" these challenges in a way that is confirmed both socially and by their inner moral self. This means accepting their personal and professional limitations, being uncertain, being fallible, and being humble. Living with the ethical challenges of surgery seems to contribute to the surgeons' confidence and vulnerability in their professional identity.
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Background
It is important for surgeons to be and to act in a right and good way towards patients, relatives, and colleagues. Studies have shown, however, that physicians often are in doubt about the best and correct actions to take for the patients in specific situations [1-3]. This question is not only a medical one, but can be understood in both action ethics and relational ethics perspectives. An action ethics perspective concerns questions as to what ought to be done in ethically difficult situations and why. In this perspective, ethics often centres on difficult ethical dilemmas and decision-making. Ethical dilemmas occur when physicians have to choose between at least two alternative and equally difficult courses of actions. Because neither of the alternatives have positive outcomes, they have to choose between two evils [4]. Ethical dilemmas can also be understood as conflicts between different courses of action that result from general and mutually exclusive ethical principles in medicine [5].
A relational ethical perspective means reflecting on the challenges we encounter in our relationships with others and how to fulfil our social roles and obligations in a good way – as a human being, a surgeon, and a colleague. It tries to answer questions such as "How can I adequately meet the challenges that confront me in the relationships in which I am involved in this situation?" [4]. The qualities that make a person a good physician are not only individual traits but they are characteristics of the relationships. One way to describe a good physician or a surgeon is to count the number of characteristics or virtues which are portrayed. According to MacIntyre [6], another way is to speak about a good physician or a surgeon. Narrative ethics focus on what life demands from us in different situations and how we ought to respond to these challenges [4,6,7].
Action and relational ethical perspectives are not interchangeable as surgeons have a dual responsibility for their actions in specific situations as well as their way of being in their relationships [4,7]. Being a good surgeon presupposes both professional competencies based on scientific and clinical knowledge and skills, and being present and showing respect and compassion for patients [4,5,8]. Physicians become involved in the patients' problems both in a professional and moral sense [9]. Traditionally, it has been assumed that compassion can impair competence and that they cannot coexist [10].
Many authors argue that the respect and trust in the physician-patient relationship have eroded in recent years in spite of the physicians' increased therapeutic capabilities [11,12]. Shorter hospital stays and organizational changes in the hospitals are said to lead to surgeons spending more time in the operating theatre and less time talking to patients and establishing a trusting relationship [7,13]. An open and honest dialogue between physicians and patients can be difficult to achieve as medicine becomes more complex, fragmented, episodic, and impersonal, according to Jones [14].
Medical problems are always existential problems too because suffering, anxiety, life, death, and cure involve the core of human existence. Physicians are working with emotionally intense issues and have to accept the possibility of failures, continuing suffering, and death on the part of their patients [12]. Meeting patients who are emotionally distressed or tragically injured can make surgery emotionally challenging [15]. Patients may also elicit emotions of anger and frustration, fear, and despair in physicians [16]. Research suggests that the delivery of bad news can be particularly troubling for both patients and physicians because of the emotional component, and that physicians experience great discomfort in such situations [17]. Physicians have to function at an optimal level despite these challenges. Straume [18] regards the physicians' vulnerability as a result of their overwhelming responsibility and experience that patients' demands often exceed the physicians' ability.
Although most clinicians are aware of the uncertainty and the limitations of medicine and their responsibility to try to reduce the likelihood of error, [19,20] the boundary between medical errors and accidents is not evident [21-23]. Physicians may have difficulty acknowledging personal errors because they can be experienced as personal defeats and thus confirm that physicians are vulnerable [20,24].
Surgeons' relationships with their colleagues have become more important as surgery has changed from relying heavily on the performance of individual surgeons to relying on a team of providers [8,25]. Several studies have found a lack of dialogue and support structures among physicians [26,27]. Physicians are said to have no tradition for open discussions about uncertainty and conflict areas in their practice, nor are they comfortable talking openly about their personal emotions and problems [12,16,24]. Adverse events are said to be generally managed by the conspiracy of silence [20,22].
The vulnerability of patients is emphasized in the literature of medical ethics. Less is written about the vulnerability of the physicians in their relationships with patients, relatives, and colleagues. Being involved may engender feelings of helplessness and vulnerability in the physician. MacLeod [28] argues that physicians have to accept their vulnerability and be able to express and share it in order to be able to live with the tensions. Little [29] suggests that understanding the peculiarities and intensity of the patient-surgeon relationship may help the surgeons understand the vulnerability of both patients and surgeons.
Studies show that physicians seem to experience uncertainty and fallibility in different ways. Less experienced female physicians in paediatric care put on an air of certainty while the more experienced gained a kind of security by allowing themselves to feel uncertain [30]. Female physicians in geriatric care seemed to accept their own vulnerability and fallibility [31]. Experienced male physicians in pediatric care related personal security to their professional experience while the less experienced thought that advances in medical knowledge and ethical guidelines would make them more secure in their work [32]. Henriksen and Hansen [33] found that general practitioners seemed to strive for the ideal of a humble attitude towards problems; being too self-confident was regarded as a threat because of the increased risk of downfall.
Few empirical studies have been found that explore the ethical challenges of surgery from the surgeons' point of view and their experience of being in ethically difficult situations. The present study is part of a comprehensive investigation of ethical reasoning among male and female physicians and nurses within surgical units. The results of this interview study with male and female surgeons will be presented in two articles. The present study describes the surgeons' experiences of being in ethically difficult situations from a relational ethics perspective. The other paper describes the ethical dilemmas as experienced by the surgeons from an action ethical perspective. The results from the interviews with the registered nurses (RNs) working in surgical units are in progress and will be addressed in a third paper.
The aim of this study is to explore the meaning of being in ethically difficult situations in surgery as narrated by male and female surgeons.
Methods
Participants and setting
Five male and five female surgeons working on surgical units at a university hospital in Norway participated in the study. All were experienced and had been working in health care from 9 to 31 years (median = 21.5), and in surgery between 5 to 21 years (median = 13). The surgeons worked full time and were on duty when the interviews were conducted. No individual characteristics will be disclosed in order to guarantee confidentiality. The surgeons gave their informed consent to participate in the study, which was also approved by the 5th Regional Ethics Committee in Norway.
Data collection
Interviews
The interviews were conducted by the first author and lasted from 35 to 75 minutes (median = 55). They were tape recorded and subsequently transcribed verbatim. The interviewees were asked to tell about one or more ethically difficult care situations that they had experienced in their work as surgeons. What constituted an ethically difficult situation was not defined, allowing the interviewees to determine what they considered ethically difficult themselves. The aim of the interviews was to obtain as many rich narratives as possible without interrupting the surgeons' narrative flow and reflection. If the surgeons did not spontaneously reflect on the events they talked about, their reflections were sought. Questions were asked when the interviewer wanted the interviewees to elaborate on their stories or had difficulty understanding the narration. These questions referred to the interviewees' thoughts, feelings, and actions [34]. Field notes were taken during the interview as aids to the interviewer's memory and in order to make it possible to understand the interview text in relation to its context, e.g. arrangements and interruptions. Nonverbal communications that seemed relevant were also noted, such as laughter and long pauses. The transcribed text was compared with the field notes and adjusted if necessary.
Data analysis
Interpretations
The method of interpretation used was inspired by the French philosopher Paul Ricoeur's phenomenological hermeneutics [35], and developed at the University of Tromsø (Norway) and Umeå University (Sweden) and has previously been used by Lindseth et al., [36] Udén et al., [1,13] Søderberg et al., [37] and Sørlie et al. [30,32,38]. This method is useful to elucidate the narratives of people's experiences. The method of interpretation proceeds through three phases, which constitute a dialectical movement between the whole and the parts of the text and between understanding and explanation [35].
Each interview was regarded as a text. However, it was not what the texts said that was a subject matter to be investigated, but rather the focus was on the ethics expressed in them or the essential meaning of ethically good phenomena (or the essential meaning missing in ethically poor phenomena)[39]. First, a naïve reading was made of all the transcribed interviews as a whole to gain a first impression of the surgeons' experiences of being in ethically difficult situations during their clinical work. The repeated naïve reading was made as open-minded as possible, without any deliberate analysis of the text. The naïve reading shows the direction the structural analysis may take. Second, a structural analysis was performed in order to validate or refute the initial understanding obtained from the naïve reading and to explain what the text was saying. The interviews were divided into meaningful parts and patterns, i.e. one sentence, parts of a sentence, or a whole paragraph with a related meaning content. The meaning units were condensed and discussed among all the authors, and themes and subthemes were identified, and presented in 'Results'. Third, a comprehensive understanding was developed, taking into account the authors' pre-understanding, the naïve reading, and the structural analysis (results). The text was read as a whole and interpreted in relation to relevant theories of ethics and results from previous investigations into the meaning of being in ethically difficult care situations [39]. The comprehensive understanding is presented under the heading 'Discussion'.
The analysis was conducted by all the authors and the interpretative agreement was considered satisfactory and to be the most useful understanding of the meaning of the surgeons' experiences of situations of ethical difficulty. The authors' interpretation was not shared or validated with the surgeons. In this study, the focus was on obtaining the meaning of the text, which cannot be validated by the interviewees. A kind of validation is accomplished by the structural analysis as the objective part of the interpretation process [39]. According to Ricoeur [35] a text has multiple but not infinite meanings. One particular phenomenological hermeneutic interpretation should therefore be seen as one of several possible interpretations and as arguments put into ongoing discourses, in this case, about ethical challenges in surgical care.
Results
Several readings of the interview texts revealed that the surgeons told about ethical challenges that confront them in their relationships with patients and colleagues and about their experiences of living with these challenges. They also reported their experiences of ethical dilemmas in surgical practice. No gender differences between the male and female surgeons were found in the analysis of the interviews. The results showed that each surgeon created many and detailed narratives. When the surgeons were asked to narrate their experiences, they did not differentiate between action and relational perspectives in their ethical reasoning. This is an analytical distinction made by the authors in order to structure the results. The authors therefore decided to separate the presentation of the results in two papers: one paper about the surgeons' experiences of being in ethically difficult situations from a relational ethics perspective and the other paper from an action ethical perspective according to the theory presented in the introduction [4]. This paper presents the ethical challenges of surgery as narrated by five male and five female surgeons.
The themes and the subthemes from the structural analysis are shown in Table 1 and presented in the text below. Direct quotations from the interviewers are included to illuminate the results.
Table 1 Themes and subthemes that emerged from the structural analysis of interviews with the surgeons.
Themes Subthemes
Dialogue with patients Openness and honesty
Involvement
Social confirmation Professional recognition
Open dialogue
Self confirmation Responsibility
Uncertainty
Fallibility
Confidence
Humility
Dialogue with patients
Openness and honesty
The surgeons emphasized the importance of dialogue with patients and especially being open and honest about all aspects of their treatment and care. Talking to patients about difficult issues before or after the operation is experienced as an important part of the surgeons' responsibility. Openness and honesty is especially important when surgeons had to tell patients that they have cancer or a fatal disease, when something had gone wrong, or the operation did not turn out as successful as expected. Using frightening words such as "death" and "cancer" is also considered to be part of an open and honest dialogue. The surgeons also felt responsible for "not involving patients with bad news they were not ready to receive, and feeling one's way in what it's right to inform about". They said that if the patients are taken seriously and are talked to in a way that they understand, there is seldom difficulty reaching a mutual understanding about diagnostics and treatment.
The patients are always told the truth about their disease, although it was experienced as an emotional burden for the surgeons to disclose bad news or present difficult decisions to the patients. "It's a burden to tell the patient that we will withdraw all active treatment. You feel a bit guilty; in a way you feel that it's your fault if the treatment does not succeed". They stressed that knowing the patient from previous meetings is important to them, as is having antennae for how the patients experience their life and the present situation. "If I do not manage to achieve what I am trying to do, I always tell [the patients] what the situation is. I never try to conceal anything. That will only torment them".
Talking openly with patients is also important for the surgeons in situations when they are in doubt about the right thing to do. They experienced relief if the patient had an answer to their doubts. They said that patients who are seriously ill usually have thought about life and death issues and have a conception of their condition, and that the question of withholding or withdrawing treatment seldom comes as a surprise to them. Some patients say that they have lived a good life and do not want an operation. Others strongly want an operation even though their prognoses are poor. "We often reach an agreement about ending a treatment that either does not lead to a meaningful life afterwards or leads to a life that would be experienced as a heavy burden".
Involvement
The surgeons said that they become personally involved with their patients, focusing on patients as persons and their quality of life as much as on their medical treatment. Being involved and knowing the patients' background and what they really want in life makes difficult ethical decisions easier to handle. The surgeons explained that caring for the patient can be felt as a personal, emotional burden. "It's not easy when people you have established a relationship with die. The only way to run away from it is to relinquish your responsibility. But that means disassociating yourself from or rejecting the patient. So you have to care, to be involved and to be a human being".
The surgeons are involved in many patients' lives and destinies and said that keeping a certain distance protects their feelings and is a way of caring for themselves. They said that keeping a certain distance is necessary in order to give the patient neutral advice and the most suitable medical treatment. The surgeons feel a responsibility to care in situations where they find it difficult, for instance when they dislike the patients' personality, behaviour, or values. They said they work hard to get involved and care for demanding and non-compliant patients.
Social confirmation
Professional recognition
Ethical challenges are discussed in both formal and informal social arenas among the surgeons. All new patients are presented at the daily morning meeting between all the surgeons, including what had been done to them and why. Only questions and short objections to the patients' diagnostics and treatments are raised at these meetings or shortly after. The surgeons arrange separate meetings to discuss problematic cases. "We assemble the nurses, the anaesthesiologists, the surgeons, and even other specialists like the nephrologists when we have patients who reside a long time in the intensive care unit. In a way we create a meeting-place for the case and discuss whether we should withdraw a life-sustaining treatment or start additional treatment for a kidney failure for instance." The informal running dialogues during the day were equally important for the surgeons. "You have to make the decision yourself, but we always discuss the problem together before difficult decisions are made. The discussions do help and are experienced as mutual support". The surgeons expressed confidence in the consensus that usually is achieved in these discussions. "I know that I would be content to receive the treatment we agree upon myself".
The surgeons emphasized the importance of having a caring relationship with their colleagues. They said that talking together and giving and receiving collegial support is necessary in order to live with the personal responsibility of being in ethically difficult situations and with the emotional burden of decision making. "People say that surgeons are a bit tough and do not talk about feelings, and that may be true. But in my experience we really care for each other. Perhaps we do not go all mushy about our feelings, but we understand when someone is in difficulty. I have experienced receiving good support in such situations. Colleagues contact you and say: "Ok, listen, a couple of years back the same thing happened to me", or: "You must not take this too hard, it could have happened to anybody". That helps".
Personal and emotional support is informally and silently shared among trusted colleagues and great value is attached to it. After having presented a difficult decision about withholding treatment at a morning meeting, one of the surgeons commented: "That same afternoon, four or five of the most experienced surgeons came to me independently, and told me that they thought that what I had done was great. They said that most surgeons were not able to do what I had done. I remember it well because I think it was so well said".
Open dialogue
The surgeons emphasized the importance of "playing with an open hand" and that openness and honesty in the relationships with colleagues presupposes a trusting atmosphere that allows everybody to feel free to voice their opinion and be listened to. They feel that it is important that everyone who is involved in the treatment and care of the patient should have an opportunity to express their opinion and to be heard before any final decision is made. Openness and honesty are particularly important when medical errors occur or when something has gone wrong during an operation. Talking about medical errors or mistakes is considered an opportunity for learning and for improving surgical routines. "You have to have an including and accepting environment that allows you to say that you could have chosen a different solution. If there is no room for you saying something like that, then there will be a tendency to conceal it. We all make mistakes and we all make wrong deliberations and sometimes choose bad solutions. We have to live with that. Therefore it's important that we try to learn from the cases where something [erratic] happens".
The surgeons focused on the necessity of dialogue and cooperation with their colleagues. "We are used to working close together in a team and it makes these difficult situations easier to handle". They found it satisfactory to work in a hospital because "there is always someone you can ask for advice when in doubt". They said they find it easier to talk to patients about difficult treatment options when the question has been discussed with experienced colleagues or senior surgeons first. This is especially important for less experienced physicians. The surgeons also make a contrast between the importance of cooperation with colleagues and the burden of being alone and being responsible decision makers.
Self- confirmation
Responsibility
The surgeons said that they experience ethically difficult situations as an important part of their everyday activities that cannot be separated from the rest. "These situations are a part of our profession that are not necessarily experienced as difficult, but are sometimes unpleasant to be in". They said they have to experience these situations personally and be involved in order to understand them and to learn to live with them.
Some ethically difficult situations are experienced as dramatic and tragic, especially when the surgeons feel the personal responsibility for saving the lives of trauma patients after major accidents. "If you are not able to cope with being in this situation and be responsible, and be the leader of the trauma team trying to save peoples' lives after a serious accident, if you cannot do that but go on wondering if you have done something wrong, then I think you will find something else to do".
The surgeons told about situations where they are alone on duty and responsible for rapid deliberations and decisions in acute and emergency situations as especially challenging. "There are no other times in this job when you feel as lonely as you do in those situations". The decision whether to continue or withdraw treatment from traumatized young patients and children is experienced as especially challenging to make alone. "It's not easy [being alone]. It's the kind of decisions you often ponder about for several days afterwards, also at home. It's the sort of decisions you really try to closely think through, and it often troubles you even after you have made a decision".
The surgeons said they have to learn to live with the unpredictable consequences of their decisions. "The practice of surgery is very specific and you feel more responsible in a way than in other areas of medicine. You have the feeling of being the direct cause when things go wrong, e.g. that you operated at the wrong time, you operated incorrectly or that you should have found the right diagnosis earlier".
Uncertainty
The surgeons spoke about "living with" the inherent uncertainty of surgery and emphasized that they can never be completely sure of the right thing to do in ethically difficult situations. They have to live and work with the uncertainty of the course of the disease, the patients' chances of survival, the risk of serious and fatal complications and the uncertainty of the patients' quality of life after extensive operations. The surgeons said they have to learn to live with the uncertainty of their deliberations and operations as there can be no right answer to ethical challenges, and no criteria to guide them when they make their decisions.
Living with uncertainty is experienced as both frightening and satisfying. The surgeons said that not knowing what to do in an uncertain situation, finding a way, and succeeding in their attempt to restore health or save life is a satisfying aspect of their work. They also commented that they have to live with their doubts and fears of being too active and the risk of knowingly imposing severe complications and a poor quality of life on their patients.
Fallibility
The surgeons said that their aim is to make all patients better. Accepting the limitation of surgery and not being able to cure a particular patient or alleviate his or her suffering is not an easy task for surgeons. They commented that it feels difficult not to be able to or not to be allowed to help patients. Sometimes they feel guilty if the treatment does not succeed, but said that they have to accept the possibility of making mistakes as an essential part of the profession. "You have to face the reality of how things are. What we are dealing with are human beings in marginal situations where things can go wrong. Everybody who is dealing with these things makes errors of deliberation and judgment. It's a part of the game that you have to live with when the margins are so tight. It would not be better if you quit. Your dearly purchased experience will be of use to nobody then".
Confidence
The surgeons said that they are focused on healing and curing and that they try to operate on the patients in most situations. "I believe that surgeons feel that it's good to do something, curing, and saving lives. That is what we have learned to do. We are in a profession that can do many useful things and that is the gratifying part of practicing surgery, – that you can identify a problem and do something about it".
The clinical experience of deliberating and choosing and finding workable solutions in clinical and ethical difficult situations makes the surgeons confident. "Most surgeons are action-oriented. If not, it's almost impossible to practice surgery, because you have to makes decisions all the time and be accountable for your decisions". Receiving social confirmation from patients, relatives, and colleagues when they succeed contributed to the surgeons' experience of confidence in their own decisions and actions. The surgeons also said that having the courage of your convictions and a set of personal ethical values is equally important in order to do a "proper and conscientious job". This means acting according to the patients' best interests and the standards of their profession. Their conscience helps them to decide which action is morally wrong, to make controversial decisions, and to voice personal, professional, and moral opinions to colleagues and patients.
Humility
The surgeons emphasized that although they deal with existential issues of life and death, they do not rule over these human conditions. "The essence is that we are not almighty. We do not save lives, we just postpone death. The only thing that is certain is that we are all going to die. You are just doing repairs and trying to help the patients to live a bit longer. " "Fortunately it's not we who rule over life and death. What we can do means less than you think and that is how it should be. I think it's important that we all acknowledge this and everyone else too".
All the surgeons said they are against active euthanasia. They also commented that a humble attitude in their work helps them acknowledge their personal and professional limitations and to recognize what they cannot and ought not do. "There is a dimension in surgery of being right about having a good reason to operate and being considerate about the benefits of carrying out an operation".
Discussion
The surgeons in this study reasoned in a relational ethics perspective, focusing on dialogue, openness, and involvement in their relationships with patients and colleagues. While the surgeons in the interviews described their experience of being in ethically difficult situations, it seems at the same time that identity was central to their experience of ethics and their enactment of moral agency. The surgeons identified themselves by telling about their ethical experience, expressing the way they are living their identity. By narrating our lived experience, we give meaning to our experiences. We understand ourselves through the stories we tell and live, as well as those told about us and by interpreting them. Personal identity is said to be constructed through the stories we tell about our lives, stories which are in turn shaped by more general institutional, cultural, or national meta-narratives that live within the culture, of surgery, medicine, and society [40-42]. Thus, it seems that the surgeons' identity has a narrative structure and is narratively derived.
Being open and honest about all aspects of patients' treatment and care is experienced as important for the surgeons. This is even true in situations when they have to disclose bad news and tell the patient that they are not able to or did not succeed in their efforts to restore health. Openness of speech is one of the spontaneous expressions of life, designated as "utterances of life" according to the writings of Løgstrup [43]. That openness and honesty are spontaneous expressions means that they are performed in an unconstrained manner and without ulterior motives.
Human life means expressing oneself with the expectation of being met by others according to Løgstrup [43]. Openness and honesty are required in trusting relationships. When openness and honesty of speech are missing, closeness and dishonesty in the relationships result [43]. Sincerity in our relationships with others is a source of satisfaction and means being open and involved and allowing oneself to be moved and impressed by others according to Pahuus [44]. It is also probable that the surgeons' social confirmation increases when the patients meet surgeons who through dialogue turn out to be caring human beings.
Although disclosing bad news to the patient is experienced as an emotional challenge by the surgeons, concealing the truth does not seem to be an option they consider. Concealing the truth from the patient is experienced as difficult. It seems that surgeons need to share their experiences by speaking openly to patients so they can live with their demanding professional life in a satisfactory way. Therefore, honesty and openness are important because the relationships with patients are important for the surgeons' lives.
The surgeons said that it is important not to hide their uncertainty and doubts from themselves or from their patients. They accepted that uncertainty is an inherent part of their profession and realized that they often have to decide in spite of lack of scientific knowledge. They also deal with problems that may have no desirable solutions. Previous studies of surgeons [21], female physicians working in pediatrics, [30] and geriatrics [31] revealed that they are aware of and accept their own uncertainty and fallibility as inevitable in their professional lives.
The surgeons said that they sometimes feel guilty when they have to disclose the bad news to patients that the treatment did not succeed. We feel guilty when we fail to do what is required of us in a situation according to Løgstrup [43], either by not answering the ethical demand of the other or by betraying something valuable in our own life – values and beliefs we hold to be good and right. The surgeons are often in situations in which they have to use their clinical competence and practical wisdom to choose between conflicting values and obligations and being responsible for their decisions and actions. They can never know for certain in a given situation if their actions are absolutely right or wrong. They have to take risks, knowing that they may not succeed in their efforts to give patients a chance of survival.
Feeling guilty is our fundamental ethical condition as human beings according to Pahuus [44] because we have only limited possibilities at hand in particular situations and in life as a whole. Choosing one solution in a situation means excluding a multiple of other desirable possibilities. Thus, feeling guilty is a side effect of trying to give meaning and direction to our lives. Reflecting on our feelings of guilt means having a dialogue with and negotiating with our inner self, thereby evaluating what we hold to be important in our life with and for others. Feeling and being guilty is a heavy burden because human existence also contains that which is final, irreparable and cannot be changed [44]. Feeling guilty can also be understood in relation to what Ricoeur [40] has called an ethics of memory – that people never will or can and must not forget the bad or good things in history. The surgeons cannot and must not forget situations in which they failed to do the right or good thing.
The surgeons said that they experience a relief when the patients give answers to their own uncertainty and doubts about the right or good thing to do. It seems as if the surgeons are relieved when patients want to decide for themselves and take responsibility for their lives, appreciating that they do not hand over this responsibility to the surgeons. The surgeons in this study do not fit into the traditional and stereotypical picture of the paternalistic and authoritarian physicians [45]. On the contrary, the surgeons in this study seem to value being in a dialogue with patients and acknowledging their autonomy. Having to take responsibility for patients' lives is experienced as an ethical challenge and a personal burden in situations when the patients are unable to express their autonomy.
The surgeons emphasized the importance of being personally involved with the patients by focusing on patients as persons and their quality of life as much as their medical treatment. However, they experienced the challenge of finding the balance between involvement and distance in the relationships, between caring for the patients and themselves, and in giving professional and neutral information. Distance based on knowledge, skills, and experience with previous cases and situations is necessary in order to help the patients in a professional manner [43,46]. Personal involvement in a situation is necessary in order to know which fundamental values are threatened in both the patients' and the surgeons' lives. The aim of a dialogue is to abolish the distance between patients and surgeons in order to establish a space for mutual understanding. Involvement does not only mean acting according to the patients' expectations because surgeons are also responsible for their professional conduct as well as their moral integrity. Professional distance means taking the other's perspective in the situation, reflecting, deliberating, and using one's experience of similar cases. The surgeons also learned from their experiences through discussions and dialogue with experienced and trusted colleagues. They may throw new light on situations and increase the surgeons' abilities to deliberate and decide in their patients' best interests [46]. Finding the 'right' balance between involvement and distance in a situation is an expression of what Aristotle [47] calls practical wisdom (phronesis) or the practical knowledge of virtuous persons. A virtuous person is able to find the middle path between two extremes. The right and good thing to do in a particular situation involves "to the right person, to the right extent, at the right time, with the right motive, and in the right way" according to Aristotle [47].
In their narratives, the surgeons focused on cooperation and the relationships with their colleagues. They experienced being listened to and together they seem to create and recreate a collegial environment where they can express their doubts and admit their errors of deliberation and decisions.
The surgeons said that they experience taking difficult decisions alone as a burden. Working in a hospital is experienced as satisfying because they appreciate working together. They seemed to accept that they are mutually dependent on each other and appreciated the support they receive in dialogue with colleagues. Interdependence and exchange is a fundamental way of human lives according to the ethics of Løgstrup [43]. He calls this "display of life" which means that human beings are fundamentally dependent on one another and that we always are giving and receiving something in our relationships with others. This also means that each individual is challenged to take care of that part of the other's life that is in one's own power.
To be seen and listened to by colleagues, be taken seriously, and comforted when something goes wrong is an answer to fundamental human needs. Openness about personal doubts and failures also make the surgeons vulnerable. The interdependency among surgeons in this study is not experienced as a burden, but as mutual support. The surgeons' acceptance and appreciation of their collegial dependency may be an answer to why surgeons are able to stand in ethical difficult situations and live with them in a satisfying way. The dialogue with colleagues reduces the surgeons' doubts, although their doubts cannot and do not disappear.
The surgeons receive social confirmation for being good surgeons through dialogues with patients, relatives, and colleagues. Their experience of being of use to society as a whole also confirms their identity as good surgeons. To be confirmed is to be seen, listened to, and accepted by other persons. A person's identity is dependent on other persons in good or bad relationships according to Ricoeur [40], as well as an inner voice from the person's own consciousness.
The surgeons in this study emphasized the importance of dialogue with patients and colleagues. All identities are socially bestowed, socially sustained, and socially transformed and people make meanings of their actions by participating in communicative contexts. We all need recognition and social confirmation from others in order to construct our identities [41,48]. Thus the self-image of a surgeon can be maintained only in social contexts in which others are willing to recognize him or her in this identity.
There is a close relationship between identity management and values in a society since human beings usually want to be something they find valuable. What constitutes morality is created, recreated and communicated in social relations [41,49]. Narratives are a display of moral identity according to Jordens and Little [42], where the speakers present themselves as experienced, knowledgeable, and ethical. Institutional settings provide the narrative auspices under which identities are articulated. Telling stories is necessary for moral agency as we must be able to account for our actions and the actions of relevant others. We also tell stories as a sort of self-examination by self-exposure. We often find out what we think and who we are by listening to what we say.
The modern self is described as multi-voiced and dialogical and moral action is determined through a process of inner dialogue. To open up to the different voices within and enter into a dialogue, one can either reach consensus with oneself or experience conflict and dissatisfaction with oneself [41,49]. The surgeons' identities are constructed through negotiation with themselves, their patients, and colleagues within a context of social organization. The view of ourselves is shaped by the values of the larger society and what collectively is tacitly deemed to be right and good actions. Conversely, one's conception of how ethical the collective group is arises through one's personal view of what constitutes right and good actions [21,49]. Surgeons attempt to reconcile their relational experiences and their actions with their identity as moral agents as they live and practice relationally with others within the institutional values of their workplace.
The modernization and secularization of western societies emphasize the ideology of individual autonomy and freedom of choice. At the same time, the way in which social structures control and restrict individual performance and freedom becomes less visible. It is not in every person's or profession's power to choose, in other words, act as his/her conscience directs [41]. The surgeons in this study seem to have a wide range of freedom to act in a way that is confirmed both from their patients, colleagues, society and by their inner selves.
According to the surgeons, their choices of actions and ways of being in relationships with patients and colleagues have to be accepted and confirmed by their inner moral selves. They speak about "having the courage of your own conviction", "taking a stand", "having a personal set of ethical values" as important in order to "live with" the ethical challenges of surgery. The surgeons in this study revealed their identities through their narratives about being in ethically difficult situations.
Being a surgeon means having the necessary specialized knowledge, skills, and experience to perform complicated, extensive, and technically advanced operations in principally uncertain and unpredictable situations. This identity is socially sustained through dialogues with patients, relatives, and colleagues. To have an identity as a surgeon is also to understand that one is vulnerable and exposed in these relationships and to know what is at stake in one's own life as well as in the lives of the patients and colleagues.
In this study uncertainty, fallibility, and humility seem to be equally important in the surgeons' identities as are their responsibility and confidence. The social importance and benefit of their work to patients and society is confirmed both socially and by their moral selves. This seems to outweigh their personal uncertainty and vulnerability. Thus, surgeons seem to appreciate their work and have learned to live with, and to a certain extent even appreciate, its most difficult dimensions. The meaning of being a surgeon is to be able to live with the tensions of contradictions, ambivalence, dilemmas, and paradoxes in their practice and to find workable solutions in these situations.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
KT participated in the design of the study, carried out the interviews, participated in the analysis and completed the manuscript.
AN read the interviews, participated in the analysis and helped to draft the manuscript.
VS participated in the design of the study, read the interviews, participated in the analysis and helped to draft the manuscript.
All authors have read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This study was supported by Sør-Trøndelag University College, Faculty of Nursing, Norway. This study is part of a larger ethical project which includes studies from different medical specialties. The authors are grateful to the participants in the study and to Ms Jill Almvang, Fulbright Scholar Marjory Schaffer, University of Oslo and Assistant Professor Stewart Clark, Norwegian University of Science and Technology, Trondheim for the linguistic revision.
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| 15737235 | PMC553983 | CC BY | 2021-01-04 16:31:58 | no | BMC Med Ethics. 2005 Feb 28; 6:2 | utf-8 | BMC Med Ethics | 2,005 | 10.1186/1472-6939-6-2 | oa_comm |
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BMC NeurosciBMC Neuroscience1471-2202BioMed Central London 1471-2202-6-121571590710.1186/1471-2202-6-12Research ArticleStatistical model of natural stimuli predicts edge-like pooling of spatial frequency channels in V2 Hyvärinen Aapo [email protected] Michael [email protected] Patrik O [email protected] HIIT Basic Research Unit and Dept of Computer Science, University of Helsinki, Finland2005 16 2 2005 6 12 12 12 10 2004 16 2 2005 Copyright © 2005 Hyvärinen et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
It has been shown that the classical receptive fields of simple and complex cells in the primary visual cortex emerge from the statistical properties of natural images by forcing the cell responses to be maximally sparse or independent. We investigate how to learn features beyond the primary visual cortex from the statistical properties of modelled complex-cell outputs. In previous work, we showed that a new model, non-negative sparse coding, led to the emergence of features which code for contours of a given spatial frequency band.
Results
We applied ordinary independent component analysis to modelled outputs of complex cells that span different frequency bands. The analysis led to the emergence of features which pool spatially coherent across-frequency activity in the modelled primary visual cortex. Thus, the statistically optimal way of processing complex-cell outputs abandons separate frequency channels, while preserving and even enhancing orientation tuning and spatial localization. As a technical aside, we found that the non-negativity constraint is not necessary: ordinary independent component analysis produces essentially the same results as our previous work.
Conclusion
We propose that the pooling that emerges allows the features to code for realistic low-level image features related to step edges. Further, the results prove the viability of statistical modelling of natural images as a framework that produces quantitative predictions of visual processing.
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Background
A number of models approach the computational modelling of primary visual cortex by using two processing stages. First, there is a linear filtering with filters that are bandpass, oriented, and spatially localized. In some models, the outputs of the linear filters are half-wave rectified, but this difference is inessential because a rectification is done in the second stage anyway. The second stage then consists of pooling together rectified outputs of the first stage, so that cells that have the same orientation and frequency, as well as similar spatial locations, are pooled together. This pooling is then essentially a summation of rectified outputs of filters of different phases. These two processing steps are assumed to roughly correspond to simple and complex cells in V1, respectively. While there is controversy of the validity of such models, see e.g. [1-3], this is probably the simplest and most succesful approach.
Recent research has seen a number of models that attempt to explain these processing stages based on statistical modelling of natural images (ecologically valid input). First, application of independent component analysis (ICA) [4] or sparse coding [5] shows that the statistically optimal linear features of natural images are very similar to those computed in simple cells in V1 [6-12]. Second, application of a variant of ICA in which some pooling is done in a second stage leads to processing that is similar to what is done in complex cells [13]. Thus, models based on natural image statistics have been able to succesfully reproduce the above-mentioned two stages, and many well-known observations on V1.
It would be most useful if we could use this modelling endeavour in a predictive manner, so that we would be able to predict properties of cells in the visual cortex, in cases where the properties have not yet been demonstrated experimentally. This would give testable, quantitative hypotheses that might lead to great advances, especially in the research in extrastriate areas such as V2, whose function is not well understood at this point.
Here, we attempt to accomplish such predictive modelling in order to predict properties of a third processing step, following the two described above. Previously, we have applied a modification of the ICA / sparse coding model on the outputs of modelled complex cells whose input consisted of natural images [14]. The modification consisted of assuming that the coefficients in the generative decomposition, as well as the values of the higher-order features, were all non-negative.
We extend our previous results in two ways. The complex cells in our previous work were all constrained to have the same frequency, which was done in order to reduce the computational load. Here, we first report a technical advance: it is not necessary to make the assumptions of nonnegativity as in [14]. Thus, we are able to use the conventional, computationally optimized ICA algorithms, in particular the FastICA algorithm [15]. We are then easily able to incorporate complex cells of different frequencies in the input without exceeding available computational resources. This enables us to study whether some kind of interaction between different frequencies emerges in the statistically optimal higher-order representation.
Results
Experiment 1: Using ordinary ICA with no constraints
As described in Methods, we input a large number of natural image patches into model complex cells that computed the sum of squares of outputs of two simple cells, one odd-symmetric and the other even-symmetric. Then, we performed independent component analysis of the complex cell outputs using the FastICA algorithm.
In the first experiment, we used only the output from complex cells in a single frequency band, f2 in Figure 1. The purpose was to show that the results in [14] can be replicated using ordinary ICA methods.
The higher-order features are represented by their basis vectors ai which show the contribution of the third-stage feature of index i on the activities of complex cells. A collection of the obtained basis vectors is shown in Figure 2 for the nonlinearity g1 (see Table 1), visualized in the same way as in [14], see Methods. We can see the same kind of emergence of collinear features as in [14]. That is, the higher-order features code for the simultaneous activation of complex cells that together form something similar to a straight line segment.
Those coefficients that are clearly different from zero have almost always the same sign in a single basis vector. Defining the sign as explained in Methods, this means that the coefficients are essentially non-negative. We thus see that the constraint of non-negativity of the basis vectors imposed in [14] has little impact on the results: even without this constraint, the system learns basis vectors which are mainly non-negative.
Other FastICA nonlinearities led to similar basis vectors. However, some led to a larger number of longer contours. Figure 3 shows the distribution of lengths for different nonlinearities. The nonlinearity g4 (robust skewness) seems to lead to the largest number of long contours.
Experiment 2: Emergence of pooling over frequencies
In the second experiment, the complex-cell set was expanded to include cells of three different preferred frequencies. In total, there were now 432 complex cells. We performed ICA on the complex-cell outputs when their input consisted of natural images. Thus, we obtained 432 higher-order basis vectors (features) ai with corresponding activities si.
We visualized a random selection of higher-order features learned from natural images in Figure 4. The visualization shows that the features tend to be spatially localized and oriented, and show collinearity as in Experiment 1. What is remarkable in these results is that many cells pool responses over different frequencies. The pooling is coherent in the sense that the complex cells that are pooled together have similar locations and orientations. A smaller number of cells is shown in more detail in Figure 5, where the coefficients in all orientation bands are shown separately.
We computed the frequency pooling measure Pi in Equation (4) of Methods for the learned basis vectors. The distribution of this measure for natural image input and white Gaussian noise input is shown in Figure 6. The figure shows that frequency pooling according to this measure was essentially nonexistent for white Gaussian noise input, but relatively strong for many basis vectors when the input consisted of natural images. To express this more quantitatively, we computed the 99% quantile for the white Gaussian noise input. Then, 59% of the basis vectors for natural image input had a pooling index Pi that was larger than this quantile. (For the 95% quantile the proportion was 63%.) Thus, we can say that more than half of the higher-order basis vectors, when learned from natural images, have a pooling over frequencies that is significantly above chance level.
To show that the pooling measure is valid, and to further visualize the frequency pooling in the higher-order features, we chose randomly basis vectors learned from natural images that have pooling significantly over chance level (Pi above its 99% quantile for white Gaussian noise). These are plotted in Figure 7. Visual inspection shows that in this subset, all basis vectors exhibit pooling over frequencies that respects the orientation tuning and collinearity properties.
The corresponding results when the input is white Gaussian noise are shown in Figure 8, for a smaller number of higher-order cells. (To make the comparison fair, these were randomly chosen among the 59% that had higher pooling measures, the same percentage as in Figure 7.) Pooling over frequencies as well as collinearity are minimal. Some weak reflections of these properties can be seen, presumably due to the small overlap of the filters in space and frequency, which leads to weak statistical correlations between complex cells that are spatially close to each other or in neighbouring frequency bands.
We also examined quantitatively whether the higher-order features are tuned to orientation. We investigated which complex cell has the maximum weight in ai for each i in each frequency band. When the input consisted of natural images, in 86% of the cells the maximally weighted complex cells were found to be located at the hot-spot (xi, yi)* (i.e., point of maximum activity, see Methods for exact definition) and tuned to the preferred orientation of the higher-order feature for every frequency f. This shows how the higher-order features are largely selective to a single orientation. When the input consisted of Gaussian white noise, only 34% of the cells were found to be orientation-selective according to this criterion.
Finally, we synthesized images from higher-order feature activities to further visualize the higher-order features (see Methods). Figure 9 shows a slice orthogonal to the preferred orientation of one higher-order basis vector (H209 in Figure 5). The intensity of the synthesized image shows no side-lobes (unnecessary oscillations), while representing a sharp, localized edge. In contrast, synthesis in the white Gaussian noise case (also shown in Figure 9) gives curves that have either side-lobes like the underlying Gabor filters, or do not give a sharp localized edge. Thus, the curve obtained from synthesis of the features learned from natural images corresponds better to the notion of an edge. We propose that the utility of pooling over frequencies is due to the broadband nature of real-world edges. Typical edges in natural images are probably not very similar to typical band-pass Gabor functions (or V1 receptive fields) which have oscillations. A proper representation of such broad-band edges would seem to require pooling over different frequencies.
Discussion
Frequency channels and edges
What is the functional meaning of the pooling we have found? We propose that this spatially coherent pooling of multiple frequencies leads to representation of an edge that is more realistic than the band-pass edges given by typical Gabor filters [16]. Presumably, this is largely due to the fact that natural images contain many sharp, step-like edges that are not contained in a single frequency band. Thus, representation of such edges is difficult unless information from different frequency bands is combined.
In terms of frequency channels, the model predicts that frequency channels should be pooled together after complex cell processing. Models based on frequency channels and related concepts have been most prominent in image coding literature in recent years, both in biological and computer vision circles. The utility of frequency channels in the initial processing stages is widely acknowledged, and it is not put into question by our results – in fact, the statistical modelling framework does show that using band-pass simple and complex cells is statistically optimal [6,13]. However, the question of when the frequency channels should be pooled or otherwise combined has received little attention [17,18]. Our results point out that a statistically optimal way is to pool them together right after the complex cell "stage", and this pooling should be done among cells of a given orientation which form a local, collinear configuration.
Related work
Several investigators have looked at the connection between natural image statistics, Gestalt grouping rules, and local interactions in the visual cortex [14,19-21]. However, few has considered the statistical relations between features of different frequencies so far. It should be noted that some related work on interactions of different frequencies does exist in the models of contrast gain control [22].
Compared to our own previous work [14], the main difference seems to be in the frequency tuning of the model complex cells. In [14], the complex cells were all constrained to have the same spatial frequency tuning – just as in Experiment 1 of the present paper. Therefore, it was impossible to obtain results related to frequency pooling. It seems that any differences in the results are not due to differences in the statistical analysis of the complex-cell outputs or the natural image data set used, because in Experiment 1 of the present paper, we essentially replicated the results of [14]. The statistical model for analyzing the outputs of complex cells was somewhat different in our earlier work: the components si and the coefficients aki were constrained to be non-negative, following proposals by [23,24]. However, this constraint seems to be immaterial, because even without imposing the constraint, the coefficients turned out to be essentially non-negative (after defining the global sign as described in Methods).
Recent measurements from cat area 18 (somewhat analogous to V2) emphasize responses to "second-order" or "non-Fourier" stimuli, typically sine-wave gratings whose amplitudes are modulated [17,25]. These results and the proposed models are related to our results and predictions, yet fundamentally different. In the model in [25], a higher-order cell pools outputs of complex cells in the same frequency band to find contours that are defined by texture-like cues instead of luminance. The same cell also receives direct input from simple cells of a different frequency, which enables the cell to combine luminance and second-order cues. This is in stark contrast to higher-order cells in our model, which pool outputs of complex cells of different frequencies. They can hardly find contours defined by second-order cues; instead they seem to be good for coding broad-band contours. Furthermore, in [17,25], any collinearity of pooling seems to be absent. This naturally leads to the question: Why are our predictions so different from these results from area 18? We suspect this is because it is customary to think of visual processing in terms of division into frequency channels – "second-order" stimuli are just an extension of this conceptualization. Therefore, not much attempt has been made to find cells that break the division into frequency channels according to our prediction. On the other hand, one can presume that the cells found in area 18 in [17,25] are different from our predictions because they use a different coding strategy from the one used in our model, perhaps related to the temporal aspects of natural image sequences [26,27].
Another closely related line of work is by Zetzsche and coworkers [28,29] who emphasize the importance of decomposing the image information to local phase and amplitude information. The local amplitude is basically given by complex-cell outputs, whereas the physiological coding of the local phases is not known. An important question for future work is how to incorporate phase information in the higher-order units. Some models by Zetzsche et al actually predict some kind of pooling over frequencies, but rather directly after the simple cell stage (see Fig. 16 in [29]).
Towards predictive modelling
The present results are an instance of predictive modelling, where we attempt to predict properties of cells and cell assemblies that have not yet been observed in experiments. To be precise, the prediction is that in V2 (or some related area) there should be cells whose optimal stimulus is a broad-band edge that has no sidelobes while being relatively sharp, i.e. the optimal stimulus is closer to a step-edge than the band-pass edges that tend to be optimal for V1 simple and complex cells. The optimal stimulus should also be more elongated [30,31] than what is usually observed in V1, while being highly selective for orientation.
Statistical models of natural images offer a framework that lends itself to predictive modelling of the visual cortex. First, they offer a framework where we often see emergence of new kinds of feature detectors – sometimes very different from what was expected when the model was formulated. Second, the framework is highly constrained and data-driven. The rigorous theory of statistical estimation makes it rather difficult to insert the theorist's subjective expectations in the model, and therefore the results are strongly determined by the data. Third, the framework is very constructive. From just a couple of simple theoretical specifications, e.g. non-Gaussianity, natural images lead to the emergence of complex phenomena.
We hope that the present work as well as future results in the same direction will serve as a basis for a new kind of synergy between theoretical and experimental neuroscience.
Conclusion
We have shown that pooling over complex cells of different frequency preferences emerges when we model the statistical properties of natural images. This is accomplished by applying ordinary ICA on a set of modelled complex cells with multiple frequencies, and inputting natural images to the complex cells. The resulting independent components, as represented by the corresponding basis vectors, code for simultaneous activation of complex cells that have similar orientations, form a collinear configuration, and span multiple frequencies. Thus, statistical modelling of natural stimuli leads to an interesting hypothesis on the existence of a new kind of cells in the visual cortex.
Methods
Data and statistical analysis
The natural images were 1008 gray-scale images of size 1024 × 1536 pixels from van Hateren's database, available at (category "deblurred") [8]. We manually chose natural images in the narrower sense, i.e. only wildlife scenes. From the source images, 50,000 image patches of size 24 × 24 pixels were randomly extracted. The mean grey value of each image patch was subtracted and the pixel values were rescaled to unit variance. The resulting image patch will be denoted by I(x, y).
The complex-cell model was similar to our previous work [14]. The filter bank consisted of a number of complex cells arranged on a 6 × 6 grid. Complex-cell responses xk to natural images were modelled with a classical energy model:
where and are even- and odd-symmetric Gabor receptive fields whose energies are pooled together in the complex cell. The complex cells had 6 × 6 = 36 different spatial locations, and at each location, four different preferred orientations and three different frequency bands. The aspect ratio was fixed to 1.5 and frequency bandwidth to 1.5 octaves, which implied an orientation bandwidth of 37°, according to the definitions in [8]. The frequency tiling of the Gabor filters is shown in Figure 1, in which all the filters W were normalized to unit norm for visualization purposes. The actual normalization we used in the experiments consisted of standardizing the variances of the complex cell outputs so that they were equal to unity for natural image input. The number of complex cells totalled K = 36 × 4 × 3 = 432. Note, however, that in Experiment 1 we only used a single frequency band.
Independent component analysis (ICA) was performed on the vector x = (x1,...,xK) using the FastICA algorithm [15]. The orthogonalization approach was symmetric. Different nonlinearities g were used, see Table 1. Thus we learned (estimated) a linear decomposition of the form
or in vector form
where the vector ai = (a1i,...,aki) gives a higher-order basis vector. The si define the values of the higher-order features in the third cortical processing stage.
Note that the signs of the basis vectors are not defined by the ICA model [4], i.e. the model does not distinguish between ai and -ai because any change in sign of the basis vector can be cancelled by changing the sign of si accordingly. Here, we defined the sign for each vector ai so that the sign of the element with the maximal absolute value was positive.
To obtain a baseline with which to compare our results, and to show which part of the results is due to the statistical properties of natural images instead of some intrinsic properties of our filterbank and analysis methods, we did exactly the same kind of analysis for 24 × 24 image patches that consisted of white Gaussian noise, i.e. the gray-scale value in each pixel was randomly and independently drawn from a Gaussian distribution of zero mean and unit variance. The white Gaussian noise input provides a "chance level" for any quantities computed from the ICA results.
Analysis of the ICA results
We visualized the resulting higher-order basis vectors ai following [14] by plotting an ellipse at each centrepoint of complex cells. The orientation of the ellipse is the orientation of the complex cell k, and the brightness of the ellipse is proportional to the aki coefficient of the basis vector ai, using a gray-scale coding of coefficient values. In Experiment 1, i.e. the case with a single frequency band, we used this method directly to visualize each higher-order basis vector in a single display. In Experiment 2, i.e. the multifrequency case, we visualized each frequency band separately.
In Experiment 2, we are interested in the frequency pooling of complex cells in different higher-order features. We quantified the pooling over frequencies using a simple measure defined as follows. Let us denote by ai(x, y, θ, fn) the coefficient in the higher-order basis vector ai that corresponds to the complex cell with spatial location (x, y), orientation θ and preferred frequency fn. We computed a quantity which is similar to the sums of correlations of the coefficients over the three frequency bands, but normalized in a slightly different way. This measure Pi was defined as follows:
where the normalization constant Cm is defined as
and likewise for Cn.
For further analysis of the estimated basis vectors, we defined the preferred orientation of a higher-order feature. First, let us define for a higher-order feature of index i the hot-spot (xi, yi)* as the centre location (x, y) of complex cells where the higher-order component si generates the maximum amount of activity. That is, we sum the elements of ai that correspond to a single spatial location, and choose the largest sum. This allows us to define the tuning to a given orientation of a higher-order feature i by summing over the elements of ai that correspond to the spatial hotspot and a given orientation; the preferred orientation is the orientation for which this sum is maximized. We also computed the length of a higher-order feature as described in [14].
It is also possible to perform an image synthesis from a higher-order basis vector. However, the mapping from image to complex-cell outputs is not one-to-one. This means that the generation of the image is not uniquely defined given the activities of higher-order features alone. A unique definition can be achieved by constraining the phases of the complex cells. We assume that only odd-symmetric Gabor filters are active. Furthermore, we make the simplifying assumptions that the receptive fields W in simple cells are equal to the corresponding basis vectors, and that all the elements in the higher-order basis vector are non-negative (or small enough to be ignored). Then, the synthesized image for higher-order basis vector ai is given by
where the square root cancels the squaring operation in the computation of complex-cell responses, and H denotes the set of indices that correspond to complex cells of the preferred orientation at the hotspot. Negative values of aki were set to zero in this synthesis formula.
Authors' contributions
A.H. conceived the basic idea and the principles of the experimental set-up, and wrote the paper. M.G. performed the experiments and elaborated the experimental set-up. P.O.H. assisted in the experiments and the writing.
Acknowledgements
A. H. was funded by the Academy of Finland, Academy Research Fellow position and project #48593. P.O.H. was funded by the Academy of Finland, project #204826. M.G. would like to thank Rodney Douglas for supporting this collaborative effort between HIIT and the Institute of Neuroinformatics, Zurich.
Figures and Tables
Figure 1 Frequency tuning of complex cells. We used three different frequency bands. The underlying Gabor filters had logarithmically spaced frequency peaks and their frequency responses overlapped at the -3dB points. Peak spatial frequencies were chosen as follows: f1 = 0.1 cycles/pixel, f2 = 0.21 cycles/pixel and f3 = 0.42 cycles/pixel. The amplitudes of the Fourier Transforms of the odd-symmetric Gabor filters are shown.
Figure 2 Basis vectors of Experiment 1. Random selection of learned basis vectors ai when the complex cells are all in a single frequency band. ICA non-linearity g was the tanh nonlinearity g1. Each patch gives the coefficients of one higher-order feature. Each ellipse means that the complex cell in the corresponding location and orientation is present in the higher-order feature, brightness of ellipse is proportional to coefficient aki.
Figure 3 Comparison of nonlinearities. Comparison of different FastICA nonlinearities in Experiment 1. The histogram gives the lengths of the contour patterns for the four different nonlinearities g1,..., g4 in Table 1.
Figure 4 Basis vectors of Experiment 2. A random selection of higher-order basis vectors ai estimated from natural images in Experiment 2. ICA nonlinearity g was the tanh nonlinearity g1. Each display of three patches gives the coefficients of one higher-order feature. Each patch gives the coefficients of one higher-order feature in one frequency band. Each ellipse means that the complex cell in the corresponding location, and of the corresponding orientation and frequency is present in the higher-order feature, brightness of ellipse is proportional to coefficient aki
Figure 5 Basis vectors in detail. Higher-order basis vectors of four selected higher-order features in Experiment 2, shown in detail. The coefficients in each orientation and frequency band are plotted separately.
Figure 6 Pooling measure. The distributions of the frequency pooling measure in Equation (4) for natural images and white Gaussian noise.
Figure 7 Basis vectors with significant pooling. A selection of higher-order basis vectors ai estimated from natural images in Experiment 2. These basis vectors were chosen randomly among those that have frequency pooling significantly above chance level.
Figure 8 Basis vectors for white Gaussian noise. For comparison, higher-order basis vectors estimated from white Gaussian noise, with each frequency band shown separately.
Figure 9 Image synthesis. Local image synthesis from the three odd-symmetric Gabor elements that have preferred orientation at the hotspot of a higher-order basis vector (H209 in Figure 5). The thick dotted curve shows the synthesis using coefficients from natural images, and the solid curves show various synthesis results using coefficients learned from white Gaussian noise input.
Table 1 Nonlinearities g used in FastICA. The nonlinearities probe the non-Gaussianity of the estimated components in different ways.
g1 (y) = tanh (y) Classic measure of sparseness
g2 (y) = y exp(-y2/2) More robust variant of g1
g3 (y) = y2 Skewness (asymmetry)
g4 (y) = exp(-y2/2) Robust variant of g3
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| 15715907 | PMC553984 | CC BY | 2021-01-04 16:03:48 | no | BMC Neurosci. 2005 Feb 16; 6:12 | utf-8 | BMC Neurosci | 2,005 | 10.1186/1471-2202-6-12 | oa_comm |
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BMC Health Serv ResBMC Health Services Research1472-6963BioMed Central London 1472-6963-5-171573332610.1186/1472-6963-5-17Research ArticleDeterminants of health insurance ownership among South African women Kirigia Joses M [email protected] Luis G [email protected] Benjamin [email protected] Germano M [email protected] Rufaro [email protected] Takondwa [email protected] World Health Organization, Regional Office for Africa, Brazzaville, Congo2 Department of Economics, University of Nairobi, Nairobi, Kenya2005 28 2 2005 5 17 17 22 5 2004 28 2 2005 Copyright © 2005 Kirigia et al; licensee BioMed Central Ltd.2005Kirigia et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Studies conducted in developed countries using economic models show that individual- and household- level variables are important determinants of health insurance ownership. There is however a dearth of such studies in sub-Saharan Africa. The objective of this study was to examine the relationship between health insurance ownership and the demographic, economic and educational characteristics of South African women.
Methods
The analysis was based on data from a cross-sectional national household sample derived from the South African Health Inequalities Survey (SANHIS). The study subjects consisted of 3,489 women, aged between 16 and 64 years. It was a non-interventional, qualitative response econometric study. The outcome measure was the probability of a respondent's ownership of a health insurance policy.
Results
The χ2 test for goodness of fit indicated satisfactory prediction of the estimated logit model. The coefficients of the covariates for area of residence, income, education, environment rating, age, smoking and marital status were positive, and all statistically significant at p ≤ 0.05. Women who had standard 10 education and above (secondary), high incomes and lived in affluent provinces and permanent accommodations, had a higher likelihood of being insured.
Conclusion
Poverty reduction programmes aimed at increasing women's incomes in poor provinces; improving living environment (e.g. potable water supplies, sanitation, electricity and housing) for women in urban informal settlements; enhancing women's access to education; reducing unemployment among women; and increasing effective coverage of family planning services, will empower South African women to reach a higher standard of living and in doing so increase their economic access to health insurance policies and the associated health services.
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Background
A health system in any country performs instrumental functions of stewardship (oversight), creation of resources (investment and training), delivering services (provision), and financing (collecting, pooling and purchasing) [1]. Ultimately, the effectiveness and efficiency with which these functions are executed determine the extent to which a health system achieves its intrinsic goals of improving health, responding to people's non-medical expectations, and fairness of financial contributions. For instance, the extent to which South Africa's post-apartheid government will be able to attain its vision of creating a caring and humane society in which all citizens have access to affordable good quality health care will depend on the performance of its national health system [2].
Prior to the 1994 democratic elections in South Africa, the health system was built on the apartheid ideology, which was characterized by racially segregated health services, geographical disparities, fragmentation, duplication and specialized hospital-centred services favouring the urban populations [3]. The health system's functions were inefficiently and inequitably performed. The health system was not effective in: improving the health status of the majority of the formerly disenfranchised South Africans; responding to their legitimate non-medical expectations; and ensuring their social protection from the impoverishing catastrophic health expenditures [4]. Consequently, provision of primary health services was for a long time neglected and inequitable [34]. The worsening poverty situation in some provinces has made the primary health services inaccessible to the majority of the population [35].
In an attempt to overcome the above-mentioned public health challenges, the post-apartheid democratic governments have introduced several reforms in the health sector: creation of a quasi-federal structure with one national and nine provincial departments of health (provincial legislatures and bureaucracies); establishment of a district health system, including expansion and upgrading of the primary care infrastructure; and health care financing [5]. The latter included: use of a population and need-based resource allocation mechanism by the National Department of Health up to 1995 (after which each province receives a block grant directly from the national treasury); the removal of public sector fees for pregnant and lactating women, children under six years of age and all those who use the public primary health care system; and the enactment of a Medical Insurance Schemes Act aimed at regulating the medical insurance schemes industry more effectively.
In spite of the abovementioned policy interventions, primary health care still remains under funded. For example, in 2003/2004 financial year, South Africa spent a total of 36.9 billion Rands (1 Rand = US$6) on health: 61.3% was spent on hospitals curative care, 16.1% on primary health care, 1.8% on HIV/AIDS treatment, 2.3% on nutrition, 4.0% on emergency services, 4.3% on administration, 2.5% on training, 1.6% on support services and 6.1% on other services [34].
According to Doherty and McLeod [6] and McIntyre et al. [7], the primary objectives of the Medical Insurance Schemes Act of 1998 are to: (i) increase the number of people covered; (ii) improve health-related cross subsidization within individual medical insurance schemes and curtail exclusion of high-risk groups, e.g. the elderly; (iii) prevent 'dumping' of medical insurance scheme members on public hospitals by requiring the schemes to cover a prescribed minimum package of hospital services for all members; and (iv) ensure effective health care cost containment.
In 2001, there were 146 registered medical schemes (i.e. those falling fully under the Act) and eight Bargaining Council schemes (i.e. those granted exemption from certain provisions of the Act), all covering a total of 7 million people [6], i.e. less than 20% of the South African population. An additional 2 million people were covered by private insurance or industry-specific health services [7]. Unfortunately, there has been no significant increase in the size of the population covered by the medical schemes since the implementation of the Act. For instance, in 2003 South Africa had uninsured population of 38.6 million people: 15.1% were from Eastern Cape province, 6.0% from Free State, 17.8% from Gauteng, 22.1% from Kwazulu Natal, 12.8% from Limpopo, 7.2% from Mpumalanga, 1.7% from Northern Cape, 8.5% from North West and 8.7% from Western Cape [34].
The objective of this study was to examine the relationship between individuals' demographic, economic and educational characteristics, and their likelihood of being insured.
Methods
Conceptual framework
There are two kinds of risks involved in health care: (i) the risk of becoming ill, with the accompanying loss in the quality of life, cost of medical care, loss of productive time during illness and, in more serious cases, death; and (ii) the risk of total or incomplete or delayed recovery [8].
Welfare economics of uncertainty predicts that individuals would like to insure against both forms of risks. The theory of expected utility, on which this study is based, assumes that each individual strives to maximize the expected value of a utility function; individuals are normally risk-averse, meaning that they have a diminishing marginal utility of income; and health risks for different individuals are basically independent, so that pooling them reduces the risk to the insurer to relatively small proportions [9].
In the South African National Health Inequalities Survey (SANHIS) [10], the respondents were asked the following question: "Does anyone in this household belong to a medical aid or health insurance scheme? 1 = Yes, 2 = No". Given the dichotomous nature of this question, we shall assume that the potential health insurance consumer faces the choice between purchase of some or no insurance. The consumer chooses between the two prospects on the basis of the utility expected from each.
The potential consumers of insurance are assumed to make decisions based on the magnitude of the perceived difference between the level of expected utility with insurance (EU1) and expected utility without insurance (EU2). We need to analyse the effect of changes in the independent variables on the difference in the level of the expected utility of the two prospects, i.e. EU1 minus EU2. If the difference were equal to zero, the consumer would be expected to be indifferent between the two prospects. However, if the difference were greater than 0, then the risk-averse consumer would be expected to opt for insurance [11].
The empirical model used in the analysis of individual household's choice between having no health insurance and having health insurance is presented in the Appendix. The variables included in the model are defined in Table 1.
Table 1 Definition of variables
Variable Variable description
Health insurance ownership 1 = if the respondent has health insurance; 0 otherwise
Health rating 1 = if self-evaluated health status is excellent, very good or good; 0 otherwise
Environment rating 1 = if the respondent feels that the environment she lives in is good, very good or excellent; 0 otherwise
Residence 1 = if the respondent resides in either metro formal area, metro transitional area, smaller city/town formal area, smaller city/town transitional area, or rural white farms; 0 = metro informal area, smaller city/town informal area, or rural – "homeland"
Income Total monthly gross income in Rand (US$≈6 Rand)
Education Respondent's education level: 1 = matriculation (standard 10 or secondary school) and above; 0 = below matriculation
Age Respondent's age in years
Age squared Respondent's age squared
Race 1 = if respondent is white; 0 if person of colour
Household size Total number of persons in a household
Occupation 1 = if a white-collar worker; 0 otherwise
Employment status 1 = if unemployed and looking; 0 = otherwise
Smoking 1 = if the respondent smokes cigarettes; 0 otherwise
Alcohol use 1 = if the respondent drinks alcohol; 0 otherwise
Contraceptive use 1 = if respondent uses a contraceptive; 0 = otherwise
Marital status 1 = if married; 0 = single, separated or divorced
Since economic theory does not provide much guidance on model specification, the choice of explanatory variables in the current study were guided by the past health insurance demand studies undertaken in the U.S.A. [12-15], Europe [16-18] and Israel [19,20].
Once again, based on past health insurance choice analysis studies, the coefficients of the variables included in equation 2 in the Appendix would, a priori be expected to assume the signs indicated in Table 2.
Table 2 Hypothesized relationships between the dependent variable (insurance ownership) and independent variables
Independent Variables Variable coefficient Expected sign Studies from which the hypothesized signs are based
Health rating B1 Negative Trujillo [23], Coasta and Garcia [28]
Environment rating B2 Indeterminate
Residence β3 Positive Liu and Chen [31]
Income β4 Positive Deb et al [22], Trujillo [23], Vera-Hernandez [26], Coasta and Garcia [28], Besley et al [32]
Education β5 Positive Deb et al [22], Trujillo [23], Vera-Hernandez [26], Coasta and Garcia [28], Besley et al [32], Liu and Chen [31]
Age β6 Positive Trujillo [23], Liu and Chen [31], Grossman [12]
Age squared β7 Negative Trujillo [23], Grossman [12]
Race β8 Indeterminate
Household size β9 Negative Deb et al [22], Vera-Hernandez [26], Besley et al [32]
Occupation β10 Positive Vera-Hernandez [26]
Employment status β11 Negative Vera-Hernandez [26], Liu and Chen [31]
Smoking β12 Indeterminate
Alcohol use β13 Indeterminate
Contraceptive use β14 Indeterminate
Marital status β15 Positive Rhine et al [21], Trujillo [23], Liu and Chen [31]
A Chi-Squared test (χ2) for independence was undertaken to test the relationship between health insurance ownership and the individual independent variables. The null hypothesis is that the two variables are independent of one another (because there is a significantly large difference between the observed data and the calculated expected data). The alternate hypothesis is that the two variables are dependent on each other (because there is not a significant difference). Thus, for health rating, the hypotheses are as follows: (i) H0: A person's health insurance ownership status and their health rating are independent or unrelated; and (ii) HA: A person's health insurance ownership status and their health rating are related (dependent on each other).
Data
The data for empirical analysis were taken from the 1994 South African National Health Inequalities Survey, a household survey of a randomly selected sample of the South African population between the ages of 16 and 64 years [10]. The full sample was 3,796 persons, out of which 3,489 were women. Our analysis focused on the latter. The data set was rich with economic, demographic, social and health characteristics of the respondents.
Results
Descriptive statistics
Table 3 presents the frequency and percentage distribution of the dependent and independent variables. Overall, 30% of the women in the sample said that they had a household member with a health insurance policy. Ninety five percent of the respondents who reported to have got a household member belonging to a medical aid or health insurance scheme lived in formal city dwellings and/or on farms owned by white South Africans.
Table 3 Frequencies and percentages for explanatory variables
Variables With insurance: frequency (%)[N = 1044) Without insurance: frequency (%) [N = 2445] Chi-square (p-value)
Health rating: 1 = Excellent/very good/good 435 (41.67) 1238 (50.63) 23.57 (p < 0.0001)
0 = Fair and poor 609 (58.33) 1207 (49.37)
Environment rating: 1 = Good/very good/Excellent living environment 491 (47.03) 1580 (64.62) 93.843959 (P < 0.0001)
0 = Fair or poor 553 (52.97) 865 (35.38)
Residence: 1 = Formal city dwellings + white farms 995 (95.31) 1625 (66.46) 325.45 (P < 0.0001)
0 = Informal dwellings +"former homelands" 49 (4.69) 820 (33.54)
Income (in Rand): No regular income 137 (13.12) 275 (11.25) 1238.93 (P < 0.0001)
1 – 950 100 (9.58) 1497 (61.23)
951 – 1900 179 (17.15) 435 (17.79)
1901 – 3800 317 (30.36) 191 (7.81)
3801 – 7600 223 (21.36) 38 (1.55)
7600 + 88 (8.43) 9 (0.37)
Education: 1 = Matriculation (standard 10) and above 566 (54.21) 2205 (90.18) 579.15 (P < 0.0001)
0 = Below matriculation 478 (45.79) 240 (9.82)
Age (in years): 16 – 25 128 (12.26) 328 (13.42) 16.53 (P = 0.0024)
26 – 35 299 (28.64) 666 (27.24)
36 – 45 284 (27.20) 586 (23.97)
46 – 55 189 (18.10) 404 (16.52)
56 and above 143 (13.70) 461 (18.85)
Race: 1 = African, Coloured & Indian 947 (90.71) 1981 (81.02) 50.87 (P < 0.0001)
0 = White 97 (9.29) 464 (18.98)
Household size: 1 – 4 household members 647 (61.97) 1043 (42.66) 123.30 (P < 0.0001)
5 – 8 351 (33.62) 1114 (45.56)
9 – 12 41 (3.93) 240 (9.82)
13 and above 5 (0.48) 48 (1.96)
Occupation: 0 = White-collar worker 326 (31.23) 168 (6.87) 357.05 (P < 0.0001)
1 = Blue-collar worker 718 (68.77) 2277 (93.13)
Employment status: 1 = Involuntarily unemployed 967 (92.62) 1933 (79.06) 95.94 (P < 0.0001)
0 = Voluntarily unemployed or employed 77 (7.38) 512 (20.94)
Smoking: 1 = If a cigarette smoker 292 (27.97) 607 (24.83) 3.78 (P = 0.0519)
0 = Not a cigarette smoker 752 (72.03) 1838 (75.17)
Alcohol use: 1 = Alcohol drinker 148 (14.18) 276 (11.29) 5.71 (P = 0.0168)
0 = Not alcohol drinker 896 (85.82) 2169 (88.71)
Contraceptive use: 0 = Uses contraceptives 213 (20.40) 399 (16.32) 8.43 (P = 0.0037)
1 = Does not use contraceptives 831 (79.60) 2046 (83.68)
Marital status: 1 = Married 627 (60.06) 1144 (46.79) 51.53 (P < 0.0001)
0 = Single, separated, divorced 417 (39.94) 1301 (53.21)
In the overall sample, the median income was 849.5 Rand and the mean was 1546.4 Rand, with a standard deviation (STD) of 1998.2 Rand. The sub-sample of the households without health insurance had an average income of 853 Rand (STD = 1073) compared to 3172 Rand (STD = 2623) in the group with insurance. Seventy-seven percent of the households with at least one member with health insurance had a monthly income of more than 951 South African Rand, compared to only 28% among the group without health insurance.
The average and median household size in the overall sample was five members, with a standard deviation of about 3. The group with health insurance had an average household size of four people (STD = 2.1) compared to 5 people (STD = 2.7) in the group without insurance. Fifty-seven percent of the sub-sample without health insurance had a household size of more than five members compared to 38% among the group with health insurance.
The group with health insurance had an average age of 40 years (STD = 12.5) compared to 41 years (STD = 14.1) in the group without insurance. The average age in the whole sample was 40.9 years (STD = 13.6) and the median was 39 years.
As alluded to in the methods section, a Chi-Squared test for independence was undertaken to test the relationship between health insurance ownership and the individual independent variables. The results are presented in the last column of Table 3. Since the computed Chi-Squared values for health rating, environment rating, residence, income, education, age, race, household size, occupation, employment status, alcohol use, contraceptive use and marital status were greater than their respective critical Chi-Squared values (at 5% significance level) we reject the null hypotheses and conclude that the row and column variables in Table 3 are not independent. Thus, for example in the case of health rating, we would accept the alternative hypothesis (HA) that a person's health insurance ownership status and their health rating are related (dependent on each other).
Regression analysis
Table 4 provides odds ratios, 'p' values, coefficients, and 't' test values. The t-test is used to test the hypothesis (i.e. H0: β = 0) about individual regression slope coefficients. The 't' values for individual variables are obtained by dividing their coefficients (e.g. βINCOME) by their standard errors (e.g. SEINCOME). For example, the coefficient for income is 0.0004727 and the standard error is 0.0000339, and given H0: β = 0, the relevant t-value is indeed 13.946 as specified in Table 4:
Table 4 Logistic model regression results
Explanatory variables Odds ratios [95% confidence interval] Coefficients 't'
Health rating 0 0.000009-0.0005 -9.676 -9.537*
Environment rating 26.76 12.43–57.60 3.287 8.404*
Residence 6.969 4.93–9.84 1.942 11.020*
Income 1.001 1.00-1.00 0.0005 13.946*
Education 2.315 1.80–2.97 0.84 6.600*
Age 1.148 1.09–1.20 0.138 5.751*
Age squared 0.999 0.99–1.00 -0.0008 -3.401*
Race 0.787 0.59–1.04 -0.239 -1.691
Household size 0.891 0.86–0.93 -0.115 -5.519*
Occupation 0.733 0.54–0.99 -0.311 -1.971
Employment status 0.518 0.38–0.69 -0.657 -4.308*
Smoking 1.633 1.29–2.07 0.49 4.052*
Alcohol use 0.617 0.45–0.84 -0.483 -3.033*
Contraceptives use 0.372 0.26–0.52 -0.988 -5.700*
Marital status 1.841 1.49–2.27 0.611 5.765*
Constant - - -4.385 -8.755
Sample size 3489
χ2(15) 1438.62
Prob > χ2 0
Pseudo-R2 0.3379
Log likelihood -1409.7041
Note: * Indicates the coefficients are statistically significant at 95% confidence level, based on a two-tailed test. On the basis of chi-square test of the log-likelihood ratio, the joint effects of estimated logistic model are statistically significant at the 0.1% level.
tINCOME = (βINCOME)/(SEINCOME) = (0.0004727)/(0.0000339) = 13.946
The decision rule is: reject the null hypothesis (H0: β = 0) if the calculated t-value, tk, is greater than the critical t-value, tc, as long as the sign of tk is the same as the sign of the coefficient implied in the alternative hypothesis (HA: β≠0). Otherwise, accept the null hypothesis (H0) that the estimated regression coefficient in question is not significantly different from zero. In the above example, the coefficient of income is statistically significant at 95% level of confidence, based on a two-sided test, since the computed t-value (13.946) is greater than the critical t-value (1.960).
The coefficient (β) of the estimated binary logit model measures the impact of a one-unit change in an explanatory variable (Ri) on the log of odds of a health insurance policy ownership, holding other explanatory variables constant. The coefficients for environment rating, residence, income, education, age, smoking and marital status are statistically significant at 95% level of confidence, and have positive signs. The latter result implies that an increase in any of these variables spontaneously impacts positively on the log of odds of health insurance policy ownership, holding other factors constant. Contrastingly, the coefficients for household size, alcohol, contraceptive use, age-squared, occupation, employment and health rating are statistically significant, and have got a negative effect on the log of odds of health insurance policy ownership.
Discussion
Health rating
Social scientists define health as a product of life expectancy (measured in years) and health-related quality of life (i.e. mobility, activities of daily living, social participation, pain, anxiety/depression, energy) [22,23]. The SANHIS data set [10] contained only self-evaluated categorical health status data. The respondents rated their current health status as either excellent, very good, good, fair or poor. The variable was re-coded into a dichotomous variable: 1 = excellent, very good, or good; and 0 if fair or poor.
An individual's stock of health determines the total amount of time he/she can spend producing commodities and money earnings [24]. Health status is an important determinant of both earnings and capacity for enjoying life. A decline in the death rate at working ages may improve earning prospects by extending the period during which earnings are received [25]. The coefficient for the health status variable took a negative sign, implying that the demand for health insurance was likely to be low among individuals who were in excellent, very good or good health. In the current study, 58.3% of the respondents in the sub-sample with a health insurance policy assessed their health status as either fair or poor. This may be a case of adverse selection [26], which results in insurance having the greatest appeal to individuals who are more likely to fall sick [9,27]. Adverse selection, depending on its extent, could jeopardize the economic viability of a health insurance scheme.
The adverse selection problem can be curbed in two main ways without compromising equity objectives, namely: (i) compulsory social health insurance (or a national health service) for a defined population through legislation – not an option for private health insurance which is voluntary by definition; and (ii) the government could step in and provide health insurance to all those at exceptionally high risk (e.g. the elderly and those with chronic diseases) and the poor who cannot afford the premiums. Commercial health insurance firms often curb adverse selection by introducing experience rating, i.e. linking insurance premium to the degree of assessed risk of falling sick (this action may have negative equity implications); and/or subjecting all those who apply for insurance cover to a thorough medical examination (this could potentially lead to cream-skimming, excluding all those with high risks of falling sick).
Economic factors
There were three economic variables, namely, income (+), occupation (-) and employment (-), where (.) is the hypothesized sign of the coefficients. The coefficients of the three variables were statistically significant and had the expected signs. High incomes, white-collar occupations and being gainfully employed are significant predictors of health insurance ownership. The proportion of people with health insurance rises considerably as one moves up the household income distribution ladder, with the coverage going from 6.3% of those in the income range 1–950 Rand to over 90.7% among those earning 7600 Rand and above per month. The trend is similar to that reported by Harmon and Nolan [16] in Ireland and Propper [17] in England and Wales. This implies that any macroeconomic interventions aimed at decreasing involuntary unemployment and boosting disposable incomes among households will spontaneously increase the probability of health insurance ownership. Thus, the post-apartheid South African government's economic programmes of black empowerment, small-scale micro financing programmes and land (and other assets) redistribution programmes are likely to increase the number of households with the ability to purchase health insurance policies.
Demographic factors
The demographic factors include: age (+), age squared (-) and household size (-), where (.) is the postulated sign of the coefficient. The coefficients for age and age squared were statistically significant at the 5% level. Economic theory predicts that as individuals advance in age, their inherited health stock depreciates at an increasing rate (a manifestation of the biological process of ageing) and they tend to increase investments in health (including health insurance) in an attempt to decrease the rate of depreciation. This is consistent with Grossman's findings [24] that because the health stock depreciation rate rises with age, it is not unlikely that unhealthy (old) people will make larger gross investments in health than healthy (young) people.
The coefficient for household size variable had a statistically significant negative effect on the likelihood of health insurance policy ownership. This finding is intuitively sensible since any increase in the household size, while holding the income constant, reduces the per capita income.
Social factors
The social factors include: education (+) and marital status (+). The coefficient for education was statistically significant, and had the expected positive sign. Respondents with at least a matriculation (secondary) level of education were two times more likely to be in possession of a health insurance policy than those with a lower level of education. This could be attributed to a positive relationship between a person's: (i) educational level and propensity to acquire skills; (ii) stock of knowledge and his/her market and non-market productivity [24] and earnings; and (iii) education and knowledge about the advantage of making regular small insurance payments to avoid the risk of catastrophic medical expenditures [28].
Marital status had a statistically significant positive effect on health insurance ownership. Married persons are more likely to have insurance cover than those who are single, separated or divorced. This finding is consistent with the result obtained by Harmon and Nolan [16], Rhine et al. [12], Trujillo [14] and Liu and Chen [28]. Married couples may have a higher demand for health insurance due to: (i) the need to protect their children [16]; (ii) higher combined income; and (iii) being more averse to the risk of catastrophic health expenditures than those who are single, separated or divorced.
Spatial and environmental factors
The spatial and environmental factors – residence (+) and environment rating (+) – had a statistically significant effect on health insurance ownership. The respondents living in formal urban settlements or rural white-owned farms had a seven times higher odds of owning a health insurance policy than those living in informal urban settlements or former rural homelands. This could partly be a reflection of the economic well being of the former group.
The respondents who felt that the environment they lived in was good, very good or excellent were twenty-seven times more likely to have a health insurance cover than those who lived in fair or poor environments. This phenomenon may be a reflection of a better socio-economic status of those living in relatively affluent, formal (and cleaner) residential areas vis-à-vis informal settlements (which are relatively deprived of all kinds of social amenities).
Behavioural factors
The behavioural factors included in the analysis were: contraceptive use (-), alcohol use (-) and smoking (+). The three had a statistically significant effect on the demand for health insurance. The coefficient for contraceptives assumed a negative sign. This implies that the use of contraceptives may not necessarily be linked with individuals' attitudes toward risk.
The coefficient for alcohol use also took a negative sign, which implies that those people who drank alcohol were less likely to purchase health insurance. On the contrary, the parameter for smoking assumed a positive sign, meaning that being a cigarette smoker, the probability of a person demanding health insurance coverage increases. In the context of the health insurance market, the latter finding could be a source of concern if it were a signal for the presence of moral hazard. Moral hazard is a potential cost of insurance in which the presence of insurance increases the tendency for losses to occur through careless, irresponsible or perhaps illegal behaviour [26].
For example, the fully insured individuals may embark on risky behaviours, such as smoking, and by so doing expose themselves to a high risk of developing various forms of cancer (throat cancer, lung cancer, etc.). Insurers attempt to control moral hazard by careful underwriting of applicants for insurance and by various policy provisions, such as deductibles (which requires an individual to pay for a certain amount of health care received before the insurance comes into effect) and co-insurance/co-payment (which requires the insured person to pay a certain percentage of eligible medical expenses in excess of the deductibles, with the insurer paying the remainder) [15,29].
This study used consumption of contraceptives, alcoholic drinks and cigarettes as proxies for consumers' attitudes toward health risks. The three may not be ideal proxies for risk attitudes; however, there were no better alternatives in the data set. If this were a study primarily designed to analyse the demand for health insurance, it would have been preferable to either proxy risk attitudes using a qualitative scale variable ranging from 1 (extremely risk-averse) to 10 (risk-lover) [11], directly estimate the revealed risk-aversion using experimental data [29], or ask the respondent to report whether he/she would consider paying for private health insurance at the point of demand [30].
Limitation of the study
The main weakness of the current study is that since the data set upon which the analysis was based was gathered for a different purpose (i.e. it was not dedicated to health insurance), it did not contain insurance-specific attributes, e.g. premiums, co-payments, deductibles, benefits covered and the quality of care in the health facilities where the insured sought care. Thus, we had a situation where important explanatory variables were left out of the estimated regression equation 2 (in the Appendix), leading to specification bias or omitted variable bias [21]. The omission of a relevant independent variable can change the estimated coefficient away from the true value of the population coefficient.
Further research
In sub-Saharan Africa, there is need for studies on the following:
• The determinants of private and social health insurance policy ownership, which include both health insurance programme attributes (e.g. premiums, co-payments, deductibles) and household socio-economic characteristics (including attitudes toward risk).
• The willingness and ability to pay for various forms of health insurance, including voluntary and non-voluntary insurance schemes [31].
• The economic viability of various forms of health insurance, e.g. social health insurance and community-based prepaid schemes.
• Design of innovative health insurance schemes; for example, within farmers' cooperative societies [36], savings and credit societies, agricultural estates, women/men developmental groups [37], civil service, etc.
• Whether the expansion of private health insurance under the current health care delivery system would yield significant public sector cost savings, and improved targeting of subsidies for the poor and preventive services [32].
• Optimal ways of curbing health insurance problems of moral hazard and adverse selection.
• Benefit-incidence analysis of alternative health insurance arrangements.
Conclusion
The environment rating, residence, income, education, age, smoking and marital status variables were all found to have a statistically significant (at 95% confidence level) positive relationship with ownership of health insurance schemes. Contrastingly, the other covariates, namely: health rating, age squared, household size, occupation, employment, alcohol use and contraceptive use had a significantly negative relationship with health insurance ownership.
There are a number of policy implications of this study:
• High incomes, white-collar occupations and being gainfully employed are significant predictors of health insurance ownership. Thus, economic development (or poverty reduction) programmes geared at: (i) improving incomes of the vulnerable segments of the South African population; (ii) reducing involuntary unemployment; and (iii) creating white-collar job opportunities will empower South African women to reach a higher standard of living and in doing so boost their economic access to health insurance policies and the relevant health services.
• Policies aimed at ensuring that the majority of South Africans attain a matriculation (i.e. secondary) education level will increase by almost two-fold, the probability of acquiring health insurance.
• The self-assessed health status was found to have a statistically negative effect on the demand for health insurance. This implies existence of adverse selection. However, this problem can be reduced through compulsory social health insurance [33]; or through state insurance for high-risk groups, particularly the poor. Since 53% of the South African population lives below the income poverty line of US$2 per day [34], implementation of the social health insurance programme [7] would increase access to basic health services by poor.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
JMK recoded the raw data and participated in the development of the conceptual framework, analysis and drafting of sections of the document. LGS, BN, GMM, RC and TM participated in the development of the conceptual framework and drafting of sections of the document. All authors read and approved the final manuscript.
Appendix: Empirical model
A binary logit model was used in the analysis of individual household's choice between having no health insurance and having health insurance. We assumed that the expected utility associated with each health insurance option is a function of a vector of its attributes (Xi) and a vector of a household's socioeconomic characteristics (Ri), plus a stochastic error term (ε). The latter component captures errors in model specification (e.g. omission of relevant variables) and errors in data measurement.
Algebraically, a household's decision process can be expressed as:
EUij = g(Xij, Ri) + ε .................................... (1);
where: EUij is the utility that ith household expects to derive from choosing jth health insurance option; j = 1 if a household has health insurance; j = 2 if a household has no health insurance; and X, R and ∈ are as defined above.
The basic assumption is that the ith household opts for 'health insurance' if EUi1 > EUi2, prefers 'no health insurance' if EUi1 < EUi2, and is indifferent between the two options if EUi1 = EUi2. Thus, the probability that ith household prefers to have health insurance is: Pi1 = P(EUi1 > EUi2). And, conversely, the probability that ith household prefers not to have health insurance is: Pi2 = P(EUi1 < EUi2).
To determine the probability of health insurance ownership, the following model was estimated:
Pij = (α + β1 HEALTH_RATING + β2 ENVIRONMENT_RATING + β3 RESIDENCE + β4 INCOME + β5EDUCATION + β6 AGE + β7 AGE_SQUARED + β8 RACE + β9 HOUSEHOLD_SIZE + β10 OCCUPATION + β11 EMPLOYMENT + β12 SMOKING + β13 ALCOHOL_USE + β14 CONTRACEPTIVES_USE + β15 MARITAL_STATUS + εi........................ (2)
where: Pij = 1 if individual 'i' owns insurance (j = 1) and equals zero otherwise (j = 0); (α) is the intercept term; (β's) are the estimated coefficients; and εi is the stochastic error term. The explanatory variables included in the model are defined in Table 1. Because of the limitations associated with linear probability model, the logit version of equation 2 was estimated, using maximum Likelihood Method.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The South African Central Bureau of Statistics provided the raw data used in the current study. AS Kochar provided commendable editorial support. We are grateful for the suggestions received from the two peer reviewers (Tim Ensor and Martin C. Mahoney) that helped to improve the quality of this paper. Rah Sabbaoth-Rapha inspired the study and provided all-round guidance.
Any mistakes remaining in the paper are those of the authors and should not be attributed to any of the acknowledged.
The article contains the views of the authors only and does not represent the opinions, decisions or stated policies of the World Health Organization or the University of Nairobi.
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| 15733326 | PMC553985 | CC BY | 2021-01-04 16:31:51 | no | BMC Health Serv Res. 2005 Feb 28; 5:17 | utf-8 | BMC Health Serv Res | 2,005 | 10.1186/1472-6963-5-17 | oa_comm |
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Nutr JNutrition Journal1475-2891BioMed Central London 1475-2891-4-41570307110.1186/1475-2891-4-4ResearchNutritional knowledge, food habits and health attitude of Chinese university students –a cross sectional study– Sakamaki Ruka [email protected] Kenji [email protected] Rie [email protected] Chuan-Jun [email protected] Naotaka [email protected] International Center for Medical Research. Kobe University Graduate School of Medicine, Kobe, 650-0017, Japan2 Seinan Jo Gakuin University Faculty of Health and Welfare, Department of Nutritional Sciences, Kitakyusyu, 803-0835, Japan3 Department of Plastic Surgery, Kobe University Graduate School of Medicine, Kobe, 650-0017, Japan2005 9 2 2005 4 4 4 2 11 2004 9 2 2005 Copyright © 2005 Sakamaki et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
We have previously shown that irregular lifestyle of young Japanese female students are significantly related to their desire to be thinner. In the present study, we examined the nutritional knowledge and food habits of Chinese university students and compared them with those of other Asian populations.
Methods
A self-reported questionnaire was administered to 540 students, ranging in age from 19-24 years. Medical students from Beijing University (135 men and 150 women) in Northern China and Kunming Medical College in southern China (95 men and 160 women) participated in this study. The parametric variables were analyzed using the Student's t-test. Chi-square analyses were conducted for non-parametric variables
Results
Our results showed that 80.5% of students had a normal BMI and 16.6 % of students were underweight with the prevalence of BMI>30 obesity being very low in this study sample. Young Chinese female students had a greater desire to be thinner (62.0%) than males (47.4%). Habits involving regular eating patterns and vegetable intake were reported and represent practices that ought to be encouraged.
Conclusions
The university and college arenas represent the final opportunity for the health and nutritional education of a large number of students from the educator's perspective. Our findings suggest the need for strategies designed to improve competence in the area of nutrition.
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Background
The increasing problem of obesity has been observed in many lower-income countries during the last decades. China has adopted an open-market policy and experienced explosive economic growth, which has led to less food scarcity at the national level and to a remarkable transition in the structure of the diet of Chinese [1]. The composition of the Chinese diet has been shifting towards a diet higher in fat and meat, and lower in carbohydrates and fiber [2]. Additionally, decreased levels of physical activity and leisure are linked to increases in the prevalence of an overweight condition, obesity and diet-related non-communicable diseases [3].
In previous reports, we examined eating habits and dietary knowledge of female students in Japan. Our results showed that irregular lifestyle was significantly related to indefinite complaint, with the majority of students having a desire to be thinner although the prevalence of students who were overweight was very low in this study sample [4]. Universities and colleges are potentially important targets for the promotion of healthy lifestyles of the adult population. However, little is known concerning the body mass index (BMI) distribution and nutritional and health-related behavior of Chinese university students. The purpose of this study was to obtain a preliminary understanding of the relative level of BMI distribution of Chinese university students and to determine the nutritional knowledge and body-shape perceptions.
Material and Methods
This study was carried out between February 2001 and April 2002. Medical students from Beijing University (135 men and 150 women) in Northern China and Kunming Medical College in southern China (95 men and 160 women) participated in this study. A sample of 540 students aged 19 – 24 years were administered a self-reported questionnaire. The questionnaire consisted of 21 questions regarding eating, drinking and smoking habits (19 questions), with 2 questions related to dieting (trying to lose weight). Self-reported height and weight were used to calculate BMI (kg/m2). The questionnaire was designed by the authors and based on a national dietary survey held by the Health and Labor Ministry of Japan. Some of the authors also traveled to China to investigate the dietary life of Chinese to facilitate questionnaire design. The questionnaire was first written in Japanese and then translated to Chinese utilizing fluent bilingual linguistic services. The translated Chinese version was back-translated to insure the original meaning was not lost. Informed consent was obtained from all participants of this study according to the Declaration of Helsinki. The statistical software package SPSS 10.0 was used for the analysis of data [5]. In this study, parametric variables were analyzed using the Student's t-test. Chi-square analyses were conducted for non-parametric variables. All analyses were two-tailed, and a 'p' value less than 0.05 was considered statistically significant.
Results
Characteristics of the sample and BMI categories
The response rate was 96% (512 / 540). The characteristics of the subjects are shown in Table 1. A total of 212 men and 300 women, with a mean age of 20 ± 1.9 years, participated in this study. The average height was 165.8 ± 7.8 cm, while the average weight was 56.9 ± 9.2 kg. Mean BMI was 20.6 ± 2.2. To analyze the distribution of BMI and health-related behavior, BMI was categorized into 4 groups according to mean BMI of ± 1 standard deviation (SD) (Figure 1). The average BMI for male students was 21.4 ± 2.5 and was highest in the categories 18.9≤BMI<21.4 (37.7%) and 21.4≤BMI<23.9 (32.5%). The average BMI for female students was 20.0 ± 1.8, with the categories 18.2≤BMI<20.0 (37.5%) and 20.0≤BMI<21.8 (31.4%) displaying high values. According to WHO BMI classifications [6], 97.1% of students were classified into the underweight or normal weight categories. 2.5% (13/512) students were overweight (BMI>25) and 0.4% (2/512) of students were obese (BMI>30). BMI values of deviations from the average sample show the presence of few extreme values.
Eating habit
The life style practices were compared by gender (see additional file 1). The majority of students (83.6 %) reported taking meals regularly, with 79.0% eating meals 3 times per day; there were no gender differences. However, a significant gender difference was found in the response relating to breakfast intake, with 66.8% of males and 82.3% of females reporting eating breakfast regularly (p < 0.0006). The frequency of snacking rate was significantly higher in females (31.1%) than in males (11.5%; p < 0.0001). The present sample demonstrated high consumption of vegetable and fruits. A total of 47.9% of students reported the consumption of colored vegetables such as spinach and carrots, and 32.5% of subjects reported eating fruit daily. Female students tend to eat more fruit than males (p < 0.0001). In addition, female students tend to eat with friends and family more frequently than males (p < 0.01). Few subjects smoke or drink alcohol. When the students eat out, female students are more likely to consider the calorie content of the menu than males (data not shown). Although 85.6% of students are aware of the concept of nutritionally balanced food, only a small number of students (7%) apply this concept when selecting food from a menu. Moreover, only 51% of students showed a desire to learn about healthy diets.
Body image and health consciousness
When subjects were asked about their history of dieting, 22.7% of respondents reported that they had dieted (see additional file 2). The proportion of female students having a dieting experience (29.8%) was more than twice as great as that of male subjects (12.7%; p < 0.0006). In total, 56% of the students selected 'thin or slim is beautiful'. The percentage by gender was, 47.4% for male and 62.0% for female students. Female students have a significantly greater desire to be thinner than males (p < 0.001). More than half of the respondents reported a desire to adopt healthier dietary habits. Moreover, a question regarding the degree of consciousness pertaining to health and diet was asked; 45.2% of male students and 48.3% of female students wish to learn about health and diet. Among female subjects, BMI<18.2 strongly showed their consciousness of health and diet (p < 0.03).
Discussion
This study aimed to determine the health, nutritional knowledge and dietary behavior of university students in China. As a result, we recorded the distribution of BMI among Chinese students and found a low prevalence of obesity, a finding that is consistent with a study of Japanese female students (BMI≥25 overweight was 5.8%, BMI>30 of obesity was 0%) [4]. In the United States, 35% of the college students are reported to be overweight or obese (BMI≥25) [7]. According to the WHO definition of obesity, BMI>30 is the cut-off point [6]. The definition is based on research of Caucasian populations. Asian populations are reported to have a higher body fat (%) at a lower BMI compared to Caucasians [8]. The WHO expert consultation reported that BMI in Asian populations is related to disease at a lower level [9]. In order to compare obesity prevalence between ethnic groups, BMI cut-off points for Asians need to be considered by well constructed and standardized body composition studies. It is notable that in China, the prevalence of overweight individuals increased from 1991 to 1997, with the increasing rate changing from 6.4 to 7.7 [10]. The proportion of energy derived from the fat of both vegetable and animal sources increased each year. A recent study revealed that energy derived from dietary fat accounted for more than 30% of the total energy [11]. Changes in dietary composition, which correspond to socioeconomic growth, may accelerate the prevalence of obesity in China.
The results of our study show that the majority of students regularly eat three times per day, and almost 80% of students eat vegetables and fruit twice per day. These eating habits ought to be encouraged. The traditional Chinese diet contains plenty of vegetables and is rice-based. The present study reported a high proportion of Chinese students eat breakfast daily. In contrast, a dietary survey of young Japanese subjects revealed a low rate of individuals engaged in regular eating patterns [12]. The skipping of breakfast has been associated with lower nutritional status and the risk of cardiovascular diseases [13]. It has also been reported that less adequate breakfast habits may contribute to the appearance and further development of obesity [14]. Therefore the importance of regular eating patterns cannot be overemphasized in nutritional education.
Our results showed that body figure perception was significantly different between female and male students. A number of researchers have investigated the relationship of body image and gender role. Women tend to desire a thinner figure, express more anxiety about becoming fat, and are more likely to diet than men [15,16]. In contrast, men have reported a desire for a heavier physique and muscularity [17]. In recent years, eating disorders have been increasing dramatically among young women. The results of our study did not confirm this suggestion to the level of statistical significance; however, it is worth pointing out that 65.0% of female students with BMI<20, which is under to normal weight range, indicated a desire to be thin. Dissatisfaction with body figure and eating disorders are closely related [18-20]. Being young, female, and dieting are identified risk factors that have been reliably linked to the development of eating disorders [21]. It was speculated that some of the students who were preoccupied with a thin body may develop eating disturbances. Thus, the promotion of healthy weight management practices should be considered when developing health education programs.
Conclusions
In conclusion, our findings reveal that the majority of students were classified into the normal BMI group, with the prevalence of BMI >30 obesity being very low in this study sample. Young female students had a greater desire to be thinner than male students. Habits involving regular eating patterns and vegetable intake were found and represent practices that ought to be encouraged. The meal and snack patterns in Chinese students were very similar to the traditional eating pattern model, although diets are changing rapidly in China and other low-income countries. The university and college arenas represent the final opportunity for nutritional education of a large number of students from the educator's perspective. Our findings suggest the need for strategies designed to improve competence in the area of nutrition, especially with respect to information relating to sources of nutrition and healthy weight management. Furthermore, public demand for health and nutritional information should be taken into consideration when implementing strategies aimed at improving the nutritional well-being of individuals.
Authors' contributions
R.S carried out questionnaire design, manuscript drafting and total coordination of the study. K.T has been involved in drafting and revision of the article. R.A contributed to the data entry and its analysis. L.CJ contributed to the questionnaire design, data collection and language translations. N.S contributed to final approval of the manuscript.
Supplementary Material
Additional File 1
Table 2 containing the results of questions related to lifestyle practices with special reference to food habit. The meal patterns, consumption of fruits and vegetables, consumption of fried foods, consumption of alcohol were assessed in male and female students. The Chi-square analyses were employed to compare the behavioral differences by gender. The evaluations of statistical significance were made at the p < 0.05.
Click here for file
Additional File 2
Table 3 contains the results of body shape perception and health consciousness of male and female students. Male and female respondents were categorized in to 4 groups respectively, according to mean BMI of ± 1 standard deviation (SD). Analyses were made between BMI groups using Chi-square analysis. The evaluations of statistical significance were made at the p < 0.05.
Click here for file
Acknowledgements
The authors express their appreciation for the invaluable partnership and support of Dr. Wang of Beijing University, Dr Zhao of Kunming Medical College and all the study participants of both institutes. We also thank Dr. Shigeki Minakami for valuable comments on the manuscript.
Figures and Tables
Figure 1 BMI distribution of Chinese university students. The BMI of male and female students was categorized into 4 groups according to mean BMI ± 1 standard deviation (SD).
Table 1 Characteristics of Participants
Total Male Female
Variable n = 512 n = 212 n = 300
Age (y) 20.4 ± 1.9 20.3 ± 1.7 20.4 ± 2.0
weight (kg) 56.9 ± 9.2 63.7 ± 8.8 52.1 ± 5.9
height (cm) 165.8 ± 7.8 172.3 ± 5.5 161.2 ± 5.6
BMI (kg/m2) 20.6 ± 2.2 21.4 ± 2.5 20.0 ± 1.8
Gender distribution in the sampled population. BMI is based on self-reported height and weight. BMI = weight [kg] / height [m]2
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| 15703071 | PMC553986 | CC BY | 2021-01-04 16:05:47 | no | Nutr J. 2005 Feb 9; 4:4 | utf-8 | Nutr J | 2,005 | 10.1186/1475-2891-4-4 | oa_comm |
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RetrovirologyRetrovirology1742-4690BioMed Central London 1742-4690-2-101571591310.1186/1742-4690-2-10ReviewMolecular strategies to inhibit HIV-1 replication Nielsen Morten Hjuler [email protected] Finn Skou [email protected] Jørgen [email protected] Department of Molecular Biology, University of Aarhus, C.F. Møllers Alle, Bldg. 130, Room 404, DK-8000 Aarhus C, Denmark2005 16 2 2005 2 10 10 22 12 2004 16 2 2005 Copyright © 2005 Nielsen et al; licensee BioMed Central Ltd.2005Nielsen et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The human immunodeficiency virus type 1 (HIV-1) is the primary cause of the acquired immunodeficiency syndrome (AIDS), which is a slow, progressive and degenerative disease of the human immune system. The pathogenesis of HIV-1 is complex and characterized by the interplay of both viral and host factors. An intense global research effort into understanding the individual steps of the viral replication cycle and the dynamics during an infection has inspired researchers in the development of a wide spectrum of antiviral strategies. Practically every stage in the viral life cycle and every viral gene product is a potential target. In addition, several strategies are targeting host proteins that play an essential role in the viral life cycle. This review summarizes the main genetic approaches taken in such antiviral strategies.
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Introduction
HIV-1 is a lentivirus belonging to the retrovirus family. The virus is diploid and contains two plus-stranded RNA copies of its genome. The approximately 9 kb RNA genome encodes at least 9 proteins, Gag, Pol, Env, Tat, Rev, Nef, Vif, Vpu and Vpr of which only the former five are essential for viral replication in vitro. HIV-1 primarily infects CD4+ T-lymphocytes and monocytes/macrophages, but also astrocytes and cells of the central nervous system (brain microglial cells) are targets. The infection spreads to the lymphatic tissue that contains follicular dendritic cells that may act as a storage place for latent viruses. Over time, virus replication leads to a slow and progressive destruction of the immune system. The development of possible methods that can delay progression of the infection or block replication of HIV-1 in infected individuals has been the subject of dedicated research efforts over the past decades. One important issue is that HIV-1 makes use of the replication machinery of the host cell, which minimizes the number of potential viral targets. On the other hand, the close host-virus relationship limits the evolutionary freedom for the viral components that interact with the host molecules.
The aim of this review is to take a comprehensive look at the molecular, intracellularly based antiviral strategies that have been reported in literature, and to discuss their potential for development into clinical protocols. We will not discuss vaccine-based strategies that recently have been reviewed in [1] and [2].
Interfering strategies against HIV-1
The inhibition strategies can be divided into two groups:
The RNA-based strategies including anti-sense RNA (or other chemically modified nucleic acids), RNA decoys (sense RNA), ribozymes, RNA aptamers, small interfering RNA (siRNA), microRNAs (miRNAs) and the protein-based strategies including transdominant negative proteins (TNPs), chimeric proteins (fusion proteins), nucleases, anti-infective cellular proteins, intracellular single-chain antibodies (sFvs) and monoclonal antibodies (Mabs). In addition, other strategies based on suicide genes, protease inhibitors and nucleoside or non-nucleoside analogues have shown to possess the ability to reduce HIV-1 replication.
The HIV-1 life cycle including the inhibiting strategies targeted against the various steps in the viral life cycle is summarized in Fig. 1 and listed in table 1. Below follows a more detailed description of the strategies taken to target individual steps of the viral life cycle. Note that strategies targeting the viral genes or mRNA directly all possess an uncertainty as to what viral function(s) are affected due to the overlapping nature of some of the reading frames [3].
Figure 1 Summarization of the HIV-1 life cycle and the inhibiting strategies targeting the different steps in the viral life cycle.
Table 1 Interfering strategy Target RNA/protein Interference site(s) Mechanism References
Anti-sense RNA Cellular CCR5 and CXCR4 co-receptors Viral entry Inhibition of CCR5 and CXCR4 gene expression 6, 18, 19
Psi-gag and U3-5'UTR-gag-env regions Pre-integration Co-packaged with genomic RNA, inhibits RT in incoming virions 6
Cellular CyPA gene Pre-integration The skipping of internal CyPA encoding exons reduces CyPA biosynthesis and thereby inhibits the reverse transcription 37
Tat/TAR interaction HIV-1 transcription Inhibits transcriptional regulation of HIV-1 gene expression 6, 7, 45, 76
Rev/RRE interaction Nuclear export Inhibits transport of unspliced and single spiced viral RNAs 6
5'UTR HIV-1 translation Inhibits the translation process 6
Psi-gag region Viral assembly Inhibits packaging of genomic RNA 6, 7, 45
5'-leader-gag region Viral assembly Inhibits the formation of Gag and Env multimeric complexes during viral assembly. 7, 18
Env and Vif encoding regions Viral assembly Inhibits env and vif gene expression 70
Nef encoding region Viral release Inhibits nef gene expression and thereby CD4 and MHC I downregulation 7
Pol encoding region Viral maturation Inhibits pol gene expression 70
RNA decoys RT enzyme Pre-integration Competes with HIV-1 RNA for the binding of RT 6
HIV-1 TAR region Pre-integration Competes with cellular tRNA3Lys for the binding to RT and primes the reverse transcription from the TAR region instead of the PBS region 6
Tat and Tat-containing RNA polymerase II transcription complexes HIV-1 transcription Inhibits Tat regulated transcription 6, 7, 18, 51
Rev protein Nuclear export Recruits Rev molecules and thereby prevents their interaction with the viral transcript 6
NC domain of the Gag protein Viral assembly Inhibits packaging by interfering with the NC domains ability to recognize the genomic RNA 6, 45
Ribozymes Cellular CCR5 and CXCR4 co-receptors Viral entry Cleaves CCR5 and CXCR4 mRNAs 6, 18
HIV-1 Gag and Pol encoding region and the U5 region Pre-integration Cleaves the viral RNA before reverse transcription is completed 6, 36
RRE and the Rev encoding region Nuclear export Cleaves the viral RNA 6, 7
U5 HIV-1 translation Cleaves off the 5'-cap structure localized on HIV-1 mRNAs 6, 7
Psi Viral assembly Cleaves HIV-1 RNAs before packaging 6, 7
Gag encoding transcripts Viral assembly Inhibits the formation of multimeric Gag and Env complexes 7, 18
SU encoding region Viral assembly Cleaves different conserved regions in the SU sequence 7
Nef encoding region Viral release Inhibits downregulation of CD4 and MHC I 7
RNA aptamers RT enzyme Pre-integration Displays high affinity and specificity for the RT enzyme and acts as templates analogues 31
Rev protein Nuclear export Possesses higher affinity for Rev than the RRE sequence and can therefore interfere with Rev function 57
siRNA Cellular CCR5 and CXCR4 co-receptors Viral entry Impairs the SU-chemokine co-receptor interaction 21, 22
CD4 protein Viral entry CD4 protein expression inhibited 23, 24
CD4-binding domain of the SU protein Viral entry Inhibits the CD4-SU interaction 26
The viral LTR region or the vif and nef encoding regions Pre-integration Guides the viral genomic RNA towards a siRNA-mediated destruction 34, 52
RT encoding region Pre-integration Inhibits RT gene expression 35
Cellular CyPA gene Pre-integration Reduces CyPA biosynthesis and thereby the reverse transcription 37
CA encoding region Pre-integration Mediates cleavage of pre-spliced viral RNA in the cytoplasm and prevents integration 23, 24, 42
Tat encoding region HIV-1 transcription Inhibits Tat transactivation 35, 49, 50
NF-κB p65 subunit HIV-1 transcription Inhibits NF-κB transcriptional activation 35, 49
3'-terminus of the nef gene HIV-1 transcription Mediates cleavage of all spliced and unspliced RNA produced from the provirus 42
Rev transcript Nuclear export Inhibits Rev mediated export of unspliced and single spliced RNAs 49, 61
Gag and Nef encoding regions HIV-1 translation Mediates cleavage of both spliced and unspliced RNA produced from the provirus 23, 24, 34, 42
shRNA/ miRNA Nef encoding region HIV-1 translation nef shRNAs act by blocking RNA stability or RNA translation 62
Transdominant negative proteins (TNPs) Interactions between Tat/TAR complex and cellular co-factors HIV-1 transcription Tat-mutants inhibit the function of the Tat protein by recruiting important cellular co-factors 7, 18, 45
Rev protein Nuclear export Rev-mutants e.g. act by preventing the interaction with cellular co-factors or by sequestering the Rev protein in the cytoplasm 7, 18, 25, 57, 58, 59
Cellular Sam68 Nuclear export Sam68 mutants inhibit Sam68 transactivation of RRE and Rev function 60
Cellular Tsg101 Viral assembly Tsg101 mutants inhibit the transport of the Gag polyprotein into multivesicular bodies 71
Vif protein Viral assembly Vif mutants block an early processing of the Gag protein 66
Cellular INI1 Viral assembly INI1 mutants e.g. interact with the integrase domain of the Gag-Pol polyprotein and interfere with prober multimerization of Gag and Gag-Pol 39
The formation of Gag and Env multimeric complexes Viral assembly E.g. interferes with complex formation 4, 6, 18
Nef protein Viral release Nef mutants e.g. inhibit CD4 downregulation 66
SU protein Viral release Overexpressed CD4 variants bind and sequester virion progeny within the cell 19
HIV-1 protease Viral maturation Pro-mutants prevent protease activation 7
Chimeric / fusion proteins SU protein Viral entry A tetrameric version of sCD4, PRO542, which is fused to the conserved region of IgG2, prevents the CD4-SU interaction 8, 13
Proviral DNA Pre-integration An IN targeted sFv-nuclease fusion protein associates with the pre-integration complex and cleaves proviral DNA after integration has occurred 7, 18
TAR element HIV-1 transcription Designed Tat-nuclease fusion proteins recognize and cleave all HIV-1 RNA transcripts 5
RRE sequence Nuclear export Designed Rev-nuclease fusion proteins recognize and cleave all HIV-1 RNAs carrying the RRE sequence 5
Rev protein Nuclear export A NS1RM-Rev mutant, with a dominant retention activity, forms mixed oligomers together with Rev and inhibits nuclear export 7, 57
The TAR and RRE elements HIV-1 transcription / nuclear export A designed fusion protein, Tev, containing the RNA binding domains of both Tat and Rev fused to a nuclease, inhibits both early and late viral gene products 5
Viral genomic RNAs Viral assembly Gag-, Vpr- and Nef-nuclease fusion proteins cleaves viral RNA, either during or after the viral assembly 5, 7
Psi-element Viral assembly A NC-nuclease fusion protein recognizes and cleaves all unspliced RNAs in the cytoplasm 5
HIV-1 protease Viral maturation An overexpressed Vpr fused to several protease cleavage sites overwhelms the protease activity by a competitive mechanism 7, 74
Nucleases Tat encoding region HIV-1 transcription Inhibits Tat transactivation 6, 7, 45
TAR element HIV-1 transcription Inhibits Tat transactivation 6, 7, 45
Chemokine ligands Cellular CCR5 and CXCR4 co-receptors Viral entry E.g. interacts directly with the co-receptors, mediates receptor blockade or mediates receptor down-regulation 8, 9, 11, 12, 13, 14, 16
Anti-infectious cellular proteins SU protein Viral entry A truncated form of CD4, sCD4, inhibits the fusion event by binding to the SU protein and thereby extending the distance to the TM protein 8, 13, 19
Intracellular antibodies (sFvs) SU protein Viral entry Inhibits the CD4-SU interaction 18
The TM pre-hairpin intermediate Viral entry Inhibits the interaction between the fusion peptide and the cell membrane 29
RT enzyme Pre-integration Inhibits RT function 7, 18
IN enzyme Pre-integration Inhibits IN function 7, 18
Tat protein HIV-1 transcription Interacts with the Tat protein and restrains it in the cytoplasm 7, 18
Rev protein Nuclear export Recruits Rev in the cytoplasm 7, 18, 25, 57
The CD4 binding region of the SU protein Viral assembly Interacts with the Env protein and restrains it in the ER 7, 18
Monoclonal antibodies (Mabs) Cellular CCR5 and CXCR4 co-receptors Viral entry E.g. inhibit the SU-chemokine co-receptor interaction, HIV-1 fusion or entry 12
Extracellular loop on CCR5 SU-chemokine co-receptor interaction Inhibits HIV-1 fusion and entry 12
Nucleoside analogues (NRTIs) RT enzyme Pre-integration Prevents the continued polymerization of the DNA chain 8
Non-nucleoside analogues (NNRTIs) RT enzyme Pre-integration Interact directly and non-competitively with the RT enzyme and inhibits its function 8
Integrase inhibitors (Oligonucleotides, dinucleotides and chemical agents) IN enzyme Pre-integration These inhibiting agents either block the catalytic function of the IN enzyme by binding to the integrase binding site located in the viral DNA, or by interacting with the catalytic core domain of the IN enzyme itself 40, 41
Protease inhibitors Protease enzyme Viral maturation Act as transition state analogous and bind to the protease more tightly than the natural substrate 11, 8, 73
Examples of other inhibiting agents Cellular CCR5 and CXCR4 co-receptor Viral entry Chemokine ligands potently inhibit the SU-chemokine co-receptor interaction 8, 9, 10, 11, 12, 13
Cellular CCR5 and CXCR4 co-receptors Viral entry Designed peptides e.g. act by disrupting helix-helix interactions, which may influence co-receptor structure, or by associating with the co-receptor surfaces and thereby inhibit the interaction with the SU protein 8, 12
Cellular CXCR4 co-receptor Viral entry AMD3100, a small organic molecule, acts by spanning the main ligand-binding cavity of CXCR4, which constrains the co-receptor in an inactive conformation 12
Cellular CCR5 co-receptor Viral entry Cyclophilin-18, a protein derived from T. Gondii acts as a CCR5 antagonist and thereby inhibits fusion and infectivity of R5 HIV-1 isolates 17
SU protein Viral entry CV-N, a 11 kDa protein with high affinity for the SU protein, inhibits the SU-CD4 interaction 15
The N- and C-peptide regions on the TM pre-hairpin intermediate Viral entry Designed N-, C-, and D-peptides interacts with the pre-hairpin intermediate and inhibit the fusion event 13, 27, 28
RT enzyme Pre-integration Small peptides, about 15–19 amino acid long, act by interfering the dimerization process of the RT enzyme 30
The Tat/TAR interaction HIV-1 transcription The TR87 compound acts by competing with Tat for binding to TAR-RNA 46
Protein /TAR RNA interaction HIV-1 transcription Pyrrolo [2,1-c][1,4]benzodiazepine-oligopyrrolo hybrids act by interrupting binding of cellular proteins and Tat to the TAR-RNA 47
Protein /TAR RNA interaction HIV-1 transcription Aromatic polyamidines carrying a Br atom inhibit cellular and viral protein-TAR RNA interactions 48
Cellular NF-κB HIV-1 transcription NF-κB activity is inhibited by minocycline, a second-generation tetracycline 38, 54
Rev Nuclear export Peptides targeted against the NES domain inhibit Rev function 57
The cellular protease furin Viral assembly Peptides mimicking a conserved target sequence inhibit furin activity and thereby cleavage of the Env protein within the ER 72
HIV-1 infected cells All A Tat-Casp3 fusion protein induces apoptosis after cleavage and activation by the HIV-1 protease 79
Virus-receptor interaction and entry
HIV-1 infection is initiated by binding of the virion gp120 surface subunit (SU protein) to the CD4 receptor. The SU protein is attached to the virus by a non-covalent binding to the gp41 transmembrane subunit (TM protein). Both SU and TM are proteolytically cleaved from the Envelope (Env) precursor protein by a cellular convertase, furin, within the endoplasmatic reticulum (ER). Both remain noncovalently attached and are targeted to the host plasma membrane by vesicular transport. The SU protein is responsible for receptor recognition on CD4+ T-lymphocytes and the TM protein mediates the fusion between the viral membrane and the host cell membrane [4,5].
Binding to CD4 induces a structural alteration in SU that exposes the binding site for a co-receptor of the chemokine family. The major co-receptors required for entry of HIV-1 are the chemokine receptor molecules CCR-5 (R5 HIV-1 isolates) and CXCR-4 (X4 HIV-1 isolates), which are used by monocytes/macrophage-tropic and T-cell tropic HIV-1 viruses, respectively [6]. When the SU protein binds to the co-receptor the result is another structural alteration exposing the N-terminal part of TM. This part, also known as the fusion-peptide, mediates the fusion between the viral and host membranes. The Env protein is also capable of mediating fusion between infected and non-infected cells by a process known as syncytium formation [4,7,8].
Current strategies are targeting particularly the CD4-SU interaction, the SU-chemokine co-receptor interaction, and the TM-mediated virus-cell membrane fusion process.
The SU-chemokine co-receptor interaction
CCR-5 and CXCR-4 co-receptors have specific chemokine ligands/antagonists that possess the ability to block the virus infection. The molecules that bind to the co-receptors can be divided into four categories: naturally occurring chemokines and their derivatives, peptides and small molecules (< 1 kDa), and Mabs, which recognize epitopes on for instance the extracellular domains of certain receptors.
Examples of chemokine ligands (beta-chemokines) that inhibit infection of R5 isolates include RANTES, a physiological ligand for the HIV-1 co-receptors CCR3 and CCR5. RANTES is actively secreted by normal T-cells. Derivatives of this peptide have been used, including aminooxypentane (AOP)-RANTES [9], and from a recent study, Nα-(n-nonanoyl)-des-Ser1 [L-thioproline2, L-α-cyclohexyl-glycine3] RANTES (PSC-RANTES) [10]. RANTES is an antagonist that besides having the ability to interact with CCR5 also has a downregulating effect on the co-receptor. RANTES can however induce chemotaxis and promote unwanted inflammatory side effects. Therefore AOP-RANTES was created by chemical modification of the amino terminus. This analogue does not promote any inflammatory side effects, and in addition it can prevent chemotaxis induced by e.g. RANTES. AOP-RANTES is a very strong antagonist that has a high affinity for CCR5, elicits rapid endocytosis of CCR5, and prevents recycling of the co-receptor back to the surface. PSC-RANTES is chemically identical to native RANTES except for the substitution of a nonanoyl group, thioproline, and cyclohexylglycine for the first three N-terminal amino acids of the native protein. This analogue acts in the same way, but has shown more potent in vitro antiviral activity than AOP-RANTES. Furthermore, it has successfully protected rhesus macaques from intravaginal exposure to a chimeric simian/human immunodeficiency virus containing an R5-tropic envelope of HIV-1 [10].
In addition to RANTES and its derivatives, the chemokine ligands macrophage inflammatory proteins 1alpha/beta (MIP-1alpha and MIP-1beta) also show an inhibiting effect by mediating a receptor blockade [8,11-13]. Examples of chemokine ligands that inhibit infection of X4 isolates include stromal cell-derived factor-1alpha (SDF-1alpha) and its derivatives that inhibit HIV-1 fusion and entry by minimizing the accessibility to the co-receptor on the cell surface and by inhibiting the SU-CXCR4 interaction [9,11-13].
The CCR5 amino-terminal domain is thought to play an important role in virus fusion and entry. This knowledge has been utilized in the development of anti-CCR5 Mabs whose epitopes include residues in the amino-terminal domain. Mabs of this kind strongly inhibit SU binding to CCR5 but only moderately inhibit HIV-1 fusion and entry [12]. Another type of Mab, the anti-ECL2 Mab whose epitopes include residues from one of the extracellular loops on CCR5 (ECL2), potently inhibits HIV-1 fusion and entry, but only moderately inhibits SU binding [12]. PRO 140, also an anti-CCR5 Mab, inhibits viral fusion with the cell membrane at concentrations that do not prevent the CCR5 chemokine receptor activity. It binds a complex epitope spanning multiple extracellular domains on CCR5, and although it acts as a weak antagonist it does not induce signaling or downregulation of CCR5. It is thought that the antiviral effect is exerted through a mechanism involving receptor blockade [14]. Mab 12G5 is a monoclonal antibody that recognizes an epitope on CXCR4. This epitope is also present in ECL2, and binding inhibits HIV-1 fusion [12,15]. A potential disadvantage of this strategy is that binding of the antibody to a receptor may trigger unwanted signal transduction [14,16].
Peptides, resembling the CCR5 transmembrane helices, inhibit HIV-1 replication and chemokine signaling by disrupting helix-helix interactions, which may influence the CCR5 structure [12]. T22 is a positively charged cyclic 18-mer antimicrobial peptide, which presumably inhibits SU-CXCR4 interaction by associating with the negatively charged surface of CXCR4 [8,12]. A truncated form of SDF-1alpha, consisting of the 16 amino-terminal residues of SDF-1alpha, also seems to possess such a blocking effect [12].
Recently, a new kind of CCR5 antagonist has been discovered in a protozoan parasite, Toxoplasma gondii [17]. This protein, cyclophilin-18 (C-18), has several potential antiviral properties including CCR5 binding, induction of the production of interleukin-12 (IL-12) from murine dendritic cells, inhibition of fusion and infectivity of R5 isolates by co-receptor antagonism and blocking of syncytia formation.
Small organic molecules, such as AMD3100, potently inhibit HIV-1 replication by an interaction with residues present on one of the CXCR4 extracellular loops, ECL2, and residues within a transmembrane helix, TM4. Upon binding to these residues AMD3100 spans the main ligand-binding cavity of CXCR4, which probably constrains the co-receptor in an inactive conformation [12].
Individuals with a homozygous deletion in the gene encoding CCR5 are healthy and protected against HIV-1 transmission, which suggests that down regulation may not pose any clinical side effects. This knowledge has led to the development of strategies that directly target the mRNA encoding CCR5 or CXC4, either by ribozymes [6,18], anti-sense RNA [6,18,19] or RNAi [20]. The latter strategy, the siRNA approach, has led to successful blocking of HIV-1 entry, protection of cells from infection and delay of virus replication [21-24]. Interestingly, it is thought that single-stranded siRNAs (the anti-sense strand of a siRNA duplex) act through the same RNAi pathway, but at a later stage than double-stranded siRNA, thereby requiring less time to exert their antiviral activity [21,25].
The CD4-SU interaction
Soluble CD4 (sCD4) is an anti-HIV-1 protein, which can be expressed and secreted from genetically engineered cells. It is a truncated form of the CD4 receptor, composed of the ectodomain that inhibits laboratory-adapted strains of HIV-1. sCD4 probably prevents the binding of the virus to the cell, by binding directly to Env, or indirectly by inducing or repressing cellular factors that influence the viral gene expression [18,19].
When sCD4 binds to SU it acts by extending the distance to TM, which inhibits the fusion. But when used against primary isolates, sCD4 was much less successful because of a lower affinity for sCD4. Surprisingly, some isolates became more infectious upon sCD4 treatment. An explanation for this may be that an interaction between the SU protein and sCD4 induces changes in SU, allowing it to bind the co-receptor with higher affinity or increased kinetics. In addition this interaction can eventually facilitate the fusion of HIV-1 with CD4- cells expressing the co-receptor [13]. This has led to the development of a tetrameric version of sCD4, PRO542, in which the SU-binding region of CD4 is fused to the conserved region of human immunoglobulin IgG2. This fusion protein has a high affinity for the SU protein and has shown promising results in phase I clinical trials [8,13].
siRNA-directed silencing of CD4 mRNA expression has been shown to specifically inhibit HIV-1 entry and thus HIV-1 replication [23,24]. However, CD4 silencing in vivo may interfere with its role in normal immune function. Thus an approach targeting the CD4-binding domain of the SU protein would be more relevant. This has successfully been achieved by expressing a 0.5 kb dsRNA containing the major CD4-binding domain of the SU protein, as the target of the env gene. By this approach it has been possible to significantly suppress the expression of the HIV-1 CA-p24 antigen in human peripheral blood mononuclear cells (PBMCs) and in HeLa-CD4+ for a relatively long period of time [26].
Strategies based on the intracellular expression of antibodies specific for the HIV-1 envelope (anti-SU) have also been shown to inhibit virus replication. This strategy is based on the usage of sFvs, containing the smallest structural domain that still possesses complete antigen and binding-site specificity of the parental antibody. They are secreted into the medium where they probably act as inhibitors by direct interaction with the viral proteins [18] to neutralize the virus [19].
Cyanovirin (CV-N), an 11 kDa protein originally isolated from cyanobacteria, potently inactivates diverse strains of HIV-1. It has a high affinity for the SU protein, and when bound it inhibits the SU-CD4 interaction. CV-N possesses the advantage that even high concentrations are non-toxic and it is an extremely stable protein. CV-N has also been coupled to a cytotoxin (Pseudomonas exotoxin), thereby selectively killing HIV-1 infected SU-expressing cells [15].
The TM-mediated virus-cell membrane fusion
As the SU protein binds to CD4, it initiates conformational changes in SU, making the interaction between the SU protein and the co-receptors more favorable. After attachment to the co-receptor further conformational changes occur in both the SU and TM proteins, thus weakening their interaction. During this process a transitory pre-hairpin intermediate of the TM protein is created, freeing the previously buried fusion peptide to interact with the host-cell membrane. This exposes the N-peptide and the C-peptide regions on the pre-hairpin intermediate that have been targets for several inhibiting strategies including synthetic C-peptides, N-peptides and sFvs.
C-peptides are based on the C terminal end of the fusion peptide, and mimics this part of the fusion peptide when it has its correct fusogenic conformation. T-20, a 36-amino acid C-peptide, is a potent inhibitor of HIV-1 infection. It acts through a dominant negative mechanism and interacts by binding to a conserved domain on the N-peptide present in the pre-hairpin intermediate. The function of this domain is to mediate a structural change, which allows the pre-hairpin intermediate to form a fusogenic hairpin state. Binding of T-20 inhibits this process and thereby impedes fusion. Disadvantages of the C-peptide strategy are the cost of C-peptide synthesis and the relatively large amounts necessary for an antiviral effect. In addition, their size makes them non-amenable to oral routes of entry and they must be injected instead [13,27,28].
The 5-Helix is a 25 amino acid N-peptide consisting of five of the six helixes constituting the C-peptide. The peptide is presumed to inhibit fusion, through binding with high affinity to the C-peptide. However because N-peptides have a strong tendency to aggregate the inhibition could also be due to their intercalation into the TM amino-terminal coiled coil [27,28].
A third kind of peptides named D-peptides have also proven effective. These peptides are small 16–18 D-amino acids residues that specifically bind to three hydrophobic pockets present at the end of the N-peptide. Since such peptides are unnatural, they are resistant to proteolytic degradation, which makes them attractive for clinical use [13,27].
Recently, a non-neutralizing antibody directed against epitopes exposed on the fusion peptide has been reported to possess antiviral effect [29]. This antibody does not neutralize HIV-1 entry when produced as a soluble protein. However, when expressed on the cell surface as a membrane-bound sFv, it is turned into a neutralizing antibody, which markedly inhibits HIV-1 replication and cell-cell fusion by a mechanism that is thought to involve an interaction with the exposed fusion peptide. This results in inhibition of the subsequent fusion process. In the same study, this sFv was targeted into the ER and trans-Golgi network of HIV-1 susceptible cell lines where it was found to significantly block the maturation process of the viral Env protein resulting in an impairment of viral assembly.
Reverse transcription and proviral integration
After fusion the viral core enters the cytoplasm and the viral RNA is copied into double-stranded cDNA. This process is mediated by the viral reverse transcriptase (RT) enzyme in a complex consisting of RT, the viral genome, and a cellular tRNA3lys. The latter acts as primer and initiates negative strand DNA synthesis by binding to the primer binding site (PBS) region, located immediately 3' to the U5 region [6,4]. RT possesses three essential activities important for replication of the virus: RNA-dependent DNA polymerase (i.e. reverse transcriptase), RNase H activity (i.e. cleaves the genomic RNA in RNA/DNA hybrids during DNA synthesis), DNA-dependent DNA polymerase activity (i.e. for synthesis of the second strand of the proviral DNA) [6,4].
Because RT is essential for viral replication it has been one of the most popular targets. This has led to the following antiviral strategies.
RT-targeted strategies
Inhibiting strategies against RT involve the utilization of nucleosides and non-nucleosides. The nucleoside analogues lack the 3'-hydroxyl group, prevent the continued polymerization of the DNA chain, and are usually named nucleoside reverse transcriptase inhibitors (NRTIs). Clinically approved examples include Zidovudine (AZT), Didanosine (ddI), Zalcitabine (ddC), Lamivudine (3TC), Abacavir succinate and Stavudine (d4T) [8].
The non-nucleoside analogues, often referred to as non-nucleoside reverse transcriptase inhibitors (NNRTIs), act at the same step in the viral life cycle as the nucleoside analogous, but by a significantly different mechanism. Instead of acting as false nucleosides, the NNRTIs bind directly and non-competitively to RT in a way that inhibits the enzyme's activity. Examples of clinically approved NNRTIs include Nevirapine, Delaviridine and Efavirenz [8].
NRTIs bind to the deoxynucleoside triphosphate-binding pocket, which is formed partly by the template-primer nucleic acid and partly by the protein surfaces. NNRTIs bind to a hydrophobic pocket exclusively present in the RT enzyme of M subtype HIV-1. When used in combination they have a more pronounced antiviral effect.
The RNA decoy strategy aimed at RT involves the expression of RNAs lacking the PBS region, thus preventing it from acting as template for reverse transcription. The RNA competes with HIV-1 RNA when RT makes the first jump during the first strand transfer [6]. Another decoy was designed to be co-packaged together with genomic RNA into new virions where it competes subsequently with genomic RNA for RT binding [6]. Also, a designed tRNA3Lys mutant containing an 11 nucleotide 3'-end complementary to the HIV-1 TAR region, shows an inhibiting effect. This mutant competes with cellular tRNA3Lys for the binding to RT and primes reverse transcription from the TAR region instead of the PBS region [6].
Other strategies against the RT enzyme involve the usage of small peptides, about 15–19 amino acids long, that inhibit RT activity by interfering with the dimerization process of the RT enzyme. The amino acid sequence corresponds to the so-called connection domain of RT, in particular a tryptophan-rich 19-mer sequence corresponding to residues 389–407, which efficiently inhibits viral replication [30]. Likewise, strategies based on intracellular expression of sFvs [7,18] and RNA aptamers [31-33] targeted against the RT enzyme are potent inhibitors of HIV-1 replication. The aptamers all recognize the same template-primer-binding cleft on RT. Some of these RNA aptamers have the potential to form pseudoknot-like secondary structures, which mimic the conformation of the template-primer when associated with the RT enzyme. Thus, these aptamers are termed template analogue RT inhibitors (TRTIs). Selectivity of the RNA aptamers is directly related to their three-dimensional structure. Utilization of the TRTI aptamers has the following benefits: 1) Aptamers have a unique specificity and a strong binding affinity for the RT enzyme. 2) Aptamers inhibit the RT enzyme competitively and will unlikely inhibit other viral or cellular proteins, thus minimizing the risk for any appreciable toxic side effects. 3) Since aptamers are expressed in the infected cell, the aptamers will be co-packaged into new virions and inhibit the next round of replication. 4) Because of the large interface of the aptamer-binding pocket, the risk of escape mutants is significantly reduced. Furthermore, mutations in essential binding domains, such as the template-primer-binding pocket, will likely impair the binding of the RT enzyme to the viral genome [31].
Anti-sense RNAs designed to be complementary to the Psi-gag and the U3-5'UTR-gag-env regions have been shown to inhibit RT in new virion particles. They are co-packaged together with the genomic RNA into the virus progeny, and inhibit reverse transcription by hybridizing to the genomic RNA [6].
siRNAs directed against several regions of the HIV-1 genome, including the viral long terminal repeat (LTR) and the accessory genes vif and nef have provided evidence that the viral genomic RNA, as it exists within the virion as a nucleoprotein reverse transcription complex, is amenable to siRNA-mediated degradation [34]. In addition, siRNAs targeted against the RT gene alone have shown potent inhibition of HIV-1 replication in MAGI cells [35].
Hammerhead ribozymes targeted against the HIV-1 gag region will cleave the viral RNA before reverse transcription is completed [6]. Hairpin ribozymes, designed to cleave a conserved site in the U5 region of the HIV-1 RNA can likewise inhibit replication [6]. Especially the tRNAVal-U5-ribozyme has shown promising results and is currently being tested in clinical trials. Moreover, hairpin and hammerhead ribozymes targeted against the HIV-1 pol region also show promising results [6,36]. In the latter strategy a hammerhead ribozyme has successfully been packaged into virions by linking it to the portion of the HIV-1 genome that provides the packaging sequence [36]. This intravirion targeting ribozyme has in the same study shown higher virus-suppressing activity than a nonpackageable counterpart.
Since host tRNA3Lys is being packaged into new virus particles, this molecule is often used when ribozymes have to be co-packaged. An example is the tRNA3Lys-hammerhead ribozymes targeted against the PBS region. Besides cleaving the HIV-1 RNA, the tRNA3Lys-ribozyme inhibits reverse transcription by competing with host tRNA3Lys for RT binding and/or for the binding to the PBS sequence. Also, when bound to the PBS, the tRNA3Lys-ribozyme is unable to prime reverse transcription [6].
In a study closely related to the earlier mentioned CCR5 antagonist, C-18, human cyclophilin A (CyPA) has been shown to be incorporated into HIV-1 during virion assembly through interaction with an exposed proline-rich loop within the capsid domain of Gag [37,38]. CyPA is required for efficient viral replication but not for cell viability meaning that its cellular function is probably being compensated for by other factors. It has been proposed that CyPA enhances HIV-1 infectivity during early post-entry events, but may also be required for viral entry. The proposed molecular interaction that underlies this enhancement is the CyPA proteins ability to mask the binding site for the human host restriction factor Ref1 and thereby counteracting its inhibitory activity, allowing reverse transcription to be completed. In an attempt to reduce CyPA biosynthesis, two different anti-sense strategies were used [37]. In one approach internal CyPA exons are skipped by means of modified derivatives of U7 small nuclear RNA (snRNA). U7 snRNA is the RNA component of the U7 small nuclear ribonucleoprotein (snRNP) involved in histone RNA 3'-end processing. By inserting appropriate anti-sense sequences into U7 snRNA it has successfully been converted from a mediator of histone RNA processing to an effector of alternative splicing. The other strategy involves the use of hairpin siRNA constructs targeting two different parts of the CyPA coding region. Both strategies greatly reduced the levels of CyPA, creating CEM-SS T-cells that sustain HIV-1 replication.
The next step is the translocation of the cDNA containing capsid into the nucleus. This process is mediated by independent pathways involving either the Vpr accessory protein, the matrix protein (MA) or the integrase (IN) protein. Vpr is thought to mediate the nuclear import of the preintegration complex through the nuclear pore complex (NPC) in non-dividing cells by interacting directly with proteins in the NPC. This transfer of viral DNA is mediated by a nuclear localization signal present in the Vpr protein. Furthermore, it has been shown that Vpr is involved in arresting HIV-infected cells in the G2 phase of the cell cycle, where the virus production has been shown to reach a maximum level [4-7]. Integration of proviral DNA is mediated by the viral IN enzyme by a process that requires the host protein integrase interactor 1 (INI1 / hSNF5). IN consists of an N-terminal zinc finger domain, a catalytic core domain, and a C-terminal domain that is important for binding HIV-1 LTR DNA [39,40]. It has two enzymatic functions; DNA cleavage and insertion of the provirus into the genome of the host [4,7]. IN recognizes short inverted repeats (att sites) at both ends of the proviral DNA and cleaves an AT overhang at the 5' end. Then it catalyzes the non-specific cleavage of the host genome and the subsequently ligation of the 5' overhang to the cellular genome [4].
Several strategies aiming at the IN function have been reported:
IN-targeted strategies
IN has no known functional analogue in human cells and is therefore an appealing target for inhibiting strategies, which generally involves the usage of oligonucleotides, dinucleotides and different kinds of chemical agents, such as dicaffeoylquinic acids (DCQAs) [40] and 2,4-dioxobutanoic acid analogous [41]. The integrase binding site in the U3 LTR region of the viral DNA contains a purine motif, 5'-GGAAGGG-3'. This motif has selectively been targeted by oligonucleotide-intercalator conjugates that interact with the viral DNA through triplex formation, thus blocking the catalytic functions of the IN enzyme [41]. Disadvantages of these compounds include the low intracellular permeability and the high mutation rate of HIV-1 that may result in nucleotide substitutions in the LTR.
The inhibiting effect of a dinucleotide, named pdCpIsodU, is due to its ability to interact with the catalytic core domain [41]. This molecule consists of a natural D-deoxynucleoside and an isomeric L-related deoxynucleoside joined together through a stereochemically unusual internucleotide phosphate bond, which makes the molecule resistant to 5'- and 3'-exonucleases. Through binding the molecule inhibits both the 3'-processing and the DNA strand transfer step.
DCQAs are non-competitive inhibitors that act by irreversible binding to the catalytic core domain. The exact chemical mechanism for this anti-IN activity is unknown, but it is thought to be caused by a simple redox-process. Two examples are 1-Methoxy-3,5-dicaffeoylquinic acid and 3,4-Dicaffeoylquinic acid. Both are relative non-toxic [40]. Finally, 2,4-dioxobutanoic acid analogous have been reported to possess potent anti-IN activity through inhibition of the DNA strand transfer step [41].
sFvs interacting with different domains on IN have been isolated, and by fusion with a nuclease, a fusion protein is created that can interact with IN in the pre-integration complex, leading to cleavage of proviral DNA. Likewise IN-specific sFvs have been shown to be inhibitory to HIV-1 replication [7,18].
Finally, siRNAs targeted against the capsid protein, p24-siRNA, is thought to interact with the gag gene in the unspliced viral RNA when present in the cytoplasm. Thereby, the viral RNA genome is cleaved before integration occurs [23,24,42].
HIV-1 transcription
Transcriptional regulation of HIV-1 gene expression is controlled by co-operative and cell-specific interactions between several host cells transcription factors, including AP-1, NF-κB, NF-AT, NF-IL-6, CREB, IRF, Sp1, LEF-1/TCF-1α, Ets-1 and USF, and the viral Tat protein [5,7,43]. The Tat protein recognizes a stem-loop structure, the trans-activation responsive element region (TAR), located in the 5'-end of the primary transcript (R region). Tat recruits a cellular co-factor, positive transcription elongation factor b (P-TEFb), composed of human cyclin T1 (hCycT1) and CDK9 (a CTD kinase). The hCycT1 component binds to the activation domain of Tat thereby increasing the affinity for TAR. This results in the formation of a Tat/TAR complex. Next, CDK9 phosphorylates the carboxy-terminal domain of the host cell RNA polymerase II, which stimulates the elongation process and thereby the overall transcriptional efficiency [4,44].
The Tat/TAR interaction is essential for activation of HIV-1 transcription and is therefore a popular target for inhibiting strategies. Another reason for choosing strategies directed against this step is that the Tat-TAR interaction is highly conserved. Thus the chance for development of escape mutants is very low, due to the fact that mutations in either Tat or TAR will cause an impaired interaction between them and thereby abolish HIV-1 replication.
One strategy is to express a Tat protein that displays a transdominant negative phenotype, which can inhibit the replication of HIV-1. These proteins act as competitors for Tat binding to an essential substrate or co-factor, or alternatively by associating with wild-type monomers to form an inactive mixed multimer. Examples include Tat proteins containing mutations in the activating domain, the protein-binding domain, or in the TAR binding domain [7,18,45]. An obvious disadvantage of this strategy is, as mentioned earlier, the mutants' ability to recruit co-factors important for maintaining of a normal cellular function. Tat function can also be impaired by using a single-chain antibody, sFv-Tat. When sFv-Tat interacts with the Tat protein, it restrains Tat in the cytoplasm, thus hindering its transcription-regulating function in the nucleus [7,18].
Several studies have shown promising results in blocking the interaction of cellular TAR RNA-binding proteins and viral Tat protein to TAR RNA. For instance these include a study in which a compound termed TR87 directly competes with Tat for binding to TAR [46], and a study involving pyrrolo [2,1-c][1,4] benzodiazepine-oligopyrrolo hybrids, which appear to interrupt protein/TAR RNA interactions and Tat-induced LTR-driven HIV-1 transcription [47], and finally a study were two aromatic polyamidines carrying a halogen atom, termed TAPB-Br and TAPP-Br, have demonstrated the potential to inhibit cellular and viral protein-TAR RNA interactions [48].
An inhibiting effect has also been observed using anti-sense-, nuclease-, or siRNA-based strategies directed against tat mRNA [6,7,35,45,49]. Notably, a recent study has demonstrated that tat siRNA delivered as pre-miRNA precursor is 80 % more effective than tat siRNA expressed as conventional short hairpin RNAs (shRNAs) [50]. Finally, anti-sense RNAs and nucleases targeting the TAR element have also shown promise as antivirals [5-7,45].
A Tat-nuclease fusion protein has been engineered by fusing the HIV-1 TAR RNA binding domain of HIV-1 Tat with the RNase H domain of HIV-1 RT. Since TAR is present at the 5' and 3' ends of all HIV RNAs, this Tat-nuclease can recognize and cleave all HIV-1 RNA transcripts. The Tat protein cycles in and out of the nucleus and the cleavage of HIV-1 transcripts should therefore take place both in the nucleus and in the cytoplasm [5].
TAR decoys represent another example of a suitable strategy. These sense RNAs act by interacting with Tat-containing RNA polymerase II transcription complexes that assemble on the HIV-1 promoter [7]. In addition the TAR decoy RNAs solely recruit the Tat protein and the cellular co-factor, P-TEFb, which is necessary for Tat-mediated transactivation [18]. To make this strategy more effective, the development of polymeric TAR decoys has been accomplished. Constructs with up to 50 TAR elements have been reported, but unfortunately these constructs also recruit essential functional cellular co-factors [6,18]. A TAR decoy based on the element of HIV-2 (TAR-2) has been shown to suppress HIV-1 replication more effective that the decoy based on HIV-1. The explanation for this is that the TAR-2 structure possesses three separate loop regions and may therefore more effectively compete with the single stem-loop structured TAR in HIV-1 for loop-binding cellular factors [51].
Besides targeting the Tat encoding regions, siRNAs have been directed against the Gag [23,24,42] and Nef [23,34] encoding regions. Both siRNAs show antiviral effects. The gag-targeted siRNA (p24-siRNA) is identical to the p24-siRNA utilized when inhibiting the pre-integration step and acts in the same manner. Since the nef gene is located in the 3' end of the HIV-1 genome and in many of the viral transcripts, a siRNA directed against the Nef encoding region will reduce the number of viral transcripts. Also a 3'-LTR directed siRNA has shown potently to suppress viral replication [42]. The 3'-LTR region was chosen, as it is in the noncoding sequence before the poly(A) tail, except for the Nef encoding RNA. Thus, by this specific siRNA approach it is possible to target both spliced and unspliced RNA produced from the provirus, whereas the p24-siRNA approach only targets the unspliced viral RNA. By combining these siRNAs a synergistic effect has been observed [23,24,34,42]. The high specificity of the RNAi approach also makes it vulnerable to inactivating mutations in the viral genome as was observed in a recent study [52]. Another promising strategy involving siRNAs includes targeting of the NF-κB p65 subunit [35]. The NF-κB p65 subunit is a key component for NF-κB transcriptional activation of HIV-1. During the early phases of HIV-1 infection in activated T lymphocytes, NF-κB binding to the HIV-1 LTR serves to stimulate the generation of at least some full-length transcripts for synthesis of Tat, which then stimulates the transcriptional elongation process. This is supported by the observation that siRNAs targeted against the NF-κB p65 subunit show a decrease of HIV-1 replication in MAGI and Jurkat cells. However, the NF-κB proteins are also critical for the regulation of immune function. They regulate the expression of a variety of genes encoding cytokines and cytokine receptors, chemokines, cell adhesion molecules, and cell surface receptors that are critical for T- and B-cell function. Therefore further studies are required before p65-siRNAs can be used in clinical trials [3,35,49]. Finally, a siRNA mediated knockdown of cellular P-TEFb has surprisingly shown to decrease HIV-1 transcription and viral replication without being lethal to the cell. It seems that there is a critical threshold of P-TEFb kinase activity that is required for cell viability and Tat transactivation. Moreover, it is suggested built-in intracellular mechanisms allow cells to cope with changes in P-TEFb protein levels [53].
Recently it has been reported that minocycline (MC), a second-generation non-toxic tetracycline, possesses the ability to inhibit NF-κB transcriptional activation and thereby viral replication in microglia [54]. These resident brain macrophages play a central role in AIDS dementia, as they are the primary targets of productive infection in the brain. Besides interrupting LTR activation, this agent also seems to influence the production of cytokines, chemokines, and other substances implicated in AIDS dementia. It appears that MC may act by increasing NF-κB complex formation, resulting in inactive homodimers. This suggests that MC possess the ability to suppress both viral production and inflammation.
HIV-1 mRNA splicing and nuclear export
Viral gene expression can be divided into an early and late phase, which is Rev-independent and Rev-dependent, respectively. In the early phase the newly transcribed mRNA is spliced by the cellular splicing machinery into multiply spliced transcripts, which mainly produces the Tat, Rev and Nef proteins. When Rev has accumulated to a critical level the mRNA production shifts from multiply spliced to the singly spliced and unspliced transcripts, characteristic of the late phase of gene expression. Rev contains an RNA binding motif that directly interacts with stem-loop IIB located within an RNA multi-stem-loop secondary structure, termed the rev response element (RRE), which is present in the env gene of all incompletely spliced viral mRNAs. The RRE can accommodate the binding of at least 8 Rev molecules, and at a certain threshold concentration of Rev protein in the nucleus, functional Rev/RRE complexes are formed, which greatly stimulate the export of unspliced and singly spliced RNA to the cytoplasm where translation can proceed. Nuclear export is mediated by cellular co-factors termed exportin 1 and Ran-GTP, which interacts cooperatively with the Rev nuclear export signal (NES) sequence [55]. The nuclear import of Rev is mediated by the cellular co-factor importin-beta that interacts directly with a NLS sequence in Rev. The host proteins B23 and p32 also interacts with the NLS region and may be involved in nucleolar localization [4,5,56-58]. The fundamental and essential function of Rev has made it a popular target for therapeutic development [57]. Fig. 2 illustrates the structure of proviral DNA and the different RNA species.
Figure 2 Schematic representation of the HIV-1 provirus and the different RNA species. Gag; group specific antigen, Gag-Pol; group specific antigen-polymerase, Env; envelope, Tat; trans-activator of transcription, Rev; regulator of expression of virion proteins, Nef; negative effector, Vif; virion infectivity factor, Vpr; viral protein r, Vpu; viral protein u, LTR; long terminal repeat.
Many of the used strategies are based on inhibiting the Rev/RRE interaction. Examples include Rev TNPs, RRE RNA decoys, anti-sense Rev/RRE RNAs, siRNAs, sFvs, nucleases, ribozymes, aptamers, chimeric proteins, and small inhibiting molecules.
The most well characterized Rev TNP is the RevM10 protein, which contains amino acid substitutions within the NES region. The ability of RevM10 to form multimeric structures and to interact with RRE is not hindered by these mutations, but the interaction with cellular co-factors is prevented leading to inhibition of Rev function [7,18,25,57-59]. Making deletions in the NLS sequence generates another kind of Rev TNP, the Rev38. The result is a mutant that accumulates in the cytoplasm where it sequesters the wildtype Rev protein by formation of inactive oligomers, thereby hindering the transport of Rev into the nucleus [18,57]. Likewise, Rev mutants lacking the ability to form multimeric structures (RevSLT26 and RevSLT40) are effective inhibitors. Of the mentioned TNPs, RevM10 is the most potent inhibitor that has been used in clinical trials [57].
RRE RNA decoys act by recruiting the Rev molecules and thereby hindering their interaction with the viral transcript. Potent inhibition has been achieved by overexpression of a 45 nucleotide chimeric tRNA-RRE transcript [7]. This type of decoy unfortunately also binds essential cellular co-factors, which has led to the design of minimal RNA decoys that only contains the Rev binding site. An example is a 41 nucleotide RRE SLIIAB RNA decoy that has been used in clinical trials [6].
sFvs targeted against Rev potently recruit Rev proteins in the cytoplasm, which accelerates the degradation of Rev [7,18,25,57]. In a comparative study the effect of monoclonal sFv targeting either the NES region or the C-terminal region of Rev was compared. The NES-specific sFv demonstrated the best antiviral effect, even though the binding affinity of the C-terminal sFv for Rev was significantly higher [57].
Additional strategies for inhibition of Rev function include, 1) RNA aptamers, possessing a higher affinity for Rev than the RRE sequence, and which recognize other epitopes than the natural RNA binding site on Rev [57], 2) peptides that recognize the NES domain [57], 3) a dominant negative mutant form of Sam68 (Src-associated protein in mitosis), whose natural function is to interact with RRE and thereby partially substitute or synergize with Rev in RRE-mediated gene expression. In particular, a C-terminally deleted mutant of Sam68 inhibits not only Sam68 transactivation of RRE, but also Rev function [60]. Unlike RevM10, which competes with wildtype Rev for binding to RRE in the nucleus, this Sam68 mutant is mainly cytoplasmic, and binds RRE very poorly. However, it retains the ability to bind Rev and the mechanism may involve trapping of Rev in the cytoplasm by direct protein-protein interaction. It is thought that the complex formation of Sam68 and Rev has a masking effect that inactivates the Rev NLS domain. Since the Sam68 NLS domain is deleted in the mutant it will not be able to substitute for the missing Rev NLS domain, thus the HIV-1 replication is inhibited. 4) chimeric proteins. One example is a construct where the Rev protein is covalently attached to a mutant form of the NS1 protein from the Influenza A virus (NS1RM-Rev). It is thought that the fusion protein and wildtype Rev form mixed oligomers, and due to the nuclear retention activity of NS1, which is dominant over the Rev-mediated nuclear export, it results in inhibition of nuclear export of viral transcripts. NS1RM-Rev has an antiviral effect equal to that of the RevM10 mutant [7,57].
As with inhibition of the Tat/TAR interaction, the anti-sense and ribozyme strategies can also be targeted towards the Rev/RRE interaction. Anti-sense directed against RRE will inhibit Rev binding, whereas anti-sense directed against the Rev encoding region will hinder the expression of Rev protein [6]. The anti-sense RNAs can be either unmodified or modified RNAs. E.g. a synthetic phosphorothioate oligodeoxynucleotide targeting Rev mRNA has antiviral activity in chronically infected cells [25]. Unfortunately, this type of anti-sense RNA strategy has shown limited success as a therapeutic agent because of unsolved problems such as efficacy, cell permeability, delivery and cost. Ribozymes targeting RRE or the Rev encoding region hinder viral replication by cleaving the targeted RNA [6,7].
By fusing the Rev protein with a nuclease it is possible to create a nuclease with affinity for the RRE and which therefore has the potential ability to specifically cleave all HIV-1 RNAs carrying the RRE sequence [5]. Also, siRNAs directed against the rev transcript potently inhibit virus replication [49,61].
Finally, strategies based on small molecules that either interact with host cellular proteins or bind to RRE, have also been applied to inhibit Rev function. Due to the ability to interact with cellular proteins, these molecules are often cytotoxic and therefore not usable for eventual clinical trials. Nevertheless they are included here because less toxic derivatives could be developed in the future. An example is Leptomycin (LMB), an antibiotic agent that interacts with the cellular protein CRM1, blocking the binding to the Rev NES domain. A clear disadvantage of LMB is, besides long-term toxicity, its ability to block the transport of other proteins with a NES domain [25,56,57]. An overexpressed truncated version of the nucleopurine, Nup214/CAN, delta-CAN, has shown a closely related mechanism of inhibition. Delta-CAN retains the skill to interact with CRM1 and inhibits Rev function in the same way [56].
Aminoglycoside antibiotics, such as neomycin B and derivatives have shown antiviral effect by binding to RRE and thereby hindering the Rev/RRE interaction. The binding of aminoglycoside antibiotics to RNA is very unspecific, and together with a low selectivity, this drug is unfortunately highly toxic for humans [25,57].
The intercalating dye, pyronin Y, completely blocks the formation of the Rev/RRE complex in vitro. But since pyronin Y also intercalates in DNA, the result is an elevated level of cell death. Other intercalating agents, derivatives of diphenylfuran, have been reported to inhibit the Rev/RRE interaction by causing a conformational change in RRE. These compounds possess the same disadvantages as pyronin Y [25].
HIV-1 translation
As illustrated in fig. 2 translation of the non-spliced RNA and single-spliced RNA result in the Gag and Gag-Pol proteins, and in the Vpu, Vif, Vpr and Env proteins, respectively. Translation of the completely spliced RNA results in the Tat, Rev and Nef proteins [4,6-8].
Designed anti-sense RNAs directed against sequences located in the 5' UTR of all HIV-1 mRNAs, would be able to hinder the ribosome-complex in completing the translation process and thereby inhibit the protein synthesis [6]. Hairpin ribozymes directed against the U5 region have shown similar antiviral responses [6,7].
A study involving nef shRNAs corresponding to nef-derived miRNAs, which recently have been demonstrated to be produced in HIV-1-infected cells [62], shows the potential to efficiently block RNA stability or mRNA translation. This may indicate that HIV-1 possess the ability to regulate its own replication. Interestingly, another study has shown that HIV-1 putatively encodes five candidate pre-miRNAs, which potentially could target a large number of cellular transcripts indicating that HIV-1 moreover may have the potential to regulate the cellular milieu [63].
Viral assembly, release and maturation
The virus particle is assembled at the plasma membrane. In this process the Gag and Gag-Pol polyproteins interact with each other by protein-protein interaction, most probably via the capsid (CA) protein domain [64]. The viral genome is packaged in a process in which the packaging signal, Psi, is recognized by the nucleocapsid (NC) protein domain of the Gag protein [65]. Another important function of the NC domain is to mediate the formation of the RNA dimer via a palindromic sequence in the dimer linkage structure (DLS) sequence, which is located in the Psi sequence [4,6]. In addition several cellular tRNAs are packaged. The assembly of the virus particle is partly regulated by the Vpu and Vif proteins. The primary function of the Vpu protein is to mediate the release of the virus particle from the cell surface, by selectively targeting the CD4 protein to a degradation pathway in the endoplasmic reticulum (ER). This permits the release of Env protein from the ER, which may otherwise be complexed with the CD4 protein, and further processing of the Env protein can then proceed [4,5]. A current thought is that Vif, besides influencing the late stages of virion assembly, may also block premature processing of Gag precursor protein by the HIV protease (Pro). This kind of temporal control of Gag processing ensures the availability of the CA, MA and NC peptides, when the assembly of the viral components takes place at the plasma membrane [4,66,67]. In addition, several studies have recently suggested that Vif may possess another important function in which it acts by overcoming the antiviral activity of a cellular cytidine deaminase, APOBEC3G (CEM15) that induces hypermutations in newly synthesized HIV-1 DNA [68,69]. The mechanism by which Vif inhibits APOBEC3G function is unclear, but it is thought to form a complex with APOBEC3G, thus preventing its viral encapsidation. Furthermore, Vif can target APOBEC3G for degradation via the ubiquitin-proteasome pathway.
After virus budding from the cell surface, maturation of the virus particle proceeds. At this stage in the virion life cycle the Gag and Gag-Pol polyprotein are proteolytically cleaved by the protease domain of Gag-Pol precursors. Cleavage of Gag results in the MA, CA, NC and CEL viral proteins. The MA proteins forms a matrix under the viral envelope, the CA proteins condenses to form a conical core surrounding the NC-coated RNA genome and the CEL protein is thought to associate with the Env protein and, in addition to mediating packaging of Vpr.
Cleavage of the Gag-Pol polyproteins, which is made by ribosomal frameshifting during translation of unspliced mRNA, results in the enzymatic proteins IN, RT and Pro. Both the Gag and Gag-Pol precursors associate with the Env protein by protein-protein interactions between the MA domain and the TM protein. After maturation the virus is ready for another round of infection [4]. Strategies directed against this final step in the viral life cycle include RNA decoys, TNPs, chimeric proteins, anti-infectious cellular proteins, sFvs, nucleases, anti-sense RNAs, ribozymes and peptides.
Viral assembly
The packaging signal Psi is a highly conserved RNA sequence and is therefore an obvious target for inhibition strategies. The Psi region contains four stem-loops located near the 5' major splice donor and the start of the gag open reading frame and is essential and sufficient for RNA packaging [45].
RNA containing a 1.43 kb region anti-sense to the Psi-gag region has been reported to inhibit packaging of genomic RNA and thus HIV-1 replication with a higher efficiency than the RevM10 mutant [6,7,45]. An antiviral effect has also been observed by targeting anti-sense RNAs against the Psi sequence, which act by hindering the NC domain of the Gag protein in recognizing the packaging signal. Likewise, ribozymes directed against the packaging signal have also been shown to have a significant effect on viral replication [7,6].
Since the Psi element is recognized by the NC domain, one strategy is to design a NC-nuclease fusion protein, which will recognize and cleave all unspliced HIV-1 RNAs. The cleavage process occurs in the cytoplasm and this will also inhibit the production of the Gag and Gag-Pol fusion proteins [5]. Expression of the gag-gene has also been reduced by using anti-sense RNAs complementary to the 5'-leader-gag region or by using ribozymes directed against the gag transcripts [7,18].
The ability of the NC domains to recognize the genomic RNA can be blocked with RNA decoys containing the Psi sequence. These decoys may form dimers with HIV-1 RNA and thereby compete with HIV-1 RNA dimers for packaging into new virus particles [6,45].
The structural proteins, Gag and Env, form multimeric complexes during viral assembly. By means of different kinds of Gag TNPs it has been possible to inhibit this step. Besides inhibiting viral assembly, these Gag TNPs also interfere with the viral release process, the uncoating of the viral genome and the reverse transcription [18]. A limitation of using Gag TNPs is that the gag-gene contains an inhibiting sequence, the CRS, which hinders the expression of the gag-gene if Rev is missing [4,6,18]. The potential effect of Env TNPs has also been tested, but this strategy shows relatively low antiviral activity when compared to Gag TNPs [18].
Given that one function of the Vif protein is to block early processing of the Gag protein, it has inspired the development of a Vif TNP that is missing the blocking function. Applying this protein means that the virus assembly is impaired, but a clear disadvantage is that HIV-1 has the potential to evolve escape mutants [66]. Similar inhibition has been observed by directing anti-sense RNAs against the Vif encoding region [70]. Another strategy relies on the INI1 protein that is the only known host protein directly interacting with HIV-1 integrase. A minimal integrase-binding fragment of INI1, S6, comprising amino acids 183–294, potently inhibits HIV-1 assembly, particle production and replication in a transdominant manner. This inhibiting effect results from direct interaction of the S6 protein with the integrase domain within the Gag-Pol polyprotein. When the S6 protein binds to integrase it is thought to interfere with proper multimerization of Gag and Gag-Pol by steric hindrance. In addition it affects maturation, blocks an interaction of the cellular assembly machinery with Gag-Pol and mediates the mislocalization of viral proteins into different sub-cytoplasmic compartments of the cell. Besides being non-toxic, another favorable feature of S6 is that it is unlikely to be immunogenic, because it is a truncated form of a host protein. In addition, virions with mutations in the S6 binding site will also be defective for interaction with INI1. This minimizes the risk for the development of HIV-1 escape mutants [39].
Recently, a specific inhibition of virus budding was demonstrated by overexpression of an amino-terminal fragment of tumour susceptibility gene 101 (Tsg101) [71]. The role of Tsg101 is to participate in the endocytic trafficking pathway. It is presumed to bind to the Gag polyprotein and subsequently mediate the transport into multivesicular bodies (MVBs), which then carry their cargo towards the cell surface.
By targeting an intracellular sFv specifically against the CD4 binding region of the SU protein, it has been possible to make cells temporarily resistant to HIV-1 infection. This sFv, named sFv105, acts by binding to the Env protein, and traps Env in the ER. This prevents the maturation process in which Env is cleaved into the SU-TM proteins. As a result, Env is prevented from reaching the cell surface [7,18]. Another way to inhibit the maturation of Env is by inhibiting the cellular protease furin [72]. Furin is a member of the mammalian subtilisin-related proprotein convertases that mediate cleavage of the Env protein at a conserved Arg-Glu-Lys-Arg sequence. Peptides that mimic this sequence have been reported to block furin activity. Especially, a polyarginine peptide has shown promising results without showing any toxic side effects on cultured cells ex vivo or in mice in vivo.
Furthermore, anti-sense RNA approaches directed against the Env message also belong to the applied strategies [70].
Multitarget ribozymes that target different sites in the SU sequence also exert antiviral effects. Some of these designed ribozymes bind and cleave up to nine different conserved regions in the SU sequence [7].
A Gag-nuclease fusion protein can be packed into new virions and thereafter, in the proceeding rounds of infection, efficiently cleave the viral genomic RNA. Since the Gag protein has many essential functions it is unlikely that HIV-1 will develop escape mutants when using the Gag-nuclease. Vpr- and Nef-nuclease fusion proteins also seem to cleave viral RNAs, either during or after viral assembly [5,7].
Viral release
A Nef TNP has shown an antiviral effect by inhibiting down-regulation of e.g. the CD4 cell surface protein. It is thought that CD4 may interact with the Env protein present on new viral particles, thus hampering viral release from the cell surface [66]. Likewise, anti-sense RNAs and ribozymes directed against a conserved 14 nucleotide region in the nef-gene also possess an antiviral effect [7].
By overexpressing different kinds of CD4 variants in the infected cell, it is possible to inhibit virus budding. The reason is most likely that the CD4 variants possess the ability to trap and restrain new virions in the cell [19].
Viral maturation
The HIV-1 protease plays an important role in virus maturation. As mentioned earlier, the HIV-1 protease cleaves the Gag and Gag-Pol polyproteins to form the structural and enzymatic proteins. Consequently, the protease is a potent target for inhibiting strategies. The current strategies involve protease inhibitors that bind to the active site of the HIV-1 protease and thereby inhibit processing of the Gag and Gag-Pol polyprotein precursors. This results in immature and noninfectious viral particles. The HIV-1 protease is an aspartyl protease and inhibitors have been designed that optimally bind to the catalytic aspartate residues and additionally to the water molecule that is critical for enzymatic action. The inhibitors are transition state analogues that bind the enzyme much more tightly than the natural substrate, making them competitive enzyme inhibitors. Examples of approved protease inhibitors include Saquinavir, Indinavir, Ritonavir, Nelfinavir and Amprenavir [8,11,73].
Other strategies that target Pro involve the application of Pro TNP [7], and the overexpression of chimeric Vpr protein in which the C-terminal region is fused to several cleavage sites recognized by the protease. This will overwhelm the protease activity by a competitive mechanism and impair protease function [7,74]. Finally, anti-sense RNAs targeting the Pol coding region inhibit this last step in the viral life cycle [70].
Combination of antiviral strategies
By combining the different antiviral strategies, the effectiveness can be increased and the chances of generating escape mutants will be minimized. Examples of combination therapies include ribozymes combined with decoy or anti-sense RNAs [6,75], and decoy RNAs combined with anti-sense RNAs [6,76]. An autoregulated dual-function anti-Tat gene is an example of the latter strategy, in which both a polymeric TAR and an anti-sense Tat are combined in one expression unit [76]. To accomplish gene expression in HIV-1 infected cells, a double-copy retroviral vector, in which gene expression is driven by the HIV-1 LTR, is used. By this approach anti-Tat gene expression is upregulated only in HIV-1 infected cells.
The RT and Pro inhibitors are preferentially not used alone to avoid the risk of generating viral escape mutants. By combining the different kinds of inhibitors (usually a combination of two nucleoside analogues with either a protease inhibitor or a non-nucleoside analogue) significant inhibition of HIV-1 is achieved. This combination strategy is also known as HAART (highly active antiretroviral therapy). Because nucleoside and non-nucleoside analogues act on two different positions on the RT enzyme they will not compete for binding and when used in combination they exhibit a more potent effect. The disadvantage concerning this strategy is the relative strong toxic effects related to these RT drugs. Another problem arises if the prescribed treatment is not exactly followed and resistant viral mutants emerge [31].
When combining strategies involving ribozymes and RNA decoys one can obtain better results than by using one of the strategies. This is clearly illustrated by the tRNAVal-RRE-SLII-U5 hairpin ribozyme, in which SLII (stem loop II) contains the Rev protein-binding site that acts as a decoy [6].
In an attempt to interfere with two essential HIV-1 activities at the same time, a double transdominant negative Tat-Rev fusion protein, Trev, has been designed. This fusion protein inhibits both Tat transactivation and Rev mediated nuclear transport [18]. A similar designed fusion protein, Tev, contains the RNA binding domains of both Tat and Rev, and can thus target both TAR and RRE within the HIV-1 RNA. Furthermore, a nuclease was fused to the Tev protein. The result is an inhibition of both early and late viral gene products. Tev contains a NLS and is therefore predicted to act primarily within the nucleus [5].
The combination of an anti-Rev sFv, which targets the Rev activation domain, and a ribozyme that targets RRE, or an RRE RNA decoy, which recruits the Rev molecules, has also shown good results [7]. Promising results have also been described by using a vector expressing three products, the U5 ribozyme, a ribozyme targeted against the Env/Rev encoding regions, and a RRE decoy respectively [18].
Strategies based upon suicide genes
Conditional expression of suicide genes in cells infected with HIV-1, e.g. by expression from a Tat dependent promoter or under Rev dependent control, has been designed in different versions. Examples of suicide gene approaches include engineering cells with a diphtheria toxin A-chain (DT-A) gene, a cytosine deaminase gene, a herpes simplex virus (HSV) thymidine kinase (tk) gene, and a herpes simplex shutoff (vhs) gene.
DT-A is a very effective cellular toxin that kills cells by blocking the protein synthesis via the ADP-ribosylating elongation factor 2 [77]. Cytosine deaminase mediates cell death through the conversion of 5-fluorocytosine to the potent cytotoxic agent 5-fluorouracil [18], and the HSV thymidine kinase mediates cell death by metabolizing nucleoside analogues, such as Ganciclovir and Acyclovir, into toxic analogues [7,18]. The latter strategy has been further explored in a study involving a live-attenuated form of HIV-1 in which the nef gene has been deleted and instead engineered to express the thymidine kinase gene [78]. This marked live-attenuated virus vector may be useful to accrue baseline information on the immunological benefits of a replicating vaccine. The safety profile of such a vaccine vector is supported by the possibility to remove cells harboring integrated proviral genomes if necessary.
Another approach involves the design of a hybrid molecule consisting of the human CD4 and a modified version of the Pseudomonas exotoxin A (CD4-PE40). This molecule binds to infected cells by a CD4-SU interaction at the cell surface and, after uptake, the exotoxin inhibits protein synthesis, thus leading to cell death [7].
Finally, the last example involves a modified apoptosis-promoting caspase-3 protein, Tat-Casp3, which acts by cleavage of the inhibitor of caspase-activated DNase, resulting in the activation of caspase-activated DNase and, ultimately, cell death. In this design the endogenous cleavage sites have been substituted with the HIV proteolytic cleavage sites, and the Pro domain of the modified Casp3 protein was removed and substituted with the Tat transduction domain. Hence, the fusion protein is only activated by the HIV protease in infected cells, resulting in apoptosis, whereas in uninfected cells it remains in the inactive zymogen form. Tat-Casp3 proteins may also be packaged inside the virion and kill the virion after it buds from the cell and/or initiate apoptosis immediately after subsequent infection of a cell [79].
Conclusion
In spite of the astonishing diversity of methods developed as antivirals against HIV-1, still many problems remain. Perhaps the most difficult problem to solve is the remarkable ability of HIV-1 to evade the different inhibiting strategies. The selection pressure enforced by the treatment may result in the selection of escape mutants that are more pathogenic than the original virus. For instance: Blocking the interaction with CCR5, which is the primarily used co-receptor, could result in usage of both the CCR5 and the CXCR4 co-receptors or CXCR4 alone. The outcome will be an accelerated reduction of CD4+ T-cells and thereby a progression of the disease [80]. The risk of evolution of a virus variant that uses new co-receptors is not unthinkable. To obtain long-term inhibition and to avoid escape mutants, it is necessary to combine the different strategies, so that several steps in the viral life cycle are inhibited at the same time.
RNAi is a very promising novel approach that in principle will provide a large number of new targets that may be combined, but unfortunately one of the biggest hurdles is the in vivo delivery problem, which needs to be addressed. A gene therapy approach may be used to make hematopoietic stem cells resistant to HIV-1, which could eventually lead to (partial) restoration of the immune system.
In spite of the advanced technology used in the different virus intervention strategies and the rapidly growing knowledge about the molecular biology of HIV-1, it has not yet been accomplished to block HIV-1 replication completely. Hopefully, scientists will succeed to push the balance of the virus-host battle in the right direction so that the immune system will be able to handle the remaining viruses, or vaccine strategies may be added to clear the virus. Independently of the path taken, it will most likely require that the latent reservoirs of virus are activated to make them more vulnerable to treatment. A combination of these directions may eventually lead to a complete eradication of the virus in infected patients.
List of abbreviations used
Aids; acquired immunodeficiency syndrome
HIV-1; human immunodeficiency virus type 1
shRNA; short hairpin RNA
siRNA; small interfering RNA
RNAi; RNA interference
miRNA; microRNA
dsRNA; double stranded RNA
Gag; group specific antigen
CA; capsid protein
MA; matrix protein
NC; nucleocapsid protein
CEL; core-envelope-link protein
Gag-Pol; group specific antigen-polymerase
RT; reverse transcriptase
IN; integrase
Pro; protease
Env; envelope
SU; surface protein
TM; transmembrane protein
Tat; trans-activator of transcription
Rev; regulator of expression of virion proteins
Nef; negative effector
Vif; virion infectivity factor
Vpr; viral protein r
Vpu; viral protein u
TAR; trans-activation responsive element
RRE; rev response element
Psi; packaging signal
DLS; dimer linkage structure
PBS; primer binding site
LTR; long terminal repeat
R; repeat
U5; unique 5'
U3; unique 3'
ER; endoplasmatic reticulum
NES; nuclear export signal
NLS; nuclear localization signal
TNP; transdominant negative protein
sFv; intracellular single-chain antibody
Mab; monoclonal antibody
TRTI; template analogue RT inhibitor
NRTI; nucleoside reverse transcriptase inhibitor
NNRTI; non-nucleoside reverse transcriptase inhibitors
HAART; highly active antiretroviral therapy
SELEX; systematic evolution of ligands by exponential enrichment
DCQA; dicaffeoylquinic acid
LMB; leptomycin
Tsg101; tumour susceptibility gene 101
MVB; multivesicular body
DT-A; diphtheria toxin A-chain
tk; thymidine kinase
RANTES; regulated upon activation, normal T-cell expressed and secreted beta-chemokine
PBMC; human peripheral blood mononuclear cell
CV-N; Cyanovirin
CyPA; human cyclophilin A
Competing interests
The author(s) declare that they have no competing interests.
Acknowledgement
We thank Ebbe Sloth Andersen for drawing Figure 1.
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| 15715913 | PMC553987 | CC BY | 2021-01-04 16:36:40 | no | Retrovirology. 2005 Feb 16; 2:10 | utf-8 | Retrovirology | 2,005 | 10.1186/1742-4690-2-10 | oa_comm |
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Curr Control Trials Cardiovasc MedCurrent Controlled Trials in Cardiovascular Medicine1468-67081468-6694BioMed Central 1468-6708-6-21573332510.1186/1468-6708-6-2ResearchPresence of factors that activate platelet aggregation in mitral stenotic patients' plasma Tengiz Istemihan [email protected] Ertugrul [email protected] Fahri [email protected] Emin [email protected] Can [email protected] Guray [email protected] Filiz [email protected] Department of Cardiology, Central Hospital, 1644 sk. No:2/2, 35010 Bayrakli, Izmir, Turkey2 Department of Hematology, Ege University Medical School, 35100 Bornova, Izmir, Turkey3 Department of Biochemistry, Kocaeli University Medical School, 41900 Derince, Kocaeli, Turkey2005 27 2 2005 6 1 2 2 20 12 2004 27 2 2005 Copyright © 2005 Tengiz et al; licensee BioMed Central Ltd.2005Tengiz et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Although the association between mitral stenosis (MS) and increased coagulation activity is well recognized, it is unclear whether enhanced coagulation remains localized in the left atrium or whether this represents a systemic problem. To assess systemic coagulation parameters and changes in platelet aggregation, we measured fibrinogen levels and performed in vitro platelet function tests in plasma obtained from mitral stenotic patients' and from healthy control subjects' peripheral venous blood.
Methods
Sixteen newly diagnosed patients with rheumatic MS (Group P) and 16 healthy subjects (Group N) were enrolled in the study. Platelet-equalized plasma samples were evaluated to determine in vitro platelet function, using adenosine diphosphate (ADP), collagen and epinephrine in an automated aggregometer. In vitro platelet function tests in group N were performed twice, with and without plasma obtained from group P.
Results
There were no significant differences between the groups with respect to demographic variables. Peripheral venous fibrinogen levels in Group P were not significantly different from those in Group N. Adenosine diphosphate, epinephrine and collagen-induced platelet aggregation ratios were significantly higher in Group P than in Group N. When plasma obtained from Group P was added to Group N subjects' platelets, ADP and collagen-induced, but not epinephrine-induced, aggregation ratios were significantly increased compared to baseline levels in Group N.
Conclusion
Platelet aggregation is increased in patients with MS, while fibrinogen levels remain similar to controls. We conclude that mitral stenotic patients exhibit increased systemic coagulation activity and that plasma extracted from these patients may contain some transferable factors that activate platelet aggregation.
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Introduction
Systemic thromboembolism represents a major complication in patients with mitral stenosis (MS), especially in those who have atrial fibrillation [1,2]. A hypercoagulable state has been reported in patients with MS and sinus rhythm [3,4]. The association between MS and higher levels of coagulation is well-known; however, the source of increased coagulation remains unclear. Increased regional left atrial coagulation activity may be involved, even when systemic coagulation assessed by peripheral blood sampling is normal [5,6]. Others have reported no significant variation in thrombogenesis, platelet activation, and endothelial dysfunction between the left atrium, right atrium and peripheral arteries or veins [7].
This study assessed systemic coagulation and platelet activities by measuring fibrinogen levels and platelet aggregation in mitral stenotic patients' peripheral venous blood and compared these measures to hematologic parameters in normal controls.
Methods
Study groups
Eighteen newly diagnosed patients with rheumatic MS were enrolled in the study. All were coded as having NYHA class I functional capacities. Two patients were excluded from this study, one due to pregnancy, the second due to current use of oral contraceptives. The study group thus consisted of 16 patients with MS (Group P, 14 female, 2 male, mean age 27.8 ± 6.5 years). Peripheral venous coagulation activity and platelet activation tests were also evaluated in 16 control patients (Group N, 13 female, 3 male, mean age 28.3 ± 6.1 years), who were normal volunteers. All participants gave informed consent.
Exclusion criteria
Patients were excluded from participating in the study for the following reasons: aortic and/or pulmonary valve disease, severe mitral regurgitation and/or left ventricular systolic dysfunction, atrial fibrillation, hypertension, diabetes mellitus, dyslipidemia, a history of renal or liver disease, malignancy, venous thrombosis, systemic or pulmonary embolism, congenital hemorrhagic disease, thrombocytopenia, thrombocytosis, acute or chronic inflammatory disease, autoimmune disease or current use of oral contraceptives or anticoagulant or anti-platelet drugs.
Echocardiography
In patients with MS, transthoracic echocardiography was performed with a 2.5-MHz transducer and a Hewlett Packard Sonos 4500 system to assess left atrial (LA) diameter, mitral valve area (MVA) and the transmitral mean pressure gradient (TMmPG). The LA anteroposterior diameter was determined using standard M-mode criteria [8], and mitral valve area was calculated according to the pressure half-time method [9].
Fibrinogen measurement
Peripheral venous blood (for measurement of fibrinogen levels) was transferred into tubes containing 1:9 trisodium citrate (0.109 M, 3.2%). Blood samples were centrifuged within 1 hour of collection at 2,500 g for 10 minutes to separate out the plasma. An STA fibrinogen kit measured fibrinogen levels in plasma, using the clotting method of Clauss. The median inter-assay and intra-assay coefficients of variation for the assays were 3.57% and 3.65% for fibrinogen, respectively.
Platelet activation tests
Blood samples were obtained from the forearm veins of participants, using standard blood drawing procedures (normal blood flow and no pressure). Several samples were drawn for different components of the study. Nine milliliters of blood was collected into special tubes containing 1 ml of 3.8% sodium citrate. Platelet count and other parameters, including white blood cell count and hemoglobin, were measured at the Ege University Hospital Hematology Laboratory using an automated hemocytometer (Cell Dyne 4000 Abbott, USA). Patients with platelet counts less than 2 × 105/ml were excluded from the study. For in vitro platelet aggregation tests, platelet-rich plasma (PRP) samples were obtained by centrifuging samples at 1,100 rpm for 10 minutes in a refrigerated centrifuge (Hettich EBA 12R, Germany). Centrifuging samples at 3,000 rpm for 10 minutes yielded platelet-poor plasma (PPP) samples. The platelet counts of both PRP and PPP samples were performed using the same hemocytometer. Platelet-poor plasma samples were added to the PRP samples to obtain a total platelet count of 2 × 105/ml. These platelet-equalized plasma (PEP) samples were analyzed to determine in vitro platelet function, using ADP, Collagen and Epinephrine (all from Bio/Data Corp. Horshram, Germany) in an automated aggregometer (Bio/Data Corp. Platelet Agregation Profiler, Horshram-Germany). Results were evaluated and calculated using maximum aggregation ratios.
PRP from healthy controls was centrifuged for 10 minutes, after which the supernatant was decanted, and normal saline added to the platelet fraction and centrifuged for 10 minutes. This procedure was repeated three times. Plasma obtained from patients with MS was added to the PPP from the control group, and platelet function tests were performed, using the same procedures mentioned above.
Statistical analyses
Chi Square tests were employed to compare categorical variables, while Mann Whitney U tests were used, where appropriate, in the univariate analysis. Platelet aggregation changes were evaluated by repeated-measures analysis of variance for intra-group comparisons. Values of p < 0.05 were considered significant. SPSS 10.0 Statistical software was used in the statistical analysis.
Results
There were no significant differences between Groups P and N for the following variables: age, gender, current smoker, platelet, white blood cell and hemoglobin count. Clinical characteristics of the study groups are shown in Table 1. The peripheral venous fibrinogen levels in patients with MS (301.9 ± 53.7 mg/dL) were not significantly different from those in control subjects (284.6 ± 55.7 mg/dL).
Table 1 Clinical features of the study groups and echocardiographic findings of patients with mitral stenosis.
Group P (n = 16) Group N (n = 16) p
Mean age (yrs) 27.8 ± 6.5 28.3 ± 6.1 NS
Male/female (n) 2/14 3/13 NS
Current smoker (n) 5 6 NS
Platelet count × 103/mm3 253.1 ± 33.5 245.6 ± 34.3 NS
WBC/mm3 5706 ± 1266 5643 ± 1229 NS
Hb (g/L) 12.4 ± 1.26 12.8 ± 1.22 NS
Fibrinogen (mg/dL) 301.9 ± 53.7 284.6 ± 55.7 NS
LA diameter (cm) 4.22 ± 0.19 - -
MVA (cm2) 1.67 ± 0.16 - -
TMmPG (mmHg) 6.5 ± 0.96 - -
Mitral regurgitation (n)
None 4 - -
Mild 11 - -
Moderate 1 - -
Group P: Patients with mitral stenosis, Group N: Control subjects, WBC: White blood cell, Hb: Hemoglobin, LA: Left atrium, MVA: Mitral valve area, TMmPG: Transmitral mean pressure gradient, NS: Non-significant.
Platelet activation tests
Adenosine diphosphate, collagen and epinephrine-induced platelet aggregation ratios were significantly higher in Group P than in Group N (84.9 ± 9.8, 76.6 ± 21.4, 80 ± 10.5 and 53.1 ± 23.2, 47.5 ± 14.5, 53.7 ± 21.3, p < 0.05, respectively). When the PPP obtained from Group P was added to Group N subjects' platelets, and platelet function tests were repeated, ADP and collagen-induced (73 ± 17.5 and 65.7 ± 17.5, respectively), but not epinephrine-induced (61.9 ± 16.2), aggregation ratios were statistically increased compared to baseline (Figure 1).
Figure 1 Induced platelet aggregation ratios in Groups P and N. Group P: Patients with mitral stenosis, Group N: Control subjects, PPP: Platelet-poor plasma, Plt: platelet, ADP: Adenosine diphosphate, COL: Collagen, EPI: Epinephrine.
Discussion
Left atrial thrombus in MS is associated with increased systemic levels of peptide by-products from the coagulation cascade [10,11]. Increases in markers of coagulation activity may also occur in the absence of thrombus, perhaps signifying that an imbalance in hemostatic regulation favoring pro-coagulant mechanisms may precede and predispose to thrombus formation [12,13]. Information about coagulation activity in patients with MS could, therefore, provide a method of identifying patients at risk of developing systemic thromboembolism.
A number of studies have reported that indexes of hypercoagulation derived from peripheral blood may not reflect intracardiac thrombogenesis. Yamamoto et al. [6] noted that fibrinopeptide A and thrombin/antithrombin III were higher in the left atrium compared to the right atrium and the femoral vein. Peverill et al. [5] found that levels of prothrombin fragments 1+2 increased in the left atrium in comparison to peripheral blood levels. Li-Saw-Hee et al. [7] reported no significant variation in indexes of thrombogenesis, platelet activation, and endothelial dysfunction between left atrium, right atrium, and the peripheral artery or vein. A number of differences between these studies and our study may, in part, account for these varying observations. All of our patients exhibited newly diagnosed, mild-to- moderate MS, and none was taking anticoagulants or anti-platelet drugs. Second, we did not investigate systemic levels of peptide by-products of the coagulation cascade or individual blood components that reflect coagulability. In our experimental design, we sought to examine the effect of mitral stenotic patients' plasma on control subjects' platelets.
In addition to protein factors, platelets play a vital role as systemic components of the hemostatic system. They are critical to the activation of intrinsic pathway factors. Because platelet activation seems to be influenced by diabetes mellitus [14], smoking [15], hypertension and the use of oral contraceptives [16], patients with these conditions were excluded from our study.
Hwang et al. [17] sought to examine a correlation among echocardiographic variables, hematologic parameters or platelet aggregability and the occurrence of spontaneous echo-contrast (SEC) in the left atrium. Platelet aggregability was evaluated with a turbidometric method, using different concentrations of activating agents. No significant difference was found in platelet aggregability between patients with left atrial SEC and patients without left atrial SEC. Neither group of patients had been receiving anti-platelet or anticoagulant therapy.
Ileri et al. [4] observed hypercoagulation in patients with mitral stenosis and sinus rhythm when SEC was present. Pongratz et al. [18] investigated the activation status of platelets in the peripheral blood of patients with atrial fibrillation. A significantly higher level of circulating platelets expressing P-selectin and CD63 and more leukocyte-platelet conjugates were found in patients positive for both SEC and left atrial thrombus or embolic events. Increased spontaneous platelet aggregation in the presence of SEC was described by Rohmann et al. [19], who measured the half-maximal formation of platelet aggregates in peripheral blood, upon stimulation with ADP.
In our study, all patients had mild-to-moderate MS, with functional capacities rated as being NYHA class I. Although peripheral fibrinogen levels were similar between Group P and N, platelet aggregation was significantly higher in Group P than in Group N. Moreover, alteration of platelet aggregation in Group N, triggered by the addition of plasma from mitral stenotic patients, may indicate the presence of possible transferable platelet activators in peripheral blood.
In conclusion, we examined systemic coagulation activity in patients with MS by measuring plasma level of fibrinogen and by performing platelet activation tests in peripheral venous blood samples. Our examinations yielded three main findings. First, mitral stenotic patients have similar fibrinogen levels in peripheral venous blood as compared to controls. Second, platelet aggregation is higher in patients with MS than in controls. Third, in comparison to controls, patients with MS have increased systemic coagulation activity and the plasma extracted from these patients seems to have some transferable factors that activate platelets.
The results from this study emphasize the complexity of the events related to platelet activation in MS. Also compounding the challenges faced in this area is uncertainty about the gold standard method for assessing platelet activation and whether platelet aggregation (ex vivo) truly reflects platelet activation in vivo [20].
Acknowledgements
This study was supported by GURVAK (Gürbüz Sağlık ve Eğitim Vakfı). Authors wish to thank Mrs. Fadime Onal for technical assistance.
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| 15733325 | PMC553988 | CC BY | 2021-01-04 16:47:32 | no | Curr Control Trials Cardiovasc Med. 2005 Feb 27; 6(1):2 | utf-8 | Curr Control Trials Cardiovasc Med | 2,005 | 10.1186/1468-6708-6-2 | oa_comm |
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Cost Eff Resour AllocCost effectiveness and resource allocation : C/E1478-7547BioMed Central London 1478-7547-3-21567033010.1186/1478-7547-3-2ResearchA cost-minimization analysis of diuretic-based antihypertensive therapy reducing cardiovascular events in older adults with isolated systolic hypertension Chen G John [email protected] Luigi [email protected] William P [email protected] Marco [email protected] Department of Public Health Sciences, Wake Forest University School of Medicine, Winston-Salem, NC, USA2 Laboratory of Clinical Epidemiology, INRCA Geriatric Department, Florence, Italy3 Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem NC, USA2005 25 1 2005 3 2 2 3 8 2003 25 1 2005 Copyright © 2005 Chen et al; licensee BioMed Central Ltd.2005Chen et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Hypertension is among the most common chronic condition in middle-aged and older adults. Approximately 50 million Americans are currently diagnosed with this condition, and more than $18.7 billion is spent on hypertension management, including $3.8 billion for medications. There are numerous pharmacological agents that can be chosen to treat hypertension by physicians in clinical practices. The purpose of this study was to assess the cost of alternative antihypertensive treatments in older adults with isolated systolic hypertension (ISH).
Method
Using the Systolic Hypertension in the Elderly Program (SHEP) and other data, a cost-minimization analysis was performed. The cost was presented as the cost of number-needed-to treat (NNT) of patients for 5 years to prevent one adverse event associated with cardiovascular disease (CVD).
Result
It was found that the cost of 5 year NNT to prevent one adverse CVD event ranged widely from $6,843 to $37,408 in older patients with ISH. The incremental cost of the 5 year NNT was lower to treat older patients in the very high CVD risk group relative to patients in the lower CVD risk group, ranging from $456 to $15,511. Compared to the cost of the 5 year NNT of other commonly prescribed antihypertensive drugs, the cost of SHEP-based therapy is the lowest. The incremental costs of the 5 year NNT would be higher if other agents were used, ranging from $6,372 to $38,667 to prevent one CVD event relative to SHEP-based drug therapy.
Conclusion
Antihypertensive therapy that is diuretic-based and that includes either low-dose reserpine or atenolol is an effective and relatively inexpensive strategy to prevent cardiovascular events in older adults with isolated systolic hypertension. Use of the diuretic-based therapy is the most cost-effective in patients at high risk for developing cardiovascular disease.
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Background
Hypertension is among the most common chronic conditions in middle-aged and older adults. Approximately 50 million Americans are currently diagnosed with this condition, and more than $18.7 billion is spent on hypertension management, including $3.8 billion for medications[1].
Treatment of hypertension can significantly decrease the risk of developing CVD [2,3]. The SHEP and other studies have demonstrated the great potential of antihypertensive treatments to significantly reduce the number of cardiovascular events in elderly patients [4-10]. This, in turn, may reduce the costs associated with this chronic condition. Based on the SHEP study, it is estimated that 24,000 strokes, 44,000 major cardiovascular events, and 84,000 admissions to the hospital could be prevented over a 5-year period [7].
Currently, primary care physicians can choose from numerous pharmacological agents to treat hypertension. The commonly used antihypertensive drug classes include diuretics, beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, alpha-blockers, and calcium channel blockers. Selection of an evidence-based therapy with demonstrated efficacy, safety, and low cost has important economic implications. The purpose of this study was to: 1) assess cost of the SHEP-based antihypertensive treatment to prevent adverse events associated with CVD, including death, stroke, myocardial infarction, and heart failure; and 2) to compare cost of the SHEP-based treatment to the costs of other commonly used antihypertensive agent treatments.
Method
The SHEP trial is a randomized, double-blind, placebo-controlled clinical trial sponsored by the National Heart, Lung, and Blood Institute and the National Institute on Aging that tested the efficacy of diuretic-based stepped-care antihypertensive drug treatment of isolated systolic hypertension (ISH) to prevent strokes [4].
Study Population
The study subjects consisted of community-dwelling men and women 60 years and older who had isolated systolic hypertension, defined as an average systolic blood pressure (SBP) ≥ 160 mm Hg and an average diastolic blood pressure (DBP) < 90 mm Hg over 2 baseline visits. The primary endpoint of the trial was combined nonfatal and fatal stroke over a 5-year period. Secondary endpoints included nonfatal myocardial infarction (MI) plus fatal coronary heart disease (CHD) and major cardiovascular disease (CVD) morbidity and mortality. A total of 2,365 and 2,371 persons were randomized into the treatment and placebo group of the study respectively.
Subjects who met the preliminary blood pressure (BP) eligibility criteria at the initial contact visit were referred to SHEP clinics for the baseline visits. At the baseline visits, subject's demographics, medical conditions, health behaviors, and cardiovascular risk factors were obtained. Methods of these measurements have been reported4. Fasting blood samples were analyzed at a central laboratory, including serum glucose, lipid levels, creatinine, uric acid, sodium, and potassium.
Of the 4,736 SHEP participants, 4,189 were included in this analysis. The 547 participants were excluded either because of missing data concerning CVD risk factors (n = 283) or with previous CHD or stroke (n = 264). These 547 excluded subjects had similar age, sex, race, and other characteristics as those who were included in this analysis.
Intervention
A stepped-care treatment approach was used, with the goal for individuals with SBP >180 mm Hg to reduce to <160 mm Hg and for those with SBP between 160 and 179 mm Hg to have a reduction of at least 20 mm Hg. All participants were given chlorthalidone, 12.5 mg/d, or matching placebo (step 1 and dose 1 medication). Drug dosage (step 1 and dose 2 medication) was doubled, 25 mg/d, for participants failing to achieve the SBP goal at the follow-up visits. If the SBP goal was not reached at the maximal dose of step 1 medication, atenolol, 25 mg/d, or matching placebo was added (step 2 and dose 1 medication). When atenolol was contraindicated, reserpine, 0.05 mg/d, or matching placebo could be substituted. When required to reach the blood pressure goal, the dosage of the step 2 drug could be doubled (atenolol 50 mg/d or reserpine 0.10 mg/d, step 2 and dose 2 medication). Potassium supplements were given to all participants who had serum concentration below 3.5 mm0l/L at two consecutive visits. The SHEP participants were followed up monthly until SBP reached the goal or until the maximum level of stepped-care treatment was reached [4,7]
Ascertainment of Outcome Events
The present analysis focused on five types of events: 1) death; 2) first-occurring major cardiovascular event, including stroke, MI, or heart failure; 3) first-occurring stroke; 4) first-occurring MI; and 5) first clinical diagnosis of congestive heart failure (CHF). The adjudication and clarification of the events was done by a panel of three physicians blinded to treatment assignment and blood pressure status. Members of the panel reviewed the documentation of new cardiovascular events over the study period and adjudicated outcome events according to predetermined criteria. [4]
Calculation of Global CVD Risk Scores
Information at the baseline on age, sex, total cholesterol, high density lipid (HDL) cholesterol, systolic blood pressure, diabetes (diabetic vs. non-diabetic), and smoking (current vs. never or past smoking) were used to calculate an a priori global score for the risk of developing future cardiovascular events, according to the Multiple Risk Factor Assessment Equation jointly proposed by the American Heart Association and the American College of Cardiology.[11] The equation assigns scores to major risk factors, using cut points that were originally developed using data on incident CHD from the Framingham study. A global CVD risk score ranging from -17 to +22 was obtained by adding the subscores. Higher values reflect a more unfavorable risk profile. Because the equation does not provide the age score for persons ≥ 75 years of age (28.5% of the SHEP study population), one additional point was assigned to men and women in this age group. Based on the global cardiovascular risk score, participants were classified into one of four CVD risk groups: low, medium, high and very high.
Calculation of Costs
The methods of economic evaluation include cost-effectiveness analysis, cost-utility analysis, and cost-benefit analysis, which can be used to assess the trade-off between costs and benefits in choices of antihypertensive treatment regimens. The primary aim of this analysis was to examine cost of the diuretic-based antihypertensive drug intervention in the SHEP trial. A cost-minimization analysis is a special type of cost-effectiveness analysis. It can be used to compare cost difference among competing alternatives of antihypertensive drug treatments when these treatments are medically equivalent. In this study, we used cost-minimization analyses to compare costs and incremental costs of NNT for 5 years to prevent one adverse event related to CVD among antihypertensive treatment regimens. The perspective of this economic evaluation was that of a national health insurance system.
We used the number-needed-to-treat as an unit of common outcome measure in the analysis. The number-needed-to-treat to prevent one adverse outcome has become a widely used measure of treatment benefits derived from the results of clinical trials. The NNT is the reciprocal of the absolute risk reduction (ARR) which is the difference between the proportions with the adverse event in the treatment and placebo groups. The 95% confidence interval of NNT was calculated based on the regression-based method described by Laupacis et al. [12]
The cost specified in the analysis includes the drug acquisition cost of SHEP treatment from the perspective of a national health insurance system. According to the SHEP treatment protocol, the stepped-care was classified into four types of drug treatments: 1) the Step 1 and Dose 1: chlorthalidone 12.5 mg/d; 2) the Step and Dose 2: chlorthalidone 25 mg/d; 3) the Step 2 and Dose 1: chlorthalidone 25 mg/d plus atenolol 25 mg/d or reserpine 0.05 mg/d; and 4) the Step 2 and Dose 2: chlorthalidone 25 mg/d plus atenolol 50 mg/d or reserpine 0.1 mg/d. Direct drug acquisition costs were calculated based on the minimum average wholesale prices (AWP) within drug manufacturers in the year 2000.[13] All drug costs were based on the AWP per unit dose. The expected cost (EC) of the SHEP drug acquisition per patient in 1 year was calculated as follows:
EC = W1 × C1 + W2 × C2 + W3 × C3 + W4 × C4
The W1, W2, W3, and W4 represent proportions of the participants using the Step 1 and Dose 1, the Step 2 and Dose 2, the Step 2 and Dose 1, and the Step 2 and Dose 2 medication, respectively. C1, C2, C3, and C4 represent the drug acquisition cost of the Step 1 and Dose 1, the Step 2 and Dose 2, the Step 2 and Dose 1, and the Step 2 and Dose 2 medication, respectively. A Monte Carlo method was performed to estimate the average cost and its standard deviation.
To compare the cost of the SHEP-based therapy to other antihypertensive drugs, it was assumed that all antihypertensive drugs in the comparisons have equal efficacy in terms of the NNT for 5 years to prevent one CVD related event. The NNT was calculated based on the method. [12]
All drug costs were expressed as dose-specific cost per patient in 1-year and/or 5-year. Using the approach, costs were calculated for each representative drug based on equipotent doses in terms of blood pressure reduction. [14] The non-SHEP based drugs, including beta-blockers (Atenolol), ACE inhibitors (Enalapril), and calcium channel blockers (Nifedipine), were selected in the analysis according to antihypertensive drug class. These drugs were considered commonly prescribed antihypertensive medications in clinical practices. [14] All costs were adjusted in 2000 constant U.S. dollars using the Consumer Price Index.
In this analysis, we focused on the drug acquisition cost for antihypertensive management. Therefore, the monitoring cost for antihypertensive treatment was not included. Total treatment cost includes antihypertensive drug cost and monitoring cost. The monitoring of treatment in ambulatory care settings including physician visits and laboratory tests have an estimated cost of $284 per patient per year. [14] Total cost of the NNT for 5 years of each drug therapy was calculated by multiplying the NNT for 5 years with the drug acquisition cost for 5 years per patient. The incremental cost is the cost of NNT for 5 years to prevent one adverse event of one alternative less the cost of the base case. In calculations of the incremental costs of the NNT for 5 years by types of outcome, the cost to prevent one stroke which was used as a base case. In calculations of the incremental costs of the NNT for 5 years by risk levels of CVD, the cost to prevent one adverse event of the very high risk level being used as a base case.
Result
Table 1 shows the expected acquisition cost of the diuretic-based antihypertensive therapies. The step 1 and dose 1 medication was the most used therapy and followed by the step 1 and dose 2 medication. The annual drug acquisition costs of the step 1 and dose 1, the step 1 and dose 2, the step 2 and dose 1 and the step 2 and dose 2 were $10.24, $20.48, and $222.45 respectively. The expected annual drug acquisition cost per patient of the SHEP treatment without potassium supplements was $83 and with potassium supplements was $91. The 5 year annual drug acquisition cost with potassium supplements per patient was $456.
Table 1 Estimated Drug Acquisition Costs of The SHEP Treatment Protocol
Drug Category Drug Cost Per Patient in 1 Year Proportion Drug Cost Per Patient in 5 years
step1 dose1 (chlorthalidone 12.5 mg/d) $10.24 0.43
step1 dose2 (chlorthalidone 25 mg/d) $20.48 0.23
step2 dose1 (chlorthalidone 25 mg/d plus atenolol 25 mg/d or reserpine 0.05 mg/d) $222.45 0.16
step2 dose2 (chlorthalidone 25 mg/d plus atenolol 50 mg/d or reserpine 0.1 mg/d) $221.93 0.17
Weighted SHEP Rx $83.29 0.91
KCL $88.33 0.09
Weighted SHEP Rx including KCL $91.24 $456
SD $101.78
Results of the 5 year NNT to prevent one adverse event and its associated cost by event type are shown in Table 2. To prevent one death, the cost for the 5 year NNT was $28,284. In other words, we need to treat 62 patients for 5 years in order to prevent one of them from death and the expected drug acquisition cost for the benefit is $28,284. The cost for the 5 year NNT to prevent one patient from one CVD event of any type is about four times lower than that of death. The cost for the 5 year NNT to prevent one MI is much higher than the cost for preventing one stroke or one CHF. Using the cost to prevent one stroke as the base amount, the incremental cost for the NNT for 5 years to prevent one MI or one CHF was $22,354 and $5,474, respectively.
Table 2 NNT and Drug Costs by Adverse Events
Event Placebo risk Treatment risk ARR NNT (95% CI) 5-Year NNT 5-year Rx Cost Per Patient Total Cost Incremental Cost
Death 0.1002 0.0858 0.0144 69 (31 – 319) 62 $456 $28,284 $13,230
CVD 0.1746 0.1147 0.0599 17 (12 – 26) 15 $456 $6,843 -
Stroke 0.0705 0.0433 0.0272 37 (24 – 76) 33 $456 $15,055 $0 (base)
MI 0.0312 0.0202 0.011 91 (48 – 740) 82 $456 $37,408 $22,354
CHF 0.0397 0.0198 0.0199 50 (33 – 103) 45 $456 $20,529 $5,474
Table 3 presents costs of the NNT for 5 years to prevent one CVD event of any type by CVD risk strata. The cost for the 5 year NNT increases as the CVD risk level decreases. It costs $20,529 for the 5 year NNT to prevent one of any type of CVD adverse events among patients in the low CVD risk group. In contrast, it only costs $5,018 for the same effect among patients in the very high CVD risk group. Using the cost of the very high CVD level as a base, if 12 patients in the high CVD level are treated, the extra cost to prevent one patient out of 12 from one CVD event is $456. The extra cost for patients in the low CVD risk group to receive the same effect is $15,511 relative to the patients in the very high CVD risk group.
Table 3 NNT and Drug Costs by CVD Risk Profile
Risk Category Placebo Risk Treatment Risk ARR NNT (95% CI) 5-year NNT 5-year Drug Cost Per Patient Total Cost Incremental Cost
1 (low) 0.1013 0.0814 0.0199 50 (18 – 59) 45 $456 $20,529 $15,511
2 (medium) 0.1476 0.0912 0.0564 18 (11 – 53) 16 $456 $7,299 $2,281
3 (high) 0.2044 0.1265 0.0779 13 (8 – 26) 12 $456 $5,474 $456
4 (very high) 0.2526 0.1699 0.0827 12 (7 – 38) 11 $456 $5,018 $0 (base)
In Table 4, the comparisons of the incremental drug acquisition cost for the 5 year NNT of the SHEP-based antihypertensive therapy to other commonly prescribed antihypertensive drugs. This analysis assumes that alternative drugs have equal efficacy to prevent CVD events. The estimated incremental net cost of the 5 year NNT to prevent one CVD event associated with use of atenolol (beta-blocker), enalapril (ACE inhibitor), terazosin (alpha-blocker), and nifedipine (calcium channel blocker) relative to the SHEP-based drug therapy ranged from $6,372 to $38,667 in older adults with isolated systolic hypertension. According to the cost ratio, it indicates that the costs of the 5 year NNT of using enalapril, terazosin, and nifedipine were up to 6.6 times more expensive compared to the SHEP-based drug therapy.
Table 4 Comparisons of Drug Acquisition Costs of 5-Year NNT Among Antihypertensive Drug Classes
Drug Class Commonly Prescribed 5-year Cost Per Patient 5-Year NNT Total Cost Incremental Cost Cost Ratio
SHEP-based drug therapy $456 15 $6,843 $0 (base) 1 (base)
Beta-Blocker Atenolol
25 mg daily $1,255 15 $18,825 $11,982 2.75
50 mg daily $1,245 15 $18,675 $11,832 2.73
100 mg daily $1,792 15 $26,880 $20,037 3.93
ACE inhibitor Enalapril
5 mg daily $2,031 15 $30,465 $23,622 4.45
10 mg daily $2,132 15 $31,980 $25,137 4.67
20 mg daily $3,034 15 $45,510 $38,667 6.65
Alpha-Blocker Terazosin
2 mg daily $2,984 15 $44,760 $37,917 6.54
5 mg daily $2,984 15 $44,760 $37,917 6.54
10 mg daily $2,984 15 $44,760 $37,917 6.54
Calcium channel blocker Nifedipine
30 mg daily $881 15 $13,215 $6,372 1.93
60 mg daily $1,762 15 $26,430 $19,587 3.86
90 mg daily $2,644 15 $39,660 $32,817 5.8
Discussion
The result of an economic evaluation essentially shows the cost per benefit gained from adapting a specific treatment. The effective and efficient use of resources has been increasingly emphasized from society, health plans, and health care providers. This cost-minimization analysis incorporating outcome data from the SHEP trial presents information treatment cost for older patients with ISH. We found that a long-term, low-dose and diuretic-based antihypertensive therapy is relatively inexpensive and effectively prevents adverse events associated with cardiovascular diseases, especially in older patients who had a high CVD risk profile.
Our findings indicate that the total and incremental treatment costs of antihypertensive drugs in ambulatory care settings range widely among drug classes as well as within drug classes. This analysis suggests that diuretic-based antihypertensive treatments are the least expensive, whereas atenolol (beta-blocker) is less costly than enalapril (ACE inhibitor) and nifedipine (calcium channel blocker), and terazosin (alpha-blocker) is the most expensive drugs in terms of the 5 year NNT to prevent one CVD event. It appears that use of the SHEP-based drug therapy offers greater economic benefits for controlling isolated systolic hypertension in the elderly than other antihypertensive drug treatments. Using a decision analysis model that simulated clinical decisions and outcomes that would occur when primary care physicians follow the JNC IV hypertension management guidelines, it was found that a newer class of calcium channel blockers can slightly increase the proportion of patients who achieve and maintain hypertension control, but at a substantially higher cost than with a generic diuretic drug. [15]
For our analyses, we presumed that all drugs offer equivalent therapeutic benefits. This assumption may have introduced a conservative bias into our primary findings. In fact, randomized controlled trials directly comparing active treatments for hypertension reported that calcium antagonists and doxazosin were inferior to low-dose diuretics or other agents in preventing cardiovascular events, suggesting that the cost-effectiveness of diuretic-based treatments may be even more favorable than estimated in the present study. [15-17] Further, in a meta-analysis of over 27,000 patients, those randomized to calcium antagonists as first-line therapy ran a greater risk of experiencing a myocardial infarction (26% higher risk), congestive heart failure (25% higher risk), and all cardiovascular events combined (10% higher) as compared to those randomized primarily to low-dose diuretics, beta-blockers and ACE inhibitors.[16] Finally, the Antihypertensive and Lipid Lowering treatment to prevent Heart Attack Trial (ALLHAT) recently reported a significantly higher risk of congestive heart failure, stroke, and major cardiovascular events in the doxazosin group than in the chlorthalidone group.[17] It is noteworthy that in this trial, only minimal differences in blood pressure control occurred between treatment groups, suggesting that the magnitude of blood pressure control represents an inadequate marker for comparing the therapeutic benefits of antihypertensive therapies.
With regard to costs projected in our study, it is noteworthy to consider that compared to the SHEP treatments, costs of treatments based on more recently developed antihypertensive agents (than reported here) are likely to be even higher than estimated in the present analyses.
The results of this study are limited to men and women 60 years and older who have isolated systolic hypertension and no presumed contraindication to any one class of antihypertensive medications. One limitation to our study relates to the fact that comparisons were based on costs of monotherapies, while combination therapies are frequently needed to control blood pressure.
The number-needed-to-treat to prevent one adverse outcome has become a widely used measure of treatment benefits in medical community, which is easy for physicians to understand. The shortcomings of NNT are that the outcome measure of an effect is with one dimension- survival probability and that it measures the specified outcome at a single point in time. Therefore, a measure of NNT can not capture an outcome in effectiveness of the intervention with two dimensions: time and survival probability. These limitations may not allow us to take time and discounting on cost and effect into account in this study.
Conclusion
Based on our findings, antihypertensive therapy that is diuretic-based and that includes either low-dose reserpine or atenolol represents a cost-effective regimen in preventing or delaying cardiovascular events in older adults. Use of the diuretic-based therapy is the most cost-effective in patients at high risk for developing cardiovascular disease. These results suggest that clinicians should consider using diuretics plus low-dose reserpine or atenolol as first-line therapy in patients with isolated systolic hypertension who are greater than 60 years old when there are no contraindications among these patients.
List of Abbreviations Used
ACE: angiotensin-converting enzyme
ALLHAT: Antihypertensive and Lipid Lowering treatment to prevent Heart Attack Trial
ARR: absolute risk reduction
AWP: average wholesale price
BP: blood pressure
CHD: coronary heart disease
CHF: congestive heart failure
CVD: cardiovascular disease
DBP: diastolic blood pressure
HDL: high density lipid
ISH: isolated systolic hypertension
JNC IV The Sixth Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure.
NNT: number-needed to treat
SBP: systolic blood pressure
SHEP: Systolic Hypertension in the Elderly Program
Conflict of Interest
The author(s) declare that they have no competing interests.
Authors' contributions
GC, LF, WM and MP participated the development of the analytic framework. GC performed all data analyses. GC, LF, WM and MP drafted and revised the manuscript. All authors approved the final manuscript.
Acknowledgements
The SHEP was supported by a contract with the National Heart, Lung, and Blood Institute and the National Institute on Aging. This study was supported by a grant NHLBI R03 HL5995-01A1 to Wake Forest University Health Sciences, Winston-Salem, North Carolina.
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| 15670330 | PMC553989 | CC BY | 2021-01-04 16:39:14 | no | Cost Eff Resour Alloc. 2005 Jan 25; 3:2 | utf-8 | Cost Eff Resour Alloc | 2,005 | 10.1186/1478-7547-3-2 | oa_comm |
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Int Semin Surg OncolInternational seminars in surgical oncology : ISSO1477-7800BioMed Central London 1477-7800-2-51573055910.1186/1477-7800-2-5ReviewColorectal cancer metastasis: in the surgeon's hands? Atkin Gary [email protected] Abhay [email protected] Ian [email protected] Department of Surgery, Barnet General Hospital, Wellhouse Lane, Herts, EN5 3DJ, UK2 Department of Surgery, Ealing Hospital, Uxbridge Rd, Middlesex, UB1 3HW, UK2005 24 2 2005 2 5 5 18 12 2004 24 2 2005 Copyright © 2005 Atkin et al; licensee BioMed Central Ltd.2005Atkin et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Lymphovascular ligation before tumour manipulation during colorectal cancer resection is termed the 'no-touch isolation' technique. It aims to reduce the intra-operative dissemination of colorectal cancer cells. Recently, the detection of circulating tumour cells has been enhanced by molecular biology techniques. This paper reviews the evidence for the no-touch isolation technique in light of the recent developments in circulating tumour cell detection.
Methods
Studies investigating the effect of colorectal cancer surgery on circulating tumour cells were identified by a Medline search using the subject headings colorectal neoplasms and neoplasm circulating cells together with the map term 'no-touch isolation technique'. Further references were obtained from key articles.
Results
Molecular biological techniques have improved the detection of circulating colorectal cancer cells. There is a trend towards reduced tumour cell dissemination with the no-touch technique compared with the conventional method. However the benefit in terms of improved patient survival remains unproven.
Conclusion
The no-touch isolation technique reduces circulating tumour cell dissemination but further work is needed to determine the significance of this with regards to patient survival.
==== Body
Background
Of the patients with colorectal cancer (CRC) undergoing surgery for resectable disease, 30–50% will subsequently develop metastases [1]. Dissemination of tumour cells is therefore thought to occur early on in the disease process. The principle of early lymphovascular ligation before manipulation of the tumour during the surgical resection of a CRC has been termed the 'no-touch isolation' technique. This was proposed by Barnes [2] as a way of reducing the incidence of liver metastases by diminishing the intra-operative dissemination of CRC cells. An early proponent of the technique was Turnbull et al [3], but their findings have not been confirmed in other studies [4].
Animal studies suggest tumour cells are shed into the circulation during resection of a primary tumour, increasing the likelihood of metastases [5]. However, the evidence in humans is not clear. This may be because the early techniques used to detect circulating tumour cells (CTC) were not sufficiently sensitive to detect the small number of cancer cells present within the blood. These detection methods relied on cytological examination of blood smears, and allowed the detection of 1 tumour cell within 100 normal cells [6].
In recent years, the question of tumour cell dissemination during surgical resection has been re-examined following the introduction of molecular biology techniques to detect CTC. These are much more sensitive, and it is now possible to detect one tumour cell in a sample of 107 normal cells [7].
This paper reviews the evidence for the no-touch isolation technique for the resection of a CRC in light of the recent developments in molecular biological techniques for CTC detection.
Methods
In performing this review, the studies looking at the effect of CRC surgery on CTC were identified by a Medline search using the subject headings colorectal neoplasms and neoplasm circulating cells together with the map term 'no-touch isolation technique' [8-16]. Further references were obtained from key articles [17-20].
We decided to review this topic by addressing three areas of uncertainty. Firstly, does the surgical manipulation of a CRC increase the level of tumour cells within the blood? Secondly, what is the biological significance of CTC in CRC? Thirdly, is the no-touch isolation technique of CRC resection associated with an improved patient survival?
To investigate the effect of tumour manipulation on CTC, we have reported the conversion, for each reviewed article, of a negative preoperative blood sample (for CTC) to a positive intra- or post-operative sample within the same patient. We have only noted conversions within the same sample source (systemic venous (SV) or portal venous (PV) blood). Where conversion rates cannot be determined, as data for individual patients are not provided, a comparison between overall preoperative and intra/post-operative positivity for CTC is given. Unless otherwise stated in the reviewed article, we have presumed the conventional method was utilised (tumour manipulation before lymphovascular ligation).
Results
A) Does the surgical manipulation of a CRC increase the level of tumour cells within the blood?
A number of experimental models have been used to investigate the effect of surgical manipulation on CTC. Nishitaki et al [5] studied 2 groups of rabbits with surgically-inoculated liver tumours. The first group had a laparotomy and manipulation of the hepatic tumour 14 days after inoculation, together with resection of the tumour. The second group had a laparotomy and resection of the tumour but without manipulation. Two weeks later the rabbits were sacrificed, and pathological examination showed significantly more hepatic venous invasion by cancer cells in the tumour manipulation group, together with significantly more lung metastases and shorter mean survival.
Romsdahl et al [21] measured the number of tumour cells by cytological examination in blood samples from the inferior vena cava of rats during manipulation of a thigh tumour. There was a manifold increase in the number of CTC during manipulation, which decreased rapidly, so that 4 minutes after manipulation 93–96% of the cells had disappeared from the circulation. In a similar experiment with mice, Liotta et al [22] showed a 10-fold increase in tumour cells in the venous effluent of a thigh tumour during manipulation.
In order for the no-touch technique to be effective in humans, early ligation of the main pedicle must eliminate passage of liberated tumour cells into the portal and systemic circulations. However, Salsbury et al [23] found the number of tumour cells in the common iliac vein dramatically increased after ligation of the inferior mesenteric vein, suggesting there was a shift of the draining venous blood into the systemic circulation following ligation. In an experimental model using dogs, Ackerman [24] showed clamping of the mesenteric veins produced an increased intestinal lymphatic and venous outflow, thereby enhancing the possibility of tumour cell dissemination. He concluded ligation of the major arterial supply, followed by the marginal vessels, is the most effective technique in minimising outflow.
On theoretical grounds, the no-touch technique would appear unsuitable for low rectal cancers, as it is necessary to mobilise tumours in the rectum before all the draining veins are controlled. In addition, a large proportion of the venous drainage occurs into the iliac system, thereby negating the effect of mesenteric pedicle ligation.
Results of reviewed articles
There have been numerous studies investigating the effect of tumour manipulation on CTC in human tumours. This review focuses on studies involving CRC. The overall conversion rates for each study (ie conversion from negative preoperatively for CTC to positive intra or post-operatively taking into account all study patients) are listed in Additional Files 1 and 2. The conversion rates taking in to account only those patients negative preoperatively for CTC are also listed.
The only study to compare directly the conventional and no-touch techniques for the resection of a CRC involved the non-random assignment of 27 patients into conventional and no-touch groups [8]. The technique of mutant-allele-specific amplification (MASA) was used to identify mutations of K-ras or p53 genes in the primary tumour of each patient. Cells containing the same mutations were then examined for in blood samples taken before, during, and after surgery. In the conventional group, 11 patients had K-ras or p53 mutations in their primary tumour. None of these patients had preoperative blood samples that contained cells exhibiting similar mutations. However, 8 (73%) did have cells containing similar mutations in the blood taken intra-operatively following tumour manipulation. In the no-touch group, 7 patients had mutations in their primary tumour. None of these patients showed similar mutations preoperatively, and only 1 patient (14%) became positive intra-operatively. From these findings, the authors conclude the no-touch technique is effective at reducing the intra-operative dissemination of colorectal tumour cells.
The other studies involving CRC have used either the conventional or no-touch surgical techniques, but have not directly compared both. Weitz et al [9] and Sales et al [10] employed the no-touch technique and found an overall conversion rate of 16% and 9% respectively. Both studies conclude the no-touch technique is effective at reducing tumour cell dissemination. Bessa et al [17] also used the no-touch technique in 50 patients who were randomly assigned to undergo open or laparoscopically-assisted colectomy. They found an overall conversion rate of 8% and 12% respectively in SV samples.
Studies employing the conventional technique of CRC resection have found overall conversion rates of 0 – 80% in SV samples [11-15,18,19] (see Additional file 2). Funaki et al [20] demonstrated the highest conversion, with 4 out of a study sample of 5 patients converting intra-operatively. This is much higher than in any other study, and although the sample size is small, the authors suggest this is evidence of tumour cell dissemination as a result of surgical manipulation. It is interesting to note 80% of these patients had rectal cancers, which require significant mobilisation before control of the venous output is obtained. Griffiths et al [18] did not detail individual patients and so the conversion rate cannot be determined, but they found 4% of patients were positive preoperatively, 50% were positive intra-operatively, and 8% postoperatively. Similarly, Ito et al [15] found significantly more patients were positive for CTC following surgery compared with preoperatively (51% v 38%, p = 0.003). Most authors conclude their results demonstrate evidence of tumour cell dissemination. However, Garcia-Olmo et al [11], who used the conventional technique and did not demonstrate a conversion in any patients, concluded the no-touch isolation technique was unnecessary.
As a number of patients were positive for CTC before surgery, a more direct assessment of the effect of tumour manipulation might be to analyse the conversion rates only for those patients negative for CTC preoperatively. This reveals a much greater conversion rate for most studies (see Additional Files 1 and 2). For example, using the conventional method, Funaki et al [20] found all 4 patients negative preoperatively converted intra-operatively. Similarly, Tien et al [13] demonstrated a conversion of 49% for SV, and 52% for PV samples. Conversely, Garcia-Olmo et al [11] used the conventional method and failed to show conversion in any patients. For the no-touch technique, the conversion rates are also greater if only the patients negative preoperatively are considered. Bessa et al [17] demonstrated the highest conversion rate, with 50% and 33% of patients converting following laparoscopically-assisted colectomy within SV and PV samples respectively.
Limitations and variations of studies under review
1) Choice of technique used to detect CTC
Before the advent of molecular biology, the detection of tumour cells within the blood depended on cytological examination of a blood smear. The incidence of circulating tumour cells during CRC resection detected by cytological examination of blood smears has been reported as 25–67% [25,26]. Griffiths et al [18] state it is easy to detect CTC within a blood sample, but this method of detection underestimates the true number of circulating cells as there is probably a considerable loss in the preparation of blood cell concentrates. Also, difficulties in interpretation arise when other large mononuclear cells mimicking cancer cells are present.
Immunocytochemistry has been used to detect CTC in a number of tumour types, including colorectal cancer [27]. This technique labels tumour cells with antibodies against specific cell components that are not expressed in haematopoietic cells. It has the advantages of preserving cellular morphology and allowing identification of cell clusters, an important drawback of molecular detection techniques [28]. It also permits simultaneous assessment of other cellular details such as proliferation activity and oncogene expression. However, it is labour intensive due to the low concentration of CTC. The sensitivity has also been questioned [29], although this can be enhanced with flow cytometry.
In recent years, molecular biological techniques have been developed for use in tumour cell detection. The polymerase chain reaction (PCR) involves detection and amplification of certain DNA mutations. A copy of the DNA containing the target sequence is made and the process is repeated many times, producing multiple copies and thereby increasing the sensitivity of detection. Mutations in the k-ras gene have often been used for detection of CTC in CRC.
However, the detection of these sequences within the blood does not necessarily imply there are circulating tumour cells, as DNA is relatively stable and may represent fragments of tumour DNA released by cell necrosis or apoptosis and persisting in the blood for some time [30]. Also, this method of detection relies on the presence of specific DNA mutations within the primary tumour, and the genetic changes associated with CRC are known to be heterogenous [28].
An alternative technique, now more commonly employed, is reverse transcriptase-PCR (RT-PCR). This detects messenger RNA (mRNA), which is used as an indicator of tissue-specific gene transcription. The technique is rapid, more easily automated than immunocytochemistry, and is capable of detecting one tumour cell in 107 normal cells [19]. RNA is rapidly degraded and so its presence in the blood suggests active expression by circulating tumour cells [13].
However, there is a lack of standardisation of RT-PCR techniques, resulting in different sensitivities and specificities between centres [31]. Illegitimate transcription (when tissue-specific genes are transcribed in non-specific cells) can occur, and the presence of non-malignant epithelial cells in the blood (for example, following venepuncture [32]) reduces the specificity. RT-PCR detects the number of copies of mRNA, not the number of CTC, so it may not accurately assess an increase in tumour cells secondary to mobilisation [13]; also it may be difficult to derive the prognostic value of CTC if the levels cannot be accurately determined in individual patients. False negatives can occur with RT-PCR, affecting the technique's sensitivity. The marker of interest may not be expressed because of tumour cell heterogeneity or because there is a poorly-differentiated subclone that has lost the ability to express the tissue-specific marker [33]. PCR inhibitors may be present within some body tissues, and it is important to note the in-vitro sensitivity reported in the reviewed articles will be higher than the true in-vivo sensitivity, as the cell lines used to determine the sensitivity are known to express strongly the marker of interest and are not affected by the presence of in-vivo PCR inhibitors.
Most of the reviewed articles that state the specificity of their technique claim a rate of 100%, indicating all the tumour samples were positive for the marker of interest, whereas none of the control blood samples (from healthy volunteers or patients undergoing resection for benign disease) were positive. However, Patel et al [14] examined 143 control subjects and found 7 (5%) were positive when a single blood sample was assessed for CTC.
2) Choice of marker used to detect CTC
There are no tumour-specific genes for CRC, and it is known malignant cells continue to express markers that are characteristic of their tissue of origin. Therefore, a range of epithelial cell markers, which are normally absent from the blood, have been used as RT-PCR targets for cancer cells derived from epithelial tissues.
The cytokeratin (CK) polypeptides are found within the cytoplasm of epithelial cells, and several CK markers have been used to detect CTC. The most common of these is CK 20, which is expressed in gastrointestinal epithelia, urothelium, Merkel cells, and tumours derived from these tissues [9]. Carcinoembryonic antigen (CEA) is a glycoprotein found on the surface of epithelial cells. It is overexpressed in nearly all colorectal cancers [34], as well as other tumours such as breast and non-small cell lung carcinoma, and has been widely used to detect CTC. Guanylyl cyclase C (GCC) is another tissue-specific marker only expressed in normal intestinal mucosa. It is a member of the guanylyl cyclase family of receptors, and its expression persists once the epithelial cell has undergone neoplastic transformation [13]. Cell surface sialylated carbohydrates, such as sialyl Lea and sialyl Lex, are associated with CRC formation and progression [35], and have been used to detect disseminated tumour cells as well as predict prognosis.
Concerns regarding marker specificity have been raised. CK 8, 18 and 19 have all been shown to be expressed in normal blood [29]. CK 20 and CEA are more specific, although they have also been seen in control samples [31,36]. The CEA family includes homologous genes expressed in granulocytes [37], and CEA may be induced by surgical stress [15] as well as occurring in the blood of patients with inflammatory conditions such as colitis [38]. GCC appears to be the most specific marker investigated. Bustin et al [31] found CK 20 transcripts in all 21 healthy volunteers studied, whereas GCC was found in only a single control.
3) Choice of blood sample used to detect CTC
The source of blood for analysis appears to be important. Most studies have investigated systemic venous samples for the presence of CTC. Animal studies suggest tumour cells become trapped in the capillary bed of the first organ encountered [39,40], and so for CRC would be seen in the liver but not in peripheral blood. Theoretically, CRC cells must pass through the liver, lungs and the microcirculation of the other tissues of the body before they pass into the systemic venous circulation. Also, any CTC should be diluted by the larger blood volume of the peripheral circulation [12]. There should be many more circulating colorectal tumour cells in the portal circulation than the systemic circulation, therefore. Griffiths et al [18] found 57% of patients were positive for CTC in PV blood during resection a colorectal tumour, as opposed to 50% of patients who were positive in their SV samples. Tien et al [13] found more PV samples were positive before, during and after surgery compared with SV samples. However, 3 of the 17 who were positive in the peripheral blood were negative in all portal samples, suggesting tumour cells were bypassing the portal circulation and entering the systemic circulation directly. It is interesting to note these 3 patients had Dukes stage C disease and it may be that tumour cells were passing directly from the lymphatics into the peripheral circulation. Bessa et al [17] found there was concordance between SV and PV samples for CEA mRNA in only 65% of cases. They also found no patients demonstrated a conversion in PV samples following open colectomy, however 8% converted in SV blood.
Due to the discrepancy in sample sources, we have only noted patients who converted within the same venous compartment. Several studies detected CTC in draining vein blood intra-operatively, and compared it against a reference SV sample taken before manipulation. It is difficult to be certain of conversion in these patients, however, as it is possible the PV samples were positive preoperatively. It is important for subsequent studies to measure CTC in both systemic venous and draining vein blood, before, during and after tumour manipulation to investigate further the discrepancy in CTC detection between the venous compartments.
The importance of multiple sampling should be considered. Glaves et al [41] suggests cancer cells are intermittently shed in to the blood, so sampling errors may occur if single samples are taken. Tien et al [13] sampled SV and PV blood twice during tumour mobilisation and found that of the 14 patients who were positive in portal blood during mobilisation, 5 (36%) were only positive in the second sample, and so would have been considered negative if a single sample was taken. In the SV samples, 3 out of 17 (18%) patients were only positive in the second sample. Mori et al [19] also performed sequential sampling intra-operatively, and found of the 5 patients positive for CTC at any of the 4 sampling time points, 2 (40%) were positive at a single time point only. Wharton et al [42] showed by increasing the sampling frequency (from once to twice), the detection of CTC is significantly increased.
Weitz et al [9] suggest another factor affecting the detection of CTC may be the degree of intra-operative blood loss and subsequent administration of intravenous fluids, thereby diluting the blood volume and reducing the likelihood of tumour cell detection. They showed the chance of intra-operative detection of tumour cells was halved by a blood loss of 0.5 l, and so excluded all patients with an intra-operative blood loss of more than 1 l from further statistical analysis. They found of the 7 patients who were positive pre- or post- but not intra-operatively, 5 (71%) had a blood loss of more than 1 litre, suggesting a possible dilution effect during the operation. In the articles reviewed, the degree of blood loss was only reported in the study by Weitz et al.
B) What is the biological significance of CTC in CRC?
The detachment of malignant cells from the primary tumour is an early step in the formation of metastases [5]. The release of tumour cells is a continuous process [43], however the metastatic process is inefficient and the presence of tumour cells within the blood does not necessarily imply the subsequent development of metastases [44]. It is poorly understood which steps in the metastatic process are responsible for the inefficiency of tumour cells to form overt metastases.
Early studies suggested fewer than 0.1% of circulating tumour cells survive in the circulation [39], and only 0.01% form metastases [45]. It was thought the majority of CTC are removed from the circulation within 24 hours [39], by elimination in the first capillary bed they encounter [46]. However, recent work using cytoplasmically-labelled tumour cells (as opposed to nuclear-labelled cells which are more vulnerable to destruction) has found the majority of cells survive in the circulation for several days following injection, and the inefficient part of the metastatic process appears to be the variable growth of the cancer cell at the secondary site [47].
The metastatic process may be enhanced by the surgical procedure itself. It is known the entrapment of tumour cells in the microcirculation of a target organ is facilitated by the presence of fibrin and platelets [48,49]. The activation of coagulation that occurs during an operation may therefore augment this process [50]. Also, surgical stress has been shown to induce immune suppression [51], thereby increasing the metastatic efficiency.
The prognostic value of CTC in CRC has yet to be fully determined. Fujita et al [52] found patients negative preoperatively for CK-19 or CK-20 had significantly fewer recurrences and better 5-year disease free survival. Nakagoe et al [53] found a high sialyl Le (x) antigen or CEA in blood draining the tumour was an independent prognostic indicator of poor survival, and Yamaguchi et al [12] found a similar finding in patients positive for both CEA and CK-20. Other studies show conflicting results, however. Bessa et al [54] found a preoperative peripheral blood sample positive for CEA did not predict prognosis, and in a comprehensive review, Tsavellas et al [29] conclude, at present, the presence of CTC cannot be considered to be a reliable indicator of prognosis in any common solid malignancy because of the lack of large standardised trials with sufficient follow up.
C) Is the no-touch isolation technique of CRC resection associated with an improved patient survival?
There are few studies directly comparing the outcomes following conventional and no-touch techniques. Turnbull et al [3] retrospectively compared 664 patients who underwent CRC resection using the no-touch technique against 232 patients with similar histological stage operated on by different surgeons employing the conventional method. They found the overall 5-year survival rate for the no-touch group was 51%, compared with 35% for the conventional group. They state the difference in mortality was due to an increased incidence of hepatic metastases in the conventional group. Subsequent criticism [4] of Turnbull's findings suggested patient selection may not have been random, and the basis of the no-touch technique incorporates an extended lymphadenectomy. The study also excluded cancers of the rectum.
Wiggers et al [4] randomly assigned 236 patients with tumours of the colon or rectum to a no-touch or conventional surgery group, and found no significant difference between the groups in terms of recurrence or survival. However, there was a trend towards fewer and later onset of liver recurrences in the no-touch group. Their recommendation was that the no-touch technique should be used for tumours where it is easily applicable.
The largest series was reported recently by Slanetz [55], who retrospectively reviewed 1863 cases of colorectal cancer resection over a period of 24 years. In 1050 cases, tumour mobilisation had occurred before regional mesenteric vessel ligation, whereas 813 cases had vessel ligation performed initially. The extent of mesenteric resection and tumour differentiation was reportedly similar between the two groups. The author reports the sequence of vessel ligation had little impact on the incidence of cancer-related deaths at 5 and 10 years, with no significant difference in survival rates between the early vessel ligation and conventional groups for colonic or rectal tumours. However, the sequence of vessel ligation did have a significant effect on the distribution of metastases. The early vessel ligation group was associated with fewer liver metastases but more systemic metastases compared with the conventional group. These findings may support the theory that after mesenteric pedicle clamping, there is a shift in draining blood into the systemic circulation [23]. There was also a significant increase in the local recurrence rate with the no-touch compared with the conventional technique (22.6% v 14.6%; p = 0.0001).
Another component of the no-touch isolation technique investigated by Slanetz is the control of intraluminal spread of malignant cells by applying bowel clamps or ligatures before tumour manipulation. He reviewed the results of 599 CRC resections in which bowel ligation prior to tumour mobilisation was used, and compared the data against 1416 resections in which bowel ligation was not performed. He found the application of bowel ligatures before tumour mobilisation significantly improved the 5-year cancer-related death rate for colon cancer (20% v 25%, p = 0.02), but not for rectal cancer. When considering colon and rectal cancer combined, bowel ligation prior to mobilisation significantly reduced the local (12% v 19%, p = 0.02) and distant (liver: 10% v 15%, systemic: 13% v 18%, p < 0.001) recurrence rates compared to resection without prior bowel ligation. These findings support the earlier work by Cole et al [56], who showed that ligatures around the bowel controlled the rate of local lymphatic and intraluminal dissemination of malignant cells.
Conclusion
It is difficult to draw any firm conclusions from the articles studied due to the lack of standardisation of sample source, CTC detection method and the small sample sizes. However, the only study directly comparing conventional and no-touch surgical techniques found a conversion rate of 73% for conventional surgery and 14% for the no-touch technique, suggesting a benefit of vascular clamping before tumour mobilisation. The overall conversion rates for the other studies employing the no-touch technique were 0–16% (see Additional file 1), compared with 0–80% for studies utilising conventional surgery (see Additional file 2). Therefore, these data suggest there is a trend towards reduced tumour cell dissemination for the no-touch isolation method. The benefit of this in terms of improved patient survival, however, remains unproven. On purely theoretical grounds, the presence of alternate lymphovascular pathways for most colorectal tumours ensures complete isolation of the tumour-bearing segment is hard to achieve, limiting the technique's efficacy. In addition, the extent of mesenteric resection, and not the surgical technique employed, is probably the most important determinant of patient outcome following CRC surgery [55].
Due to the lack of consensus regarding the best technique for detection, the biological importance of CTC has not been fully determined. With the introduction of molecular biological techniques, the sensitivity for detection has improved considerably. The hope is that accurate detection of occult circulating malignant cells would help to stage the disease and predict prognosis, as well as monitor the response to therapy and highlight early the possibility of recurrence. However due to the differences in methodology and conclusions of studies investigating this area, the detection of CTC cannot, at present, be used to dictate therapeutic strategy.
Despite the methodological heterogeneity of the articles reviewed, we believe the introduction of highly sensitive methods of CTC detection has forced a re-analysis of the role of the no-touch isolation technique in CRC surgery, and the stages of CRC resection using the no-touch technique would appear to offer a sensible, systematic approach to the surgical management of large bowel tumours. However, further work needs to be done to investigate the remaining areas of uncertainty. In particular, future developments in CTC detection must ensure more automation and greater standardisation of techniques between centres. RT-PCR appears to offer the greatest sensitivity, and techniques detecting multiple markers that are more tumour-specific should be investigated [57,58]. The true biological importance of CTC in CRC needs to be assessed further, and finally, the actual benefit of the no-touch technique can only be determined by large scale randomised clinical trials utilising multiple venous sampling in patients matched for age and stage of disease.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
GA researched the topic and prepared the manuscript, IM conceived the article and AC critically analysed and formatted the manuscript.
Supplementary Material
Additional File 1
Conversion rates for studies employing the no-touch isolation technique of CRC resection
Click here for file
Additional File 2
Conversion rates for studies employing the conventional technique of CRC resection
Click here for file
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| 15730559 | PMC553990 | CC BY | 2021-01-04 16:38:37 | no | Int Semin Surg Oncol. 2005 Feb 24; 2:5 | utf-8 | Int Semin Surg Oncol | 2,005 | 10.1186/1477-7800-2-5 | oa_comm |
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Theor Biol Med ModelTheoretical Biology & Medical Modelling1742-4682BioMed Central London 1742-4682-2-61573332810.1186/1742-4682-2-6ResearchVolume-based non-continuum modeling of bone functional adaptation Wang Zhengyuan [email protected] Adrian [email protected] Medical and Clinical Informatics Group, Bioinformatics Institute, #07-01 Matrix, 30 Biopolis Street, 138671 Singapore2005 28 2 2005 2 6 6 28 9 2004 28 2 2005 Copyright © 2005 WANG and MONDRY; licensee BioMed Central Ltd.2005WANG and MONDRY; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Bone adapts to mechanical strain by rearranging the trabecular geometry and bone density. The common finite element methods used to simulate this adaptation have inconsistencies regarding material properties at each node and are computationally demanding. Here, a volume-based, non-continuum formulation is proposed as an alternative. Adaptive processes corresponding to various external mechanical loading conditions are simulated for the femur.
Results
Bone adaptations were modeled for one-legged stance, abduction and adduction. One-legged stance generally results in higher bone densities than the other two loading cases. The femoral head and neck are the regions where densities change most drastically under different loading conditions while the distal area always contains the lowest densities regardless of the loading conditions. In the proposed formulation, the inconsistency of material densities or strain energy densities, which is a common problem to finite element based approaches, is eliminated. The computational task is alleviated through introduction of the quasi-binary connectivity matrix and linearization operations in the Jacobian matrix and is therefore computationally less demanding.
Conclusion
The results demonstrated the viability of the proposed formulation to study bone functional adaptation under mechanical loading.
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Background
Much research effort has been devoted to understanding the functional adaptation of bone under physiological loading ever since the idea of bone functional adaptation was proposed by Wolff more than one hundred years ago [1-14]. Various computational models have been put forward in the past decades and the methods describing the changing rate of bone density corresponding to strain energy density, with finite element implementation, have become the most popular of them [6,15-29].
The common finite element approach is to take the element densities as the state variables and define elements with either constant or varying densities, then update the material densities for the next step of computation according to the computed strain energy density [22,23,26,27]. With more and more powerful desktop computers and commercial finite element analysis software available, this approach is widely used today. Yet some specific problems of this approach are not well addressed so far, although decades have passed, and the numerical results are inevitably affected.
One common problem is the inconsistency of material densities on element boundaries [30]. During the updating of material densities in each step, different elements may take different densities due to strain energy density, thus often leading to conflicting material properties at the boundaries shared by more then one element. Since this conflict affects the integration points, which always come from the element boundaries, the errors are carried forward and cannot be eliminated by smoothing techniques. So it is not surprising that, if the program is allowed to run for a certain time, most of the elements tend to become either saturated or completely resorbed, leading to checker-board patterns especially in the proximal area of the femur [30].
Some effort has been made trying to solve this problem. For example, a node-based variant of the finite element method was tried with focus on the densities of the nodes rather than densities of the whole elements [21,30]. The node densities are then interpolated across the whole elements before the next step of computing begins. The results are improved, but the stress and strain quantities are still conflicting at the nodes.
Other previous work has used Voronoi structures [31] to study the effects of crack growth on trabecular bone, tapered strut models [32] to study the ageing effect through a parametric approach or continuum FEM [33] to compute the strain energy density in order to overcome individual drawbacks of the common method described. Their potential impact on the formula proposed here is discussed below.
The long existing problems and the limitations of assuming a continuum drive this new effort to explore the possibility of a non-continuum formulation of bone functional adaptations through nodal analysis in the hope of eliminating the errors present in the previous approaches. In the proposed non-continuum formulation, neighboring nodes are connected by struts that are defined with invariant material densities with respect to time but strut volumes are defined as state variables indicating different configurations of bone structure. The updating of strut volume will depend on the strain energy density in the strut in the previous step. As a result, there is no conflict either in density or in strain energy density. The shift of state variables from bone densities to bone volumes not only eliminates the errors inherent to the density-based finite element approaches but also transforms the continuum formulation to a non-continuum formulation [34]. The advantages of a volume-based non-continuum formulation may be appreciated by looking at the bone volume ratios in osteoporotic bones. The ever-increasing resolution of modern imaging techniques now allows to take a much closer look at the trabecular structure of the bone. In the trabecular network, trabeculae with different lengths and thicknesses are connected with each other to form a scaffold serving both mechanical and biological functions [35,36]. They are well connected in normal bones but poorly connected in osteoporotic bones in addition to reduced thickness. To characterize the trabecular structure, two terms are often used: bone volume/tissue volume (BV/TV) ratio and bone material orientation [15,25]. Although the cortical bone is densely packed with mineralized material, the trabecular bone dominates the inside space of the bone, highly exposed to bone marrow, highly distributed in volume, and highly involved in bone remodeling. The ratio of trabecular bone volume over tissue volume can be below 30% in osteoporotic bones, which means most space is taken up by void or bone marrow and this questions the appropriateness of a continuum formulation [35]. Besides elimination of the errors mentioned earlier, the small physiological range of bone deformation during normal activities allows linearization operations in the volume-based non-continuum formulation. This saves computation time and alleviates the high demand on hardware resources.
The proposed volume-based non-continuum formulation shows computational advantages in modeling bone functional adaptations and has much potential for clinical applications in this field.
Results
Changes in trabecular structure
Fig. 1, 2, 3 and 4 show the node-based representation of bone adaptation results according to loading cases of one-legged stance, abduction, adduction and the combined loading case respectively.
Figure 1 Adaptation Results: one-legged stance. Results of bone functional adaptation. The color bar shows percentage of actual bone density against maximum bone density, which is 1.74 g/cm3. The density of bone structure is not indicated by the number of sample nodes selected in that region, but by the density (converted from volume) of each node, which is expressed as degree of "red" in this illustration.
Figure 2 Adaptation Results: abduction. Results of bone functional adaptation. The color bar shows percentage of actual bone density against maximum bone density, which is 1.74 g/cm3. The density of bone structure is not indicated by the number of sample nodes selected in that region, but by the density (converted from volume) of each node, which is expressed as degree of "red" in this illustration.
Figure 3 Adaptation Results: adduction. Results of bone functional adaptation. The color bar shows percentage of actual bone density against maximum bone density, which is 1.74 g/cm3. The density of bone structure is not indicated by the number of sample nodes selected in that region, but by the density (converted from volume) of each node, which is expressed as degree of "red" in this illustration.
Figure 4 Adaptation Results: the combined loading case. Results of bone functional adaptation. The color bar shows percentage of actual bone density against maximum bone density, which is 1.74 g/cm3. The density of bone structure is not indicated by the number of sample nodes selected in that region, but by the density (converted from volume) of each node, which is expressed as degree of "red" in this illustration.
The nodal density is a percentage relative to its maximum value (0~100%). It is 'converted' based on volume information (BV/TV) for the purpose of easy visual inspection. As suggested by Zhu [34], the value of 1.74 g/cm3 is used as the maximum value in the present formulation.
In the combined loading case and one-legged stance, the nodal densities are generally high in the proximal area where a considerable number of nodes reach the highest density due to the relatively high load, while in the large distal area, the densities become lower. In the case of abduction, the external load is the smallest out of the three loading cases and the nodal density in this case seldom reaches the maximum. Although the high densities also appear in the proximal area of femoral head, the densities are lower than those of other loading cases. In the large distal area, the node densities generally range at very low levels. In the case of adduction, the highest densities still appear in the proximal area of femoral head, but in the large diaphyseal area, the densities range at very low levels.
In summary, one-legged stance and the combined loading case generally result in higher bone densities than the other two loading cases due to higher mechanical loading. The femoral head and neck are the regions where densities change most drastically under different loading conditions while the distal area always contains the lowest densities regardless of the loading conditions.
Program convergence and bone fracture probability
Starting from a uniform material distribution, the program computes the strain energy density, adjusts the strut configurations, and continues on to the next iteration. Fig. 5 shows the convergence of the model, which demonstrates the adapting process of the model evaluated with the normalized error residue against the final solution. The solution vector starts with a state of uniform material distribution, it then moves toward the final state with uniform strain energy density in the solution space. As the residue of solution drops, the residue of bone volume/tissue volume ratio also drops toward trivial while the program progresses.
Figure 5 Program Convergence. Program convergence evaluated by residues between successive solutions.
A prediction of bone fracture risk has been proposed using stress range levels [37] from which analysis of the material fatigue strength under given loading conditions can be derived. The material is subjected to a range of stress levels due to external load, then the stress leads to fatigue damage and finally leads to collapse of the material. This prediction method was used to estimate the fracture probability of the simulation described here. The BV/TV ratios, stress levels and fracture probability are shown in Fig. 6, 7 and 8 respectively. While the program progresses toward the final solution, the volume of bone material used, indicated by the BV/TV ratio, increases by a few percents then slowly decreases again; meanwhile, the stress level moves down to a low level in the final phase and accordingly, the estimated fracture probability drops from around 90% to around 2% in the final step. It is safe to say that the program finally simulates a reasonable configuration of bone internal structure as a result of the physiological adaptation process.
Figure 6 BV/TV Evolution During Adaptation Process. BV/TV evolution during adaptation process.
Figure 7 Evolution of Apparent Normal Stress Level Within Bone Tissue. Apparent stress level evolution during adaptation process.
Figure 8 Fracture Probability. Bone facture probability during adaptation process.
Discussion
A volume-based non-continuum formulation has been developed that describes the adaptation of bone to various mechanical loading situations. In the finite element approach to bone adaptation simulation, the integration of the entries in the coefficient matrix can be a heavy computing task [22,23,26,27]. In the model proposed here, this is alleviated through the introduction of the simpler connectivity matrix and Jacobian matrix [38]. In daily physiological activities, bone deformations are small and the Jacobian matrix can be linearized.
As mentioned in the introduction, one common problem is the inconsistency of material densities or strain energy densities on element boundaries [30]. Since all the integration points come from the boundaries, the resulting errors will essentially affect the computation. In the improved node-based implementation of the finite element method, the stress and strain quantities are still conflicting at the nodes. In the volume-based non-continuum formulation proposed here, this conflict is eliminated. The material density and strain energy density are all consistent in each individual strut, and the computing becomes less demanding.
As described above, previous work [31-33] addresses some of the weaknesses of the common FEM model. Makiyama [31] employed the "Voronoi structure" to study the effects of crack growth on trabecular bone. The method for generating a Voronoi structure could be quite useful when it calibrates the artificially constructed structure against the physical trabecular structure scanned from a patient. This might then serve as the starting state of bone configuration before adaptation begins. Moore [39] proposed a model to replace the partially damaged trabecula with another trabecula reduced in thickness. If this concept is combined with that of the strut structure, one may also derive the model proposed here, that is, a strut model with either varied modulus due to bone mineralization or adaptive cross-section/volume or even tapered struts as proposed by Kim [32].
Hip fracture is one typical manifestation of osteoporosis, and the results obtained by the simulation indicate that considerable changes of bone structure take place in the regions of femoral head and neck, where the stress level is normally higher than that of distal regions. The variations in stress level as shown in Fig. 7 reflect the adaptive process of the bone internal structure and different structural configurations will yield different stress levels in spite of little change in bone volume / tissue volume ratio.
In the current literature, the time scale for adaptive processes is not very well defined. This general lack of knowledge poses a problem for any experimental proof of concept – while the numbers of strain repetition can be predefined, they must be done within biologically suitable time frames. If a given strain comes too sudden, the bone may break instead of remodel; if the strain is applied over too long a period, it may not be a sufficient stimulus to activate adaptive processes. The lack of well-defined temporal constraints, however, is common. Kim's approach [32] is very interesting in as much as it may allow to integrate the effect of time in the model proposed here. At present, however, there is not sufficient data available to allow to integrate time effects of the clinically interesting mid-range scale, i.e. weeks to months. Kim's model looks at the process of ageing of 35 years and more.
The simulation model presented here may, beyond theoretical calculations, be applied to look at two clinical questions. Firstly, the simulation can be adjusted so that a realistic density distribution is the starting point, and outcomes following certain loading conditions, such as a predefined number of load cycles can then be predicted. Secondly, the program can integrate the measured bone density of a given patient to estimate the fracture risk based on stress level calculations.
Conclusion
By eliminating the common inconsistencies at each node, the formulation presented here shows good numerical performance and successfully predicts reasonable bone structure changes under different loading conditions. It is viable to serve as an alternative method apart from the traditional finite element based approached to study bone adaptations. In conclusion, the volume based non-continuum formulation is a new approach to bone adaptation study and has its own advantages.
Methods
Volume-based representation of the trabecular bone structure
In the volume-based non-continuum formulation used here, the trabecular structure is represented by a connected strut system and each strut can take different sizes according to the mechanical loading requirements, that is, strain energy density. The strut representation is shown in Fig. 9, which resembles a small volume of the trabecular structure. In this setting, the BV/TV ratio can be directly obtained from the ratio of the strut volumes over the unit volume, and material orientation can be obtained though the resultant of the vectorial material components of the struts, as described by equation (1) and (2).
Figure 9 Bone Structure Decomposition. Representation of bone material by struts. Each strut can assume different geometric dimensions and material properties. Apparent mechanical property of bone material is based on the strut configuration.
where vi is the volume of the i-th strut in the j-th basic unit, Vj is the volume of the j-th basic unit, Rj is the material orientation of the j-th basic unit, is the orientation of the i-th strut in the j-th basic unit and N is the number of struts in the j-th basic unit.
For the formulation proposed here, the bone structure is decomposed into and represented by a connected network of struts. These struts are a mathematical abstraction of the physical bone structure, from which the BV/TV ratio can be derived. The thickness of a strut is adapted during the bone adaptation process in mimicry of the physiological processes.
Volume adaptation under mechanical loading
The bone mass will vary under mechanical loading. In engineering, the general relationship between varying mass, density and volume is described as:
Since the density here is taken as constant regarding time, the second term on the right hand side, , simply vanishes and the mass variation is realized through volume variation under mechanical loading. Based on the density-based adaptation proposed by Zhu X. et al (39), which is stated as:
the volume-based adaptation can thus be stated as:
where βi = Ui / ρik, which is a comparative coefficient describing the comparison of a given mechanical stimulus in each sensor cell with the reference value k, and Ui represents the strain energy density for the I-th sensor unit; N is the number of sensor cells and fi(x) is the spatial influence function; B(t) is a remodeling coefficient; α indicates the remodeling power of strain energy density [34].
Non-Continuum formulation
With the whole bone represented by a volume-based strut system, the non-continuum formulation can be noted as follows:
where A is a connectivity matrix describing the connecting relationship between the struts, α is the linearized Jacobian matrix, is the nodal displacement vector to be solved for and is the loading vector derived from the external mechanical load. The generalized conjugate residue method is used to solve this formulation [38,40].
Connectivity matrix A is the matrix to show the relationship between connected struts with the entries of 1, -1 or 0. A strut starts from the node with the index corresponding to the entry 1 and ends at the node with the index corresponding to -1. It is further illustrated in Fig. 10 and equation (7).
Figure 10 Connectivity of Struts. Physical connectivity relationship between struts is indicated by the connectivity matrix.
Finally, the different loading conditions to be applied are shown in Fig. 11.
Figure 11 Loading cases. Quantitative information of different loading cases. Four loading cases are considered: one-legged stance, abduction, adduction and the combined loading case (weighted based on their respective daily occurrence cycles).
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
Zhengyuan Wang developed the formulation and partly prepared the manuscript, Adrian Mondry participated in the adaptation controls and partly prepared the manuscript.
Acknowledgements
This project is supported by the BioMedical Research Council of Agency for Science, Technology and Research, Singapore. Thanks also go to SMA5211 lecturers from Singapore-MIT Alliance, for helpful advice on nodal formulation.
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| 15733328 | PMC553991 | CC BY | 2021-01-04 16:39:26 | no | Theor Biol Med Model. 2005 Feb 28; 2:6 | utf-8 | Theor Biol Med Model | 2,005 | 10.1186/1742-4682-2-6 | oa_comm |
==== Front
J NeuroinflammationJournal of Neuroinflammation1742-2094BioMed Central London 1742-2094-2-81573332110.1186/1742-2094-2-8ResearchAstrocyte production of the chemokine macrophage inflammatory protein-2 is inhibited by the spice principle curcumin at the level of gene transcription Tomita Michiyo [email protected] Brita J [email protected] Christopher P [email protected] Thomas J [email protected] Department of Medicine, University of North Dakota School of Medicine & Health Sciences, 501 North Columbia Road, Grand Forks, ND 58201, USA2 Boston University, 140 Commonwealth Avenue, Chestnut Hill, MA 02467, USA3 Loyola University – Chicago, 6525 North Sheridan Road, Chicago, IL 60626, USA4 Research Service, Fargo VA Medical Center, 2101 Elm Street, Fargo, ND 58102, USA2005 25 2 2005 2 8 8 21 1 2005 25 2 2005 Copyright © 2005 Tomita et al; licensee BioMed Central Ltd.2005Tomita et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
In neuropathological processes associated with neutrophilic infiltrates, such as experimental allergic encephalitis and traumatic injury of the brain, the CXC chemokine, macrophage inflammatory protein-2 (MIP-2) is thought to play a pivotal role in the induction and perpetuation of inflammation in the central nervous system (CNS). The origin of MIP-2 in inflammatory disorders of the brain has not been fully defined but astrocytes appear to be a dominant source of this chemokine.
Curcumin is a spice principle in, and constitutes approximately 4 percent of, turmeric. Curcumin's immunomodulating and antioxidant activities suggest that it might be a useful adjunct in the treatment of neurodegenerative illnesses characterized by inflammation. Relatively unexplored, but relevant to its potential therapeutic efficacy in neuroinflammatory syndromes is the effect of curcumin on chemokine production. To examine the possibility that curcumin may influence CNS inflammation by mechanisms distinct from its known anti-oxidant activities, we studied the effect of this spice principle on the synthesis of MIP-2 by astrocytes.
Methods
Primary astrocytes were prepared from neonatal brains of CBA/CaJ mice. The cells were stimulated with lipopolysaccharide in the presence or absence of various amount of curcumin or epigallocatechin gallate. MIP-2 mRNA was analyzed using semi-quantitative PCR and MIP-2 protein production in the culture supernatants was quantified by ELISA. Astrocytes were transfected with a MIP-2 promoter construct, pGL3-MIP-2, and stimulated with lipopolysaccharide in the presence or absence of curcumin.
Results
The induction of MIP-2 gene expression and the production of MIP-2 protein were inhibited by curcumin. Curcumin also inhibited lipopolysaccharide-induced transcription of the MIP-2 promoter reporter gene construct in primary astrocytes. However MIP-2 gene induction by lipopolysaccharide was not inhibited by another anti-oxidant, epigallocatechin gallate.
Conclusion
Our results indicate that curcumin potently inhibits MIP-2 production at the level of gene transcription and offer further support for its potential use in the treatment of inflammatory conditions of the CNS.
MIP-2astrocytescurcumingene transcriptionchemokinesinflammation
==== Body
Background
Curcumin (1,7-Bis 94-hydroxy-3-methoxyphenyl)-1,6 heptadiene-3, 5-di-one) is a spice principle in and constitutes approximately 4% of turmeric and is responsible for curry's characteristic yellow color. As is true of other naturally occurring polyphenolic compounds, such as caffeic acid phenyl ester, rosmaric acid and resveratrol, curcumin possesses antioxidant properties which may reduce the production of free radicals and improve cell viability under conditions of enhanced oxidative stress[1,2]. Curcumin also has anti-inflammatory properties which include the capacity to inhibit 5- and 8-lipoxygenases and cyclooxygenases[3,4], is chemopreventive as evidenced by its capacity to abrogate 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced DNA synthesis and tumor promotion in mouse skin[5], antiproliferative as shown by its suppressive effect on the growth of C6 glioma cells[6], and anti-metastatic as suggested by its ability to inhibit angiogenesis in vivo[7].
Curcumin's immunomodulating and antioxidant activities suggest that it might be a useful adjunct in the treatment of neurodegenerative illnesses characterized by inflammation such as Alzheimer's disease[8]. Relatively unexplored, but relevant to its potential therapeutic efficacy in neuroinflammatory syndromes is the effect of curcumin on chemokine production. An active role for chemokines has been demonstrated in the pathogenesis of a variety of central nervous system (CNS) disorders accompanied by inflammation. In neuropathological processes associated with neutrophilic infiltrates, such as experimental allergic encephalitis (EAE) and traumatic injury of the brain, the CXC chemokine, macrophage inflammatory protein-2 (MIP-2) appears to play a pivotal role in the induction and perpetuation of inflammation in the brain[9,10]. In EAE, for example, elevated levels of MIP-2 mRNA and protein preceded infiltration of the CNS by polymorphonuclear leukocytes (PMNs). Similarly, in traumatic brain injury, the kinetics of MIP-2 expression paralleled the recruitment of neutrophils to the inflammatory site[10] and, in experimental bacterial meningitis, neutralization of MIP-2 with a monoclonal antibody attenuated infiltration of the CNS with PMNs[11]. The origin of MIP-2 in inflammatory CNS disorders has not been fully defined, but in EAE astrocytes, appear to be the dominant source of this chemokine[9] and are likely to contribute significantly to MIP-2 production in other neuropathological states as well.
To explore the possibility that curcumin may influence CNS inflammation by mechanisms distinct from its antioxidant and known anti-inflammatory activities, we examined the effect of this spice principle on the synthesis of MIP-2 by astrocytes. Our results indicate that curcumin potently inhibits MIP-2 production at the level of gene transcription and offer further support for its potential use in the treatment of inflammatory conditions of the CNS.
Methods
Mice
Six to eight-week-old CBA/CaJ mice were purchased from Jackson Laboratories (Bar Harbor, ME) and bred in our animal facility.
Materials
Curcumin, epigallocatechin (EGCG) and E. coli lipopolysaccharide (LPS; O55B1) were purchased from Sigma, (St Louis, MO). Rabbit anti-cow glial fibrillary acidic protein polyclonal antibody was obtained from Dako Corp. (Carpinteria, CA).
Preparation and culture of astrocytes: Astrocytes were prepared from the brains of neonatal (3 to 7-day-old) CBA/CaJ mice by a modification of the method of Pousset et al[12]. Briefly, four brains were combined, homogenized in 0.25% trypsin through a sterile screen (pore size; 100 μM), incubated for 5 min at 37°C and centrifuged at 400 × g. The pellet was suspended in Hank's Buffered Salt Solution (HBSS) and debris was removed by gravity sedimentation on ice for 3 min. The supernatant was collected, centrifuged and the pellet was washed twice with culture medium consisting of DMEM containing 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 1 mM L-glutamate and penicillin/streptomycin (Gibco BRL, Grand Island, NY). The cells were plated on 35 mm dishes and cultured at 37°C in a humidified atmosphere contain 5% C02. After 16 hours, plates were washed to remove non-adherent cells and debris. For experiments in which mRNA or MIP-2 protein were quantified, adherent cells were cultured until they reached confluence. For transfection experiments, adherent cells were cultured until they were nearly confluent. Medium was refreshed in all astrocyte cultures every 2–3 days. The preparations were >98% glial fibrillary acidic protein positive, as measured by flow cytometric analyses using a EPICS XL flow cytometer[13].
Cell viability determination: The effect of curcumin on the viability of astrocytes was assessed by measuring cytosolic lactate dehydrogenase (LDH) leakage into the media as detailed earlier[14]. Briefly, astrocytes were incubated with curcumin (10-4 M to 10-6 M) for up to 48 hours, the supernatants were then harvested and LDH was measured by colorimetric assay using a kit from Sigma diagnostics.
mRNA and protein analyses: Confluent cultures of astrocytes were incubated with LPS (10 ηg to 5 μg/ml) for varying periods of time in the presence or absence of curcumin (10-4 M to10-7 M). After 4 hours of culture, cells were harvested and mRNA was isolated as previously reported[14]. MIP-2 mRNA levels were determined using semi-quantitative polymerase chain amplification (PCR) as described earlier[14] using the primers: 5'-TGCCGGCTCCTCAGTGCT-3' (forward) and 5'-GCCTTGCCTTTGTTCAGTATCTTTTG-3' (backward). In other experiments, the effect of EGCG on induced MIP-2 mRNA production was determined by culturing astrocytes with LPS in the presence or absence of varying doses of the catechin (10-3M to 10-4M). To assess the effect of curcumin on MIP-2 protein production, astrocytes were cultured with LPS in the presence or absence of curcumin (10-5M) for 16 hours. Supernatants were then harvested and MIP-2 levels were determined by enzyme linked immunosorbant assay (ELISA; R&D systems, Minneapolis, MN).
Preparation of the reporter gene, pGL3-MIP-2: A 537 base pair MIP-2 fragment was prepared by amplifying rat genomic (kidney) DNA using the primers: 5'GCCCACCGAGTCTCTGTTTC3' (forward) and 5'GTTGGTGGCCAGCAGGAGGA3' (backward), then digesting with Rsa I/Nco I. The fragment, which corresponded to base pairs -539 to -2, relative to adenine (assigned +1) in the translation initiation codon of the MIP-2 gene (accession number AJ49888), was ligated to a Sma I/Nco I digested, promoterless luciferase reporter vector, pGL3-Basic (Promega, Madison, WI). The direction of the insert was confirmed by restriction endonuclease digestion and its fidelity determined by sequence analyses as previously described[15]. The MIP-2 promoter-reporter gene construct, pGL3-MIP-2 is shown in Figure 1.
Figure 1 PGL3-MIP-2. A 537 bp fragment, which corresponds to base-pairs -539 to -2 (relative to adenine [assigned +1] in the translation initiation codon of the MIP-2 gene) was generated and inserted into a promotorless luciferase reporter vector, pGL3-Basic.
Transfection experiments: Astrocytes were transfected cells using a modification of the method of Franzoso et al[16]. Briefly, 1.5 μg of DNA containing either pGL3-MIP2 or pGL3-basic were incubated in HBS solution (137 mM NaCl, 5 mM KCl, 0.88 mM Na2HPO4, 20 mM Hepes) containing 250 mM CaCl2 for 10 minutes at room temperature. The mixtures were added in 2 mL of media to astrocytes that were nearly confluent. After a 16-hour incubation in a humidified atmosphere at 37 C° containing 5% CO2, cells were washed to remove debris and cultured for an additional 24 hours. LPS plus or minus curcumin (10-4M to 10-7M) was then added and transfected cells were further cultured for 24 hours. At the conclusion of culture, cells were harvested, cell lysates were prepared, and lysates were analyzed using a luciferase assay system (Promega, Madison, WI) in accordance with the manufacturer's instructions.
Results and discussion
To determine whether mechanisms apart from its well-documented anti-oxidant activity might provide possible neuroprotection against inflammation-mediated injury, we investigated the effect of curcumin on astrocyte production of the chemokine MIP-2 in response to LPS. In initial experiments, we found that optimal MIP-2 production occurred when confluent astrocyte cultures were stimulated with 5 μg/ml of LPS during a 16-hour culture (data not shown). Culturing such astrocytes with a dose of curcumin (10-5M) that had no effect on viability as measured by LDH release (data not shown), abrogated LPS-stimulated MIP-2 production (Figure 2).
Figure 2 LPS-induced MIP-2 production is inhibited by curcumin. Confluent astrocytes were cultured in medium alone or were stimulated with LPS (5 μg/ml) in the presence (LPS + Curcumin) or absence (LPS) of curcumin (10-5 M). The supernatants were collected after 16 hours and MIP-2 protein (in picograms/ml) was measured by ELISA. Data are the mean ± standard deviation of 4 experiments. Mean MIP-2 production in the medium and LPS + curcumin groups differ significantly from that in the LPS group (p < 0.001 by Student's t test). Mean MIP-2 production does not differ significantly between the medium and LPS + curcumin groups (p > 0.2 by Student's t test).
The effect of curcumin on LPS-induced production of MIP-2 mRNA was examined next. Preliminary experiments showed that optimal message for MIP-2 in response to LPS occurred after 4 hours of culture in astrocytes (data not shown). As was true for MIP-2 protein, culture of astrocytes with curcumin (10-5M) markedly inhibited chemokine gene expression in response to LPS (Figure 3).
Figure 3 LPS-induced MIP-2 mRNA expression is inhibited by curcumin. Confluent astrocyte cultures were stimulated with LPS (5 μg/ml) in the presence or absence of varying doses of curcumin (10-5-10-7M) or vehicle (0.05% ethanol), mRNA was then extracted, reverse transcribed and amplified using a mouse MIP-2 primer.
To determine whether curcumin inhibits MIP-2 gene transcription, a construct was created in which 537 base pairs of the MIP-2 promoter, spanning nucleotides -539 to -2 of the MIP-2 gene (see Methods), were fused to a promoter-less luciferase reporter gene (pGL3-MIP-2, Figure 1). As shown in the representative experiment in Figure 4, curcumin abrogated LPS-stimulated MIP-2 gene expression in transiently transfected astrocytes. In three separate experiments, essentially complete inhibition of LPS-induced MIP-2 gene expression (100%, 92%, 94%) was observed with curcumin in doses of 2 μM.
Figure 4 Curcumin inhibits the activity of MIP-2 at the level of gene transcription. Confluent astrocyte cultures were transfected with 1.5 μg of pGL3-MIP-2 or pGL3-Basic and stimulated with LPS (5 μg/ml) in the presence or absence of varying amounts of curcumin (10-4-10-6M). Transfected cells were harvested and luciferase activity in the cell lysates was quantified. The dose of curcumin is shown in log scale. Results are representative of three experiments.
As a specificity control, the effect of EGCG, a catechin present in green tea with potent anti-oxidant activity, was examined on MIP-2 gene expression in astrocytes. In contrast to curcumin, EGCG in doses as high as 10-3 M had no effect on LPS-stimulated MIP-2 mRNA expression (Figure 5). The results suggest that the inhibitory effect of curcumin on MIP-2 production may not be due to its anti-oxidant properties.
Figure 5 Curcumin but not EGCG inhibits MIP-2 mRNA expression. Confluent cultures of astrocyte were stimulated with LPS (5 μg/ml) in the presence or absence of varying doses of curcumin (10-5-10-7M), EGCG (10–3), or appropriate vehicle (0.05% ethanol for curcumin, DMSO for EGCG). mRNA was then extracted, reverse transcribed and amplified using mouse MIP-2 primers.
The study presented herein shows for the first time that curcumin is a potent inhibitor of inducible MIP-2 production by astrocytes, which are a major source of this chemokine in the brain[9]. In transient transfection experiments of astrocytes, virtually complete inhibition of MIP-2 inducible gene expression was observed with 2 μM curcumin. Since blood levels of curcumin approximating 2 μM were shown by Yang, et al[17] to block amyloid aggregation in a transgenic model of Alzheimer's disease, we believe that our data may have in vivo relevance.
Transfection experiments in macrophages using a promoter, reporter-gene construct that contains canonical NFκB and NF-IL-6 cis-acting elements demonstrate that inhibition of MIP-2 by curcumin occurs at the level of gene transcription. The importance of either of these elements in the regulation of inducible MIP-2 gene expression in astrocytes remains to be determined. In some systems, inhibition of NFκB per se by curcumin is sufficient to abrogate gene expression. Thus, curcumin and its hydrogenated metabolites were shown to completely suppress transcription of nitric oxide synthase through down regulation of IκBkinase and NFκB activation in macrophages[18]. However, considering the fact that NFκB activation is linked to multiple upstream signaling pathways[19] and that curcumin has been shown to suppress a number of inflammatory signaling cascades[20], inhibition mediated by this spice principle may be quite complex and highly variable, depending on the cell type and the activating stimulus.
Inhibition of chemokine production represents a novel, potential mechanism by which curcumin may confer neuroprotection in CNS disorders characterized or accompanied by leukocytic infiltration. As stated above, MIP-2 is a dominant, driving force in the pathogenesis of many CNS disorders that are associated with infiltration of neutrophils in the brain[9,10]. Experimentally, recruitment of neutrophils to the CNS is followed by a breeching of the blood-brain barrier that is especially severe after administration of MIP-2[21] and may further contribute to inflammation by causing indiscriminate entry of leukocytes into the brain. The possible contribution of inflammatory infiltrates to neuronal injury is best illustrated by experimental studies in which MIP-2 activity was neutralized. For example, administration of anti-MIP-2 antibody to rats infected with Hemophilus influenza type b abrogated the influx of neutrophils to the meninges, ventricular system, and the periventricular areas of the brain and substantially decreased neuronal damage[11].
In addition to astrocytes, microglial cells and endothelial cells may be potential sources of MIP-2 production in pathological states of the brain. Stimulation of brain microvascular endothelial cells with tumor necrosis factor alpha (TNFα), induces the release of MIP-2 within 4 to 8 hours of in vitro culture[22]. Since TNFα levels in the brain are significantly elevated in traumatic brain injury (TBI), it remains possible that cytokine-mediated release of MIP-2 by endothelial cells, particularly those which comprise the blood brain barrier, may predispose to intracerebral neutrophil accumulation and neuronal injury in TBI. Similarly, in a model of hypoxia/reoxygenation, large increases in MIP-2 mRNA and protein were demonstrated in microglial cells suggesting a possible mechanism to account for PMN accumulation and inflammation in cerebral ischemia.
Apart from its ability to inhibit MIP-2 production, curcumin's pleotropic antiinflammatory and anti-oxidative properties suggest its possible use in diseases of the brain accompanied by inflammation. Thus, LPS stimulation transcriptionally upregulates inducible nitric oxide synthase and cyclooxygenase-2 genes in microglia. This leads to the synthesis of nitric oxide (NO) and prostaglandins (PGs), respectively, and the possible formation of neuron-damaging free radicals, such as peroxynitrite. Curcumin abrogates the production of both NO and PGs in LPS activated microglial cells[20]. In a recently completed Phase I clinical trial, oral curcumin at a daily dose of 3.6 grams was, in general, well-tolerated and decreased inducible PGE2 production in blood samples taken 1 hour after dose on days 1 and 29 of treatment by approximately 60%[23]. Consistent with its possible use in neurodegenerative diseases associated with oxidative stress injury, curcumin has been reported to decrease oxidative damage and amyloid deposition in a transgenic mouse model of Alzheimer's disease[24], and to reverse Aβ-induced cognitive deficits and neuropathology in rats[25].
In summary, the capacity of curcumin to inhibit astrocyte production of MIP-2, together with its broad immunosuppressive activities, strongly support the potential use of this spice principle in the treatment of inflammatory diseases of the CNS.
List of abbreviations
EAE, experimental allergic encephalitis; EGCG, epigallocatechin gallate; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; MIP-2, macrophage inflammatory protein-2; NFκB, nuclear factor kappa B; NO, nitric oxide; PG, prostaglandin;
pGL3-MIP-2, a reporter gene construct containing the MIP-2 promoter; PMN, polymorphonuclear leukocyte; TBI, traumatic brain injury; TNFα, tumor necrosis factor alpha.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
MT participated in experimental design, acquisition of data, supervised all experiments, and carried out isolation of astrocytes, ELISA and transfection assays.
BH isolated and amplified the MIP-2 gene promoter, and generated the MIP-2 promoter construct, pGL3-MIP-2.
CS participated in culture of astrocytes and PCR analysis of MIP-2 gene.
TS conceived of the study, participated in its design, and helped to draft the manuscript.
All authors read and approved the final manuscript.
Acknowledgements
This study was, in part, supported by the North Central Chapter of the Arthritis Foundation.
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| 15733321 | PMC553992 | CC BY | 2021-01-04 16:38:21 | no | J Neuroinflammation. 2005 Feb 25; 2:8 | utf-8 | J Neuroinflammation | 2,005 | 10.1186/1742-2094-2-8 | oa_comm |
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Respir ResRespiratory Research1465-99211465-993XBioMed Central London 1465-9921-6-151570520610.1186/1465-9921-6-15ResearchCorrelation of exhaled breath temperature with bronchial blood flow in asthma Paredi Paolo [email protected] Sergei A [email protected] Peter J [email protected] Department of Thoracic Medicine, National Heart and Lung Institute, Faculty of Medicine, Imperial College, London, UK2005 10 2 2005 6 1 15 15 4 11 2004 10 2 2005 Copyright © 2005 Paredi et al; licensee BioMed Central Ltd.2005Paredi et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
In asthma elevated rates of exhaled breath temperature changes (Δe°T) and bronchial blood flow (Qaw) may be due to increased vascularity of the airway mucosa as a result of inflammation.
We investigated the relationship of Δe°T with Qaw and airway inflammation as assessed by exhaled nitric oxide (NO). We also studied the anti-inflammatory and vasoactive effects of inhaled corticosteroid and β2-agonist.
Δe°T was confirmed to be elevated (7.27 ± 0.6 Δ°C/s) in 19 asthmatic subjects (mean age ± SEM, 40 ± 6 yr; 6 male, FEV1 74 ± 6 % predicted) compared to 16 normal volunteers (4.23 ± 0.41 Δ°C/s, p < 0.01) (30 ± 2 yr) and was significantly increased after salbutamol inhalation in normal subjects (7.8 ± 0.6 Δ°C/ s, p < 0.05) but not in asthmatic patients. Qaw, measured using an acetylene dilution method was also elevated in patients with asthma compared to normal subjects (49.47 ± 2.06 and 31.56 ± 1.6 μl/ml/min p < 0.01) and correlated with exhaled NO (r = 0.57, p < 0.05) and Δe°T (r = 0.525, p < 0.05). In asthma patients, Qaw was reduced 30 minutes after the inhalation of budesonide 400 μg (21.0 ± 2.3 μl/ml/min, p < 0.05) but was not affected by salbutamol.
Δe°T correlates with Qaw and exhaled NO in asthmatic patients and therefore may reflect airway inflammation, as confirmed by the rapid response to steroids.
asthmanitric oxidetemperaturebronchial blood flowinflammation.
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Asthma is an inflammatory disease of the airways. Vasodilatation is a critical feature of inflammation, and angiogenesis and vascular remodelling are features of chronic inflammatory diseases, such as asthma [1]. The increased vascularity of the airways in asthma [2] is partly due to the elevated number of vessels associated with angiogenesis and partly due to vasodilation caused by the release of vasodilator mediators, such as, histamine, bradykinin [3], and nitric oxide (NO) [4]. In a recent study we have found that patients with asthma have higher increases of exhaled breath temperature (Δe°T) compared with normal subjects and that this is correlated to the concentration of exhaled nitric oxide (NO) [5]. Therefore, we suggested that patients with asthma have high Δe°T. and that this may be due to airway inflammation and elevated levels of NO. In the present study we hypothesise that elevated Δe°T may result from increased heat exchange in the airways due to elevated bronchial blood flow (Qaw) caused by inflammation and airway remodelling. To test this, we investigate the relationship between airway inflammation as assessed by exhaled NO, with Qaw and Δe°T measured non-invasively. Qaw is an expression of bronchial blood flow, whereas Δe°T reflects the rate of temperature increase in the exhaled breath. We hypothesised that Qaw changes may contribute to the levels of Δe°T and we studied their relationship considering that, potentially, minor changes of bronchial blood flow may not affect exhaled breath temperature.
Elevated levels of exhaled NO in asthma [6,7] are likely to be due to the activation of the inducible form of NO synthase (iNOS) by inflammatory cytokines [8] and therefore, reflect airway inflammation. Because NO also regulates bronchial vascular tone [9] and may increase bronchial blood flow [10,11] we measured its concentration in the exhaled breath as a marker of inflammation and we analysed its relationship with Qaw and Δe°T.
The measurement of Qaw is non-invasive and was standardised and adapted from a previously validated technique [12,13]. Δe°T and bronchial blood flow (Qaw) were also measured non-invasively allowing us to make repeated measurements and to study the interactions of these two markers and exhaled NO.
The inhalation of corticosteroids has been shown to have an acute vasocostrictive effect on the bronchial circulation [14] and the measurement of Qaw has been advocated to assess airway steroid sensitivity. In order to evaluate further the correlation of Qaw and Δe°T with inflammation, we studied the acute effect of inhaled budesonide on these parameters and their reciprocal changes. Furthermore, to validate our methods, we also evaluated the vasodilating effect of the short-acting β2-agonist salbutamol.
Methods
Patients
Nineteen asthmatic patients were studied (7 male, age 40 ± 5 yr, FEV1 74 ± 6 %, 9 patients were on inhaled steroid treatment and 10 patients had mild persistent asthma and were on β2-agonist inhalers only). These two groups of patients were chosen because they are representative of the larger majority of asthmatic patients; in addition this allowed us to verify cross sectionally the effect of inhaled steroids on bronchial blood flow and exhaled NO.
We also examined 16 control subjects (age 30 ± 2 yr, 8 male) recruited from our outpatient clinic and from volunteers (Table 1). Most of these subjects had previously taken part in other published studies. The diagnosis of asthma was established in each patient according to American Thoracic Society criteria [15]. Patients with acute chest infection or disease exacerbation during the month before enrolment were excluded. Patients with history of diabetes, liver disease, heart failure, lung cancer, or alcohol/drug abuse were not eligible for the study. All subjects were life-long non-smokers. All asthmatic patients refrained from using β2 agonists and corticosteroids for at least 12 h prior to the study. The tests were carried out at ambient temperature between 23 and 25C° and humidity 60% and 65%.
Table 1 PATIENT CHARACTERISTICS
Asthma not steroid treated (n = 10) Steroid treated asthma (n = 9) Normals (n = 16)
Age (years) 37 ± 7 45 ± 7 30 ± 2
Sex (M/F) 4/6 3/6 8/8
FEV1 (% predicted) 93 ± 10 75 ± 11 97 ± 9
Smokers 0 0 0
Ex smokers 0 0 0
Therapy:
Inhaled β-drenergics 10 10 0
Theophylline 0 0 0
Inhaled steroids 0 10 0
Oral steroids 0 0 0
Definition of abbreviations:; FEV1 = forced expiratory volume in one second. Values are means ± SEM.
Study design
The study was approved by the Brompton and Harefield NHS trust Ethics Committee. After a clinical examination was carried out, Δe°T, Qaw and exhaled NO were measured at least after one hour of rest in the laboratory. This was followed by spirometry.
Eight asthmatic subjects and eight normal volunteers agreed to have Δe°T and Qaw measured after the inhalation of budesonide 400 μg and salbutamol 200 μg or placebo, which were administered in three different visits. The measurements were repeated every 15 minutes for 1 h after budesonide and placebo inhalation and at baseline and every 10 minutes for half an hour after the inhalation of salbutamol.
Exhaled breath temperature measurement
During a flow and pressure controlled single breath exhalation exhaled breath temperature gradients were measured as previously described [5]. As previously shown [5], during a flow and pressure controlled exhalation from total lung capacity through a 2.77 mm mouthpiece[16] exhaled breath temperature was measured by a fast response (1 ms) high accuracy (0.015 ± 0.027°C), thermometer (Picotech Ltd) interfaced with a computer by a single channel Picotech Oscilloscope (model ADC 42, resolution 12 bits) allowing online recording of exhaled breath temperature.
In a preliminary study exhaled breath temperature tracings were analyzed mathematically. The tracings proved to have an exponential rise and the point at 63% of the total temperature increase was chosen to study the slope of the curves because it represents two time constants of the maximal °T change and therefore allows a better mathematical characterization of the tracings before plateau.
The time constant of the thermometer response was found by measuring the time for the temperature to reach 63% of the final reading. This avoided large errors in estimating when the asymptotic final reading had been reached.
The exhaled breath temperature changes exponentially with time. The shape of the curve depends upon the time constant (T). In one time constant the response reaches 63% of its final change.
The response is of the form
Where V2 is the final value, V1 the initial value, and t is the time after exhalation was started. As t > infinity, the exponential term > 0. Hence the response rises asymptotically to its final value. When performing experiments to determine the time response of a system, it is difficult to tell at which time the final value is obtained, since there will be a small change in the response for a relatively long time as the asymptote is approached. Jitter and noise in the signal will also add to this problem. It is easier to estimate the asymptotic value and find the time at which a certain percentage of this is reached. Various percentages are possible, the most commonly used in physics and biology is the time constant, although in some applications (particularly electronic engineering) the rise time to 95% of the final value is used.
The 63% arises because when t = T, the expression above becomes
(V2-V1)(1-1/e) = (V2-V1)(1-0.37) = 0.63(V2-V1)
In n time constants the percentage reached is 100 × (1-1/en)
which, approaches 98% after about 5 time constants.
The rate of temperature increase (Δe°T) calculated between the beginning of exhalation and 63 % of the total temperature increase (a/b, where "a" is 63% of Δ°T and "b" the time to reach "a") proved to be the more reproducible parameter to characterize the curves.
We evaluated the effect of different exhalation flow rates, distance of the thermocouple from the edge of the mouthpiece and ambient temperature on Δe°T and end-expiratory plateau temperatures. We found that Δe°T but not plateau temperatures were elevated at low (2–3 L/min) compared to higher (5–6 L/min) exhalation flow rates (6.25 ± 0.4°C/s and 4.45 ± 0.8°C/s, p < 0.01) in 8 normal subjects. When the thermocouple was inserted close to the edge of the mouthpiece (1 cm) the Δe°T was significantly higher (7.15 ± 0.2°C/s) compared to when it was located farther (2 cm) (4.45 ± 0.8°C/s, p < 0.01). There was a tendency for faster Δe°T when the subjects were starting exhaling from higher baseline ambient temperatures but this was not significant for temperature changes within ± 3°C (5.05 ± 0.8°C/s at 28°C and 4.45 ± 0.8°C/s at 22°C p > 0.05). The volume ventilation did not influence the Δe°T value. The difference in exhaled breath Δe°T and plateau temperatures measured during two successive collections at five minutes intervals (single session variability) was 4.4%, while between sessions variability (n = 6, one day interval) was 6.8%. The reproducibility of the test was confirmed by the Bland and Altman test [17].
Exhaled NO measurements
Exhaled NO was measured using a modified chemiluminescence analyzer (model LR2000; Logan Research, Rochester, Kent) as previously described [18].
Bronchial blood flow
We modified a previously validated soluble inert gas uptake method to measure Qaw, using acetylene rather than the potentially explosive dimethylether [12,13]. The subjects were sitting in front of a valve system inhaling through a mouthpiece (with nose clips on) initially room air and then a gas mixture from a Teflon bag containing 35% O2, 0,3% acetylene, 5% sulphur hexafluoride, CO 3% and a balance of nitrogen. During the exhalation the concentration of acetylene was measured directly online by a mass spectrometer, and Qaw was calculated from the Fick principle (dilution of acetylene concentration) (Figure 1), the area under the curve (AUC) being inversely proportional to the bronchial blood flow. Qaw was expressed as μl/ml/min representing the volume of blood per volume of dead space per time.
Figure 1 Method for the measurement of bronchial blood flow (Qaw). Subjects inhale 60% of their vital capacity from a reservoir containing acetylene 0.3% and then exhale into a mass spectrometer (Panel A). Panel B shows a tracing of acetylene, the area under the curve, corresponding to the conducting airways, is proportional to airway blood flow.
Previous studies have used dimethyl ether instead of acetylene for the measurement of bronchial blood flow. In the current study we used acetylene because of the higher explosive potential of dimethylether when in contact with O2. This method was previously validated in the sheep where the invasive inoculation of radioactive micro spheres directly into the bronchial circulation provided similar results [19]. The use of micro spheres is considered the Gold Standard for the measurement of blood flow, however, this method is invasive and presents limitations such as the recirculation of the radioactive spheres.
Acetylene and diethyl ether present similar blood solubility and affinity for haemoglobin when measured at the same temperature [20]. Gas exchange efficiency is largely dependent on solubility. Because these two gases have similar physicochemical characteristics we assume that they can be used interchangeably. This is further confirmed by the finding of a similar range of bronchial blood flow and similar response to corticosteroids (vasoconstriction) and beta 2 agonists (vasodilatation) in this study compared to previously published studies which used the dimethyl ether method.
Statistics
Comparisons between groups were made by one-way analysis of variance (ANOVA).. Data were expressed as means ± standard error of mean. The relationship between the exhaled breath temperature, Qaw, NO and FEV1 were tested with the linear correlation coefficient.
Results
Bronchial blood flow (Qaw)
Qaw was elevated to a similar extent in patients with mild persistent and moderate asthma (on regular inhaled corticosteroids and β2-agonists) (46.0 ± 51 μl/ml/min) and patients with mild intermittent asthma (on β2-agonists as needed only) (52.64 ± 3.0 μl/ml/min, p > 0.05) compared to normal subjects (31.56 ± 1.6 μl/ml/min, p < 0.01, Figure 2, Panel A). Qaw was correlated with exhaled NO (r = 0.57, p < 0.05, Figure 3, Panel A) and Δe°T (r = 0.52, p < 0.05, Figure 3, Panel B).
Figure 2 Levels of bronchial blood flow (Qaw) (Panel A) and exhaled breath temperature gradients (Δe°T) (Panel B) in normal subjects (□) and patients with asthma (•).
Figure 3 Correlation of bronchial blood flow (Qaw) with exhaled nitric oxide (NO) (Panel A) and exhaled breath temperature gradients (Δe°T) (Panel B) in patients with asthma.
Exhaled air temperature
Δe°T was higher in asthmatic patients (7.27 ± 0.6 Δ°C/ sec) compared to normal subjects (4.23 ± 0.41 Δ°C/ sec, p < 0.01, Figure 2, Panel B) and was not statistically different in steroid treated (7.56 ± 0.99 Δ°C/ sec) compared to untreated patients (6.83 ± 0.78 Δ°C/ sec, p > 0.05).
Exhaled NO
NO levels were elevated in asthmatic subjects not on steroid treatment (15.6 ± 2.8 ppb) compared to steroid treated patients (7.5.6 ± 2.3 ppb, p < 0.05) and normal subjects (4.7 ± 0.3 ppb, p < 0.05).
Effect of budesonide and salbutamol inhalation
Bronchial blood flow
In asthmatic patients Qaw was significantly reduced 30 minutes after the inhalation of budesonide compared to baseline (53.0 ± 5.0 μl/ml/min and 21.3 ± 2.32 μl/ml/min respectively, p < 0.05 Figure 4 Panel A) and returned to baseline levels at 60 minutes (52.6 ± 4.0 μl/ml/min). In normal subjects there was a tendency for lower Qaw after the inhalation of budesonide but such changes were not significant (30.3 ± 5.0 μl/ml/min and 26.3 ± 3.0 μl/ml/min at baseline and at 30 minutes respectively, p > 0.05, n = 5, Figure 4 Panel A).
Figure 4 Acute effect of budesonide inhalation (400 μg) (Panel A) and salbutamol (200 μg) (Panel B) on bronchial blood flow (Qaw).
In 8 normal volunteers Qaw was increased after salbutamol inhalation (32.3 ± 8.1 μl/ml/min and 55.0 ± 3.0 μl/ml/min at baseline and at 10 minutes respectively, p < 0.05), while this effect was not present in asthmatic patients (54.0 ± 7.0 μl/ml/min and 52.0 ± 6.0 μl/ml/min, p > 0.05, n = 5, Figure 4 Panel B).
Placebo had no effect on Qaw in subjects with asthma (54.0 ± 3.0, 56.1 ± 4.5 and 53.1 ± 8.1 μl/min/ml/ at baseline, 30 and 60 minutes respectively, p > 0.05) nor in normal subjects (34.0 ± 3.0,38.1 ± 4.5 and 35.1 ± 8.1 μl/min/ ml/ p > 0.05)
Exhaled breath temperature
The inhalation of budesonide was associated with a tendency for a decrease of Δe°T in asthmatic patients (from 10.17 ± 2 Δ°C/ sec at baseline to 8.6 ± 3 Δ°C/sec 30 minutes after inhalation Figure 5 Panel A), even though this decrease was not significant; the changes in Qaw and Δe°T were correlated in asthmatic patients (r = 0.78, p < 0.05). In normal subjects, no significant changes of Δe°T were found at any of the time points after budesonide inhalation (3.5 ± 2 Δ°C/sc at baseline, 3.5 ± 1 Δ°C/sc at 30 minutes, 3.53 ± 2 Δ°C/sc at 60 minutes, p > 0.05).
Figure 5 Acute effect of budesonide inhalation (400 μg) (Panel A) and salbutamol (200 μg) (Panel B) on exhaled breath temperature gradients (Δe°T).
In 5 normal volunteers Δe°T was increased after the inhalation of 200 μg of salbutamol (3.50 ± 0.29 Δ°C/ sec and 7.8 ± 0.6 Δ°C/ sec, p < 0.01), while this effect was not present in asthmatic patients (10.1 ± 0.44 Δ°C/ sec and 9.67 ± 0.51 Δ°C/ sec, p > 0.05, n = 5, Figure 5 Panel B)
Δe°T was unchanged in subjects with asthma (9.17 ± 2 and 10.23 ± 3.5 Δ°C/ sec at baseline and 10 minutes respectively, p > 0.05) and in normal subjects (3.50 ± 0.29 Δ°C/ sec and 4.20 ± 0.32 Δ°C/ sec p > 0.05) after the inhalation of placebo.
Discussion
This is the first study to show that elevated levels of Qaw and Δe°T are correlated with one another and with airway inflammation as assessed by exhaled NO. We propose that high levels of NO generated in asthmatic patients as a result of airway inflammation may cause vasodilatation of the bronchial circulation contributing to increased heat exchange. This is supported by the demonstration that lower Δe°T levels, after the inhalation of corticosteroids, are correlated with reduced levels of bronchial blood flow. We propose that these non-invasive measurements may be useful to evaluate airway inflammation and may provide a tool to assess steroid sensitivity.
Angiogenesis and microvascular remodelling are features of chronic inflammatory diseases, such as asthma [21]. As inflammatory diseases evolve, the microvasculature undergoes progressive changes in structure and function. Blood vessels enlarge and proliferate supplying inflammatory cells in chronically inflamed tissues. Because of these changes, asthmatic patients have increased vascularity of the airway mucosa which is related to the severity of the disease [2]. Airway vascular remodelling and inflammation maybe responsible for increased bronchial blood flow [22] and exhaled breath temperature gradients in asthmatic patients [5].
In a previous study [23] we have proved that patients with asthma have elevated Δe°T compared to normal subjects and because we found a significant correlation with exhaled NO we suggested that this was due to airway inflammation. In the present study we hypothesised that Δe°T is elevated in asthma as a result of increased bronchial blood flow and we studied the relationship between Qaw and Δe°T and airway inflammation as assessed by exhaled NO. Even though the patients enrolled in this study were significantly older than the control group, our previous studies [5,24] indicate that that age does not affect Δe°T. We studied airway inflammation measuring exhaled NO and we investigates its relationship with Qaw which has also been suggested as a marker of inflammation. We also studied the interaction of these two parameters after inhaled corticosteroids β2 agonists.
We confirmed previous data showing elevated levels of Qaw in asthmatic subjects compared to normal volunteers, using a modified method developed by Onorato et al [25]. In the current study we preferred the use of acetylene over dimethyl ether because the latter is highly explosive when in contact with oxygen. Gas exchange efficiency is largely dependent on solubility, because these two gases have similar physicochemical characteristics we assume that they can be used interchangeably. Furthermore, the measurement of Qaw in this study presented the same response pattern and timing to the inahaltion of steroids [12] and beta agonists [26] as previously showed using the dimethyl ether method confirming that the method presented in our study is an acceptable measurement of Qaw.
The method used in this study for the measurement of Qaw produces results which include the contribution of the dead space and trachea blood flow to the total bronchial blood flow. We acknowledge that the trachea may not be the main site of inflammation in asthma, but inflammation in asthma extends from the larynx to the terminal bronchioles and the tracheal mucosa certainly appears inflamed in many asthmatic patients. When tracheal inflammation occurs there will be a good separation between normal subjects and patients with asthma because the measurements of bronchial blood flow and exhaled breath temperature will particularly reflect the contribution of this part of the respiratory tract
For the first time we have shown, using non-invasive methods, that changes in bronchial blood flow can alter exhaled breath temperature indicating that the bronchial circulation may control airstream temperature. Furthermore, in this study not only we have shown a correlation between Qaw and Δe°T but we have also shown that these measurements respond similarly to steroids and beta 2 agonists. Therefore, patients with asthma have a significantly faster rise of breath temperature and Qaw and these are correlated. We presume that this is due to the increased vascularity of the bronchial vessels [27] and elevated blood supply and therefore heat transfer across the bronchial wall. Hyperaemia and hyperperfusion are consistent features of tissue inflammation, therefore, the finding of increased exhaled air temperature and bronchial blood flow in asthmatic patients may be due to the elevated levels of exhaled NO which is a marker of inflammation and a potent bronchial vasodilator. Even though the correlation between Qaw and Δe°T may appear to be weak, it is noteworthy that the acute changes of these variable was significantly correlated, reinforcing the hypothesis that elevated breath temperature gradients in asthmatic patients may reflect increased bronchial blood flows.
We were able to show a positive correlation between exhaled NO and Δe°T. NO is a gas produced by several types of pulmonary cells, including inflammatory, endothelial and airway epithelial cells. Elevated levels of exhaled NO in asthma [6], and interstitial lung disease [28] are likely to be due to the activation of the inducible form of NO synthase (iNOS) and therefore may reflect airway inflammation, alternatively NO maybe produced by the bronchial epithelium. In addition the activity of iNOS, the inducible enzyme responsible for the synthesis of NO, is temperature-dependent [29], therefore elevated airway temperatures in patients with asthma [5] may induce further synthesis of NO. NO is a potent vasodilator and may play a role in the regulation of bronchial vasomotor tone [10], so that elevated levels of NO may lead to vasodilatation and increased bronchial blood flow as shown by the correlation between exhaled NO and Δe°T. Unfortunately, in the current study, we were unable to establish the contribution of NO produced by the bronchial vasculature compared to the pulmonary circulation.
In this cross-sectional study we could not show any differences of Δe°T or Qaw in corticosteroid-treated compared to untreated asthmatic patients, despite the efficacy of steroids in reducing bronchial blood flow [12]. This is consistent with previous studies published by our group and others showing that steroid-treated asthmatic patients have similar Δe°T [5] and Qaw [22] compared to untreated patients. One hypothesis is that the vasoconstrictive action of inhaled corticosteroids may have been balanced by β2 induced vasodilatation resulting in minimal changes in bronchial artery diameter and blood flow and therefore no changes of Qaw and Δe°T. However, these results must be confirmed by placebo controlled studies.
In contrast to the effect of chronic treatment with corticosteroids, the acute inhalation of budesonide caused a significant temporary reduction of Qaw which returned to baseline one hour after inhalation. This is also consistent with a previous publication [22], notably, in the current study Qaw and Δe°T and their interaction were studied simultaneously in the same group of patients for the first time. Corticosteroids may cause vasoconstriction by numerous mechanisms. They can potentiate the vasoconstrictor actions of noradrenalin and angiotensin II by upregulating their vascular receptors [30]. Furthermore, corticosteroids may inhibit the synthesis of NO [31], thus causing vasoconstriction. In addition to these mechanisms of action, corticosteroids may also have a very rapid action (less than 5 minutes) by inhibiting noradrenalin uptake in bronchial blood vessels [32]. The glucocorticoid-induced vasoconstriction in asthmatics seems to be accompanied by a greater α1-adrenergic vasoconstrictor response [26]. This adds further support to a α1-adrenergic steroid interplay in the regulation of vascular tone.
Further studies are required, investigating the dose response relationship for inhaled steroids would provide valuable information.
The cardinal signs of inflammation are rubor (redness), calor (heat), tumor (swelling), dolor (pain), and impaired function (functio laesa). Exhaled breath temperature and bronchial blood flow may reflect rubor and calor in the airways and therefore may be markers of tissue inflammation and remodelling as confirmed by the positive correlation between Δe°T, Qaw and exhaled NO which was shown for the first time in this study. Measurement of exhaled breath temperature and bronchial blood flow may provide means of detecting airway inflammation and vascular remodelling in a non-invasive way.
Acknowledgments
This study was supported by the National Heart and Lung Institute, London UK No Part of the research presented was founded by tobacco industry sources
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| 15705206 | PMC553993 | CC BY | 2021-01-04 16:36:26 | no | Respir Res. 2005 Feb 10; 6(1):15 | utf-8 | Respir Res | 2,005 | 10.1186/1465-9921-6-15 | oa_comm |
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Virol JVirology Journal1743-422XBioMed Central London 1743-422X-2-131572768510.1186/1743-422X-2-13ResearchMutational study of sapovirus expression in insect cells Hansman Grant S [email protected] Kazuhiko [email protected] Tomoichiro [email protected] Katsuro [email protected] Naokazu [email protected] Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan2005 23 2 2005 2 13 13 28 1 2005 23 2 2005 Copyright © 2005 Hansman et al; licensee BioMed Central Ltd.2005Hansman et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Human sapovirus (SaV), an agent of human gastroenteritis, cannot be grown in cell culture, but expression of the recombinant capsid protein (rVP1) in a baculovirus expression system results in the formation of virus-like particles (VLPs). In this study we compared the time-course expression of two different SaV rVP1 constructs. One construct had the native sequence (Wt construct), whereas the other had two nucleotide point mutations in which one mutation caused an amino acid substitution and one was silent (MEG-1076 construct). While both constructs formed VLPs morphologically similar to native SaV, Northern blot analysis indicated that the MEG-1076 rVP1 mRNA had increased steady-state levels. Furthermore, Western blot analysis and an antigen enzyme-linked immunosorbent assay showed that the MEG-1076 construct had increased expression levels of rVP1 and yields of VLPs. Interestingly, the position of the mutated residue was strictly conserved residue among other human SaV strains, suggesting an important role for rVP1 expression.
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Introduction
The family Caliciviridae is made up of four genera, Sapovirus, Norovirus, Lagovirus, and Vesivirus, which contain sapovirus (SaV), norovirus (NoV), rabbit hemorrhagic disease virus, and feline calicivirus strains, respectively. Human SaV and NoV strains are agents of gastroenteritis. The prototype strain of human SaV, the Sapporo virus, was originally discovered from an outbreak of gastroenteritis in an orphanage in Sapporo, Japan, in 1977 [1]. Chiba et al. identified viruses with the typical animal calicivirus morphology, called the "Star of David" structure, by electron microscopy (EM). SaV strains were recently divided into five genogroups (GI to GV), of which GI, GII, GIV, and GV strains infect humans, while GIII strains infect porcine species [2]. The SaV GI, GIV, and GV genomes are each predicted to contain three main open reading frames (ORFs), whereas SaV GII and GIII have two ORFs. SaV ORF1 encodes for non-structural proteins and the major capsid protein (VP1). SaV ORF2 (VP2) and ORF3 (VP3) encoded proteins of yet unknown functions. The NoV genome is organized in a slightly different way than the SaV, since ORF1 encodes all the nonstructural proteins, ORF2 encodes the capsid protein (VP1), and ORF3 encodes a small protein (VP2).
Human SaV and NoV strains are noncultivable, but expression of the recombinant VP1 (rVP1) in a baculovirus expression system results in the self-assembly of virus-like particles (VLPs) that are morphologically similar to native SaV [3,4] In a recent NoV expression study, a single amino acid substitution in the rVP1 gene affected VLP formation but not rVP1 expression [5]. In a different study, inclusions of NoV ORF3 and poly(A) sequences in a construct increased the expression levels of NoV rVP1 and the stability of VLPs when compared to constructs without these sequences [6]. Recently, cryo-EM analysis of SaV VLPs and X-ray crystallography analysis of NoV VLPs predicted the SaV shell (S) and protruding domains (subdomains P1 and P2) that were based the NoV domains [7,8]. Chen et al. also described strictly and moderately conserved amino acid residues in the capsid protein among the four genera in family Caliciviridae.
The purpose of this study was to compare the time-course expression of two different SaV rVP1 constructs in a baculovirus expression system by Northern blotting, Western blotting, enzyme-linked immunosorbent assay (ELISA), and EM. Our novel results have indicated that nucleotide point mutations increased the yields of SaV VLPs in insect cells, offering an alternative explanation for the increased expression levels of rVP1 and yield of VLPs.
Results
Wt, MQG-1076, and MEG-1076 constructs
Expression of SaV rVP1 in a baculovirus expression system results in the self-assembly of VLPs [4]. However, during PCR amplification nucleotide point mutations occurred in our initial MQG-1076 construct, at nucleotide positions 4 and 1076 in VP1, which resulted in two amino acid substitutions at residues 2 and 358, respectively, and a silent nucleotide mutation at position 1895 in VP2 (Fig. 1). Despite these two substitutions the MQG-1076 construct formed VLPs morphological similar to native SaV (data not shown). In order to further investigate these substitutions we expressed another construct (MEG-1076 construct) having only one substitution, at residue 358 in VP1 (Fig. 1). This construct also formed VLPs. Finally we expressed a construct (Wt construct) without these nucleotide point mutations, i.e., having the native sequence. The Wt construct also formed VLPs, however the expression level of rVP1 was noticeably lower than those of the MQG-1076 and MEG-1076 constructs in which had similar levels (data not shown). In order to compare expression levels, we infected Wt and MEG-1076 recombinant baculoviruses each at a multiplicity of infection (MOI) of 14.5 in 2.7 × 106 confluent Tn5 cells in 1.5 ml of Ex-Cell 405 medium followed by incubation at 26°C. RNA transcription and rVP1 expression experiments were run in parallel for the Wt and MEG-1076 constructs.
Figure 1 Schematics of the SaV constructs, Wt, MEG-1076, and MQG-1076, containing the rVP1, rVP2, and poly(A) sequences. Each construct began at the predicted AUG start. The triangles show the positions of the nucleotide point mutations. The black triangle had an amino acid substitution in the VP1, whereas the open triangle in the VP2 gene did not change amino acid sequence. An RNA probe (anti-VP1) was used to monitor the transcription of rVP1 mRNA in which contained the native sequence, i.e., lacking the mutation at 1076.
Northern blot analysis
Total RNA was extracted from the cells at 1, 2, 3, 4, 5, 6, 7, and 8 days postinfection (dpi) for Wt and MEG-1076 constructs. Equal amounts (500 ng) of total RNA were added to a 2% agarose gel containing formaldehyde and stained with SYBR Gold (Fig. 2A). The rVP1 mRNA was then analysed by Northern blot with a probe specific for the VP1 gene (native sequence) corresponding to the VP1 position 157 to 1283 (Fig. 1). The rVP1 mRNA transcript was predicted to be approximately 2300 nucleotides long. As shown in Figure 2B, rVP1 mRNA was detected for each construct. This result showed that the insert sequence and some part of the baculovirus vector, approximately 300 nt, was transcribed, although the exact location(s) on the vector has yet to be determined. Nevertheless, the MEG-1076 construct had increased band intensities, indicating an increased steady-state level, when compared to those of the Wt construct (Fig. 2B). For the Wt construct, rVP1 mRNA was detected at 1 dpi, peaked at 2 dpi, decreased at 3 and 4 dpi, and then decreased to undetectable levels at 5, 6, 7, and 8 dpi. For the MEG-1076 construct, rVP1 mRNA was detected at 1 dpi, peaked at 2 dpi, had steady-state levels at 3 and 4 dpi, and then decreased at 5 dpi but could still be detected at 6, 7, and 8 dpi. These results indicated that the MEG-1076 rVP1 mRNA also had greater stability when compared to those of the Wt rVP1 mRNA.
Figure 2 Northern Blot analysis of Wt and MEG-1076 rVP1 mRNA. The total RNA was purified from the cells at 1, 2, 3, 4, 5, 6, 7, and 8 dpi. (A) The relative amounts of total RNA for each construct. (B) The steady-state levels of rVP1 mRNA with an anti-VP1 probe specific for the VP1 gene, corresponding to the VP1 nucleotide position 157 to 1283.
Western Blot analysis
Western blot analysis was used to compare the expression levels of Wt and MEG-1076 rVP1. The culture medium was separated from the cell lysate 1, 2, 3, 4, 5, 6, 7, and 8 dpi as described in the Materials and Methods. Equal volumes of culture medium and cell lysate at each dpi were used for both constructs. Proteins were separated by SDS-PAGE, electrotransferred to PVDF, and detected with a 1:3000 dilution of hyperimmune rabbit Mc114 VLP antiserum. A band at the predicted rVP1 size (60 K) was first detected in the culture medium at 2 and 4 dpi for MEG-1076 and Wt constructs, respectively, which increased each day thereafter as evidenced by an increase in band intensity (Fig. 3A). As indicated by increased band intensities, the MEG-1076 construct expressed increased levels of rVP1 (60 K) than those of the Wt construct. Similarly, these results were reproduced using different MOIs in order to address the variability in virus stock quality (data not shown).
Figure 3 Western blot analysis of Wt and MEG-1076 rVP1. Confluent Tn5 cells were infected with Mc114 recombinant baculoviruses at MOI of 14.5 and incubated at 26°C. The culture medium, including the cells, were harvested 1, 2, 3, 4, 5, 6, 7, and 8 dpi as described in the materials and methods. (A) The cell culture medium was concentrated by ultracentrifugation, resuspended in 20 μl of Grace's medium, and 5 μl was mixed with loading dye and loaded into each well. (B) The cell lysate was separated from the culture medium, resuspended in 200 μl of Grace's medium, and 5 μl was mixed with loading dye and loaded into each well.
A thin band of approximately 55 K was also detected in the culture medium that appeared at 4 and 5 dpi for Wt and MEG-1076 constructs, respectively, and increased each day thereafter. In a different experiment, we determined the amino acid sequence of the MQG-1076 upper and lower bands by an Edman's degradation method. We discovered that the first three amino acid residues were MQG for both the upper and lower bands. This result indicated that the 55 K bands for these constructs were likely truncated or C-terminal deleted forms of rVP1. A thin band of 60 K was detected at every dpi in the cell lysate for the MEG-1076 construct (Fig. 3B), however the intensity of this band did not increase to the same extent as the MEG-1076 60 K band in the culture medium (Fig. 3A). This suggested that immediately after translation the majority of rVP1 was rapidly exported from the cells to the culture medium, though a fraction accumulated within the cells. This may also explain why no 60 K bands were detected in the cell lysate for Wt construct.
The VP2 amino acid sequence was the same in all constructs. We did not detect rVP2 during the time-course expression of the MQG-1076 construct using the antiserum raised against E. coli expressed VP2 (data not shown).
Antigen ELISA and EM analysis of Wt and MEG-1076 VLPs
An antigen ELISA system was used to compare the yields of Wt and MEG-1076 VLPs at 1, 2, 3, 4, 5, 6, 7, and 8 dpi. The ELISA incorporated hyperimmune rabbit (capture) and guinea pig (detector) antisera raised against purified Mc114 VLPs [4]. The ELISA first detected VLPs at 2 and 3 dpi for MEG-1076 and Wt constructs, respectively (Fig. 4). For both constructs, the yields of VLPs increased each day thereafter, however the MEG-1076 construct had increased yields of VLPs than those of the Wt construct at 4, 5, 6, 7, and 8 dpi, approximately 6-fold increase. EM was used to verify the VLP formation of each of these constructs. We first detected VLPs at 4 dpi in the culture medium for both constructs and the numbers of VLPs increased each day thereafter (data not shown).
Figure 4 Antigen ELISA analysis of Wt and MEG-1076 VLPs. The ELISA used hyperimmune rabbit (capture) and guinea pig (detector) antiserum raised against Mc114 VLPs. For the antigen ELISA, purified Mc114 VLPs were used as the positive control at concentrations ranging from 500 ng to 0.24 ng.
Amino acid analysis
The MEG-1076 construct contained a nucleotide point mutation in which resulted in an amino acid substitution at position 358 in VP1. We aligned 21 different VP1 amino acid sequences of SaV GI, GII, and GV strains and found this residue was strictly conserved, but more importantly, there was a strictly conserved amino acid motif at this site, NGDV (data not shown). However, when we included a porcine SaV GIII strain and a recently identified SaV GIV strain (PEC and Hou-7, respectively), only the GD site was strictly conserved, though several other amino acids nearby were also strictly conserved (Fig. 5). Further analysis of other SaV GIV strains are clearly needed in order to examine the possibility that the NGDV motif was moderately conserved in other human SaV strains. Figure 5 also showed that the predicted SaV P2 domain had very few conserved amino acid residues. Apart from the strictly conserved GD motif, the only other strictly conserved motif in the P2 domain was at the 5' end.
Figure 5 VP1 amino acid alignment of SaV GI, GII, GIII, GV, and GV strains. We originally aligned 21 SaV GI, GII, and GV sequences but to simplify the figure we used one representative strain from each genogroup. The green bar shows the SaV P2 domain predicted by Chen et al. [7]. The asterisks indicate conserved amino acids. We originally aligned 21 different VP1 amino acid sequences of SaV GI, GII, and GV strains and found the residue (N) at position 358 (yellow) was strictly conserved (data not shown), but SaV GIII and GIV strains (PEC and Hou-7, respectively) had other residues at this position. The alignment of the five SaV genogroups showed the amino acid motif, GD, was strictly conserved (red) and several other amino acids surrounding the residue at position 358 were also strictly conserved (red).
Discussion
Expression of the human SaV rVP1 in a baculovirus expression system was first reported in 1997 [9]. In that study, the full-length VP1 gene, ORF2, and poly(A) sequences were included in a construct (Sapporo strain, GI). The second human SaV reported to form VLPs was with a construct (Houston/90 strain, GI) using only the VP1 sequence, i.e., lacking ORF2 and poly(A) sequences [10], while the third human SaV reported to form VLPs used a construct (Parkville strain, GI) with only VP1 and ORF2 sequences, i.e., lacking poly(A) sequence [7]. We recently expressed human SaV GI, GII, and GV rVP1 with constructs (Mc14, C12, and NK24 strains, respectively) that included ORF2 and poly(A) sequences [4]. Additional information on human SaV rVP1 expression is lacking, although it appeared that the yields of human SaV VLPs were typically low for these three genogroups.
In this study, we compared the time-course expression of two different Mc114 SaV rVP1 constructs in a baculovirus expression system (Fig. 1). The MEG-1076 construct had two nucleotide point mutations, one in the VP1 gene in which resulted in an amino acid substitution, and one in the VP2 gene in which was silent. Although both constructs formed VLPs morphological similar to native SaV, the levels of transcription, translation, and VLP formation were clearly different. As shown in Figure 2B, the MEG-1076 rVP1 mRNA had increased steady-state levels and greater stability when compared to those of the Wt rVP1 mRNA. This difference was understood to be due to the nucleotide mutations in the MEG-1076 construct, since a similar result was observed in a NoV expression study [6]. Bertolotti-Ciarlet et al. found that a nucleotide point mutation in a NoV rVP1 construct (ORF2-AUG → ACG-ORF3+3' UTR construct, represented in bold) had decreased levels of rVP1 mRNA at 36 hours post-infection, by approximately 50%, when compared to a construct without the mutation (ORF2+ORF3+3' UTR construct). Bertolotti-Ciarlet suggested that the RNA secondary structure or changes in the mRNA stability could be responsible for the different steady-state levels, but this was not proven.
Also, the MEG-1076 construct had increased levels of rVP1 expression and yields of VLPs in the culture medium when compared to those of the Wt construct (Fig. 3A). On the other hand, the concentration of rVP1 in the cell lysate remained more or less the same during the time-course expression for the MEG-1076 construct. And for the Wt construct, rVP1 was not detected in the cell lysate, although this may have been related to the low expression levels (Fig. 3B). Our results showed that the MEG-1076 construct had a 6-fold increase in yields of VLPs in the culture medium (Fig. 4), which corresponded to approximately 80 μg of CsCl purified VLPs from 200 ml of culture medium (at 6 dpi), but less than 5 μg of CsCl purified VLPs in the cell lysate (data not shown). These results suggested that either (i) immediately after translation the majority of rVP1 was exported from the cells to the culture medium where the majority of VLPs were folded but a fraction were simultaneously folded within the cells or (ii) VLPs were folded within the cells and then the majority of VLPs were immediately exported from the cells to the culture medium, though a fraction remained within the cells.
In a recent NoV expression study, a single amino acid substitution in the rVP1 gene affected VLP formation but not rVP1 expression [5]. In that study, a (native) histidine residue at position 91 (relative to NoV Snow Mountain Virus strain amino acid VP1 sequence) was found to be essential for VLP formation and a construct with a substituted (mutant) arginine residue at this position failed to form VLPs despite expressing rVP1. Interestingly, that study found a single amino substitution was critical for the formation of VLPs, whereas our results showed that a single amino acid substitution was beneficial, i.e., increased the yields of VLPs. Bertolotti-Ciarlet found that inclusions of NoV ORF3 and poly(A) sequences in a construct increased the expression levels of NoV rVP1 and the stability of VLPs when compared to constructs without these sequences; and suggested that expression of other caliciviruses (NoV and SaV) rVP1 that resulted in low yields or unstable VLPs may be due to constructs that lacked the VP2 gene [6]. An alternative explanation was that point mutations influenced steady-state levels of mRNA and stability, which in turn influenced VLP formation. In our case, one or two nucleotide point mutations caused an enhancement of transcription, leading to increased yields of SaV VLPs in insect cells. Furthermore, many of these studies that expressed calicivirus rVP1 in insect cells only examined rVP1 expression and yields of VLPs but not rVP1 mRNA transcription [11-14]. However, another reason for the increased yields of VLPs may be associated with adaptation of SaV rVP1 to the baculovirus expression system and insect cells, since a similar result was observed with porcine enteric calicivirus in primary kidney cells [15].
Although the growth rate and replication efficiency of the recombinant baculoviruses themselves and differences in the levels of virus replication might account for such variation, we observed similar results using other MOIs, that is, the MEG-1076 construct continued to express greater yields of VLPs than the Wt construct (data not shown). Another explanation may have been differences in the extents to which these baculoviruses induce apoptosis and all these may result from features in the baculovirus skeleton rather than from the inserted SaV sequence. Such effects might for instance affect the number of adherent cells harvested or the degradation rates of both proteins and RNAs. However, we found that the MQG-1076 construct, developed from a separate experiment, had similar expression levels to that of the MEG-1076 construct (data not shown), which may eliminate the possibility that the baculovirus skeleton played a role in the increased yields of VLPs. On the other hand, we could not demonstrate whether the nucleotide mutations in VP1 and/or in ORF2 affected the transcription, a construct with only one of these mutations would be needed. Nevertheless, our results indicate that translation was exclusively affected by the single amino acid substitution in VP1. Therefore, the final increase in yields of VLPs may have been coupled at multiple levels, involving one or both of the nucleotide mutations in VP1 and VP2.
We did not detect rVP2 during the time-course expression of the MQG-1076 construct (data not shown). The Wt and MEG-1076 constructs had an identical amino acid sequence, which would suggest a similar negative-result. NoV studies have found that inclusion of VP2 increases the stability of VLPs, though the expression level of NoV rVP2 was low [6]. These results may suggest that (i) SaV rVP2 was expressed at undetectable levels, (ii) SaV rVP2 was not expressed in the insect cells, or (iii) SaV rVP2 was degraded in the insect cells. The SaV GI, GIV, and GV genomes are each predicted to encode a third ORF (ORF3) overlapping the VP1 gene, whereas SaV GII and GIII have only two ORFs. The functions of SaV ORF2 and ORF3 still remain unknown.
The amino acid substitution (N → S) for the MEG-1076 construct occurred in the VP1 gene at residue 358. This asparagine residue was recently identified as a moderately conserved residue among the caliciviruses capsid proteins [7], but more importantly, the residue was strictly conserved among 21 different SaV GI, GII, and GV strains and belonged to a strictly conserved amino acid motif, NGDV (Fig. 5). However, when we included SaV GIII and GIV strains (PEC and Hou-7, respectively) we found that only the GD amino acids were strictly conserved though several other amino acids nearby were also strictly conserved (Fig. 5). These data further suggested that this site played an important role in the regulation of SaV VLP formation.
Recently, the cryo-EM analysis of SaV was determined and compared to NoV X-ray crystallography structure [7]. Chen et al. analysed 30 different VP1 amino acid sequences of calicivirus strains belonging to the four genera in the family Caliciviridae and identified strictly and moderately conserved residues, and predicted the P1 and P2 domains of SaV VP1 based on NoV X-ray crystallography structure. Based on these predictions, the residue at position 358 (amino acid sequence) was found as a moderately conserved residue among the caliciviruses. This arginine residue was predicated to be in the P2 domain, which is defined as the outer most protruding domain for NoV and thought to provide strain diversity [16]. Further high-resolution structural analysis of SaV VLPs is clearly needed in order to determine the precise domains and regions of SaV. However, our expression results have indicated that only approximately 80 μg of purified VLPs from 200 ml of culture medium was possible (data not shown), thus in order to determine the X-ray crystallography structure of SaV, a minimum increase in expression level of about 20-fold would be required: a challenging feat.
Materials and methods
Virus strain, RNA extraction, cDNA synthesis
SaV GI Mc114 strain (GenBank accession number, AY237422) was isolated from a male infant seven months of age from the McCormic Hospital, Chiang Mai, Thailand on the 7th May 2001 [17]. RNA extraction and cDNA synthesis were performed as previously described [18].
PCR and sequencing
Our initial SaV rVP1 construct (MQG-1076 construct) was amplified with ExTaq DNA polymerase. However, this construct was later found to have two nucleotide point mutations in ORF1 at positions 4 (GAG → CAG) and 1076 (AAT → AGT) and one nucleotide point mutation in ORF2 at position 1895 (GTG → GTA) (relative to the VP1 start and represented in bold). Primer and PCR errors likely introduced these mutations. These three nucleotide point mutations resulted in two amino acid substitutions in the VP1 gene, one at the second residue, where glutamic acid (E) → glutamine (Q), and one at residue 358, where asparagine (N) → serine (S). The nucleotide point mutation in ORF2 did not result in an amino substitution. Despite the two amino acid substitutions, the MQG-1076 construct formed VLPs. We designed another construct (MEG-1076) using the pDEST8-MQG-1076 as template but with a new sense primer and used KOD-plus DNA polymerase according to the manufacture's instructions (Toyobo, Japan). The MEG-1076 construct had the same nucleotide point mutations at positions 1076 in VP1 and 1895 in VP2 as the MQG-1076 construct but not at nucleotide 4 in VP1 (Fig. 1). Lastly, we designed a third construct with the native sequence (Wt construct) using KOD-plus DNA polymerase and the original cDNA [4]. PCR-amplified fragments were cloned into the Gateway Expression System (Invitrogen, Carlsbad, Calif.) as previously described [4]. The insert sequences of the pDONR8 plasmids were confirmed, including the partial upstream and downstream sequences on the plasmids in which were found to be identical for the Wt and MEG-1076 constructs. Sequencing was performed as previously described [18].
Expression of rVP1 in insect cells
Recombinant bacmids were transfected into Sf9 cells (Riken Cell Bank, Japan) and the recombinant baculoviruses was collected as previously described [4]. The expression of the rVP1 constructs were analyzed by infecting recombinant baculoviruses at a MOI of 14.5 in 2.7 × 106 confluent Tn5 cells in 1.5 ml of Ex-Cell 405 medium followed by incubation at 26°C. The total culture medium was harvested 1, 2, 3, 4, 5, 6, 7, and 8 dpi. The culture medium was centrifuged for 10 min at 3,000 × g, and further centrifuged for 30 min at 10,000 × g. The VLPs in the culture medium were further concentrated by ultracentrifugation for 2 h at 45,000 rpm at 4°C (Beckman TLA-55 rotor), and then resuspended in 30 μl of Grace's medium. The cell lysate from the first centrifuge was resuspended in 200 μl of Grace's medium and stored at 4°C.
Northern blotting
Total RNA was prepared from the attached cells at 1, 2, 3, 4, 5, and 6 dpi with 1 ml of Isogen (Nippon Gene, Japan). For 7 and 8 dpi, the cell culture medium (containing unattached cells) was collected and centrifuged for 5 min at 3,000 × g, the supernatant removed, and then the cells were dissolved with 1 ml of Isogen. The cells were stored at -80°C. RNA was purified by a chloroform/ ethanol method (Nippon Gene, Japan). Briefly, RNA was mixed with chloroform, centrifuged at 12,000 × g for 15 min at 4°C, and the aqueous layer collected. This was repeated once, and then the aqueous layer collected and mixed with isopropanol and stored overnight at -20°C. The solution was mixed, centrifuged at 12,000 × g for 15 min at 4°C, and the supernatant discarded. The pellet was resuspended in 80% ethanol, centrifuged at 12,000 × g for 15 min at 4°C. This was repeated once, and then the pellet air-dried and resuspended in 25 μl of TE, and stored at -80°C. The amounts of purified RNA were determined spectrophotometrically (Bio-Rad, USA). The same amounts (500 ng) of total RNA were loaded for each construct and each dpi onto a 2% denaturing agarose gel containing formaldehyde. The amounts of total RNA were compared using SYBR Gold staining (Invitrogen, USA). RNA was transferred to a positively charged nylon transfer membrane (Hybond-N+; Amersham Biosciences, Ireland) under vacuum (VacuGene XL; Pharamacia LKB, Sweden) and analyzed by Northern blotting according to the DIG Northern Starter Kit (Roche, USA), except for a minor modification. Briefly, a RNA probe corresponding to Mc114 VP1 position 157 to 1283 (anti-VP1) was generated from a PCR fragment (native sequence) according to the manufacture's instructions (Roche, USA). Hybridization was performed overnight at 68°C with anti-VP1 in 10 ml of ultrasensitive hybridization buffer (Ambion, Canada). After hybridization, immunological detection was performed according to the manufacture's instructions (Roche, USA).
Western blotting, ELISA, EM, and protein sequencing
Western blotting, ELISA, and EM were used to examine rVP1 expression as previously described [4]. However, it should be acknowledged that the hyperimmune rabbit and guinea pig antisera were raised against the MQG-1076 VLPs. Protein sequences were determined by an Edman's degradation method.
Amino acid alignment
VP1 nucleotide sequences were translated using Genetyx software (software development Co. Version 11.2.2) and submitted to online ClustalW at DDBJ . In total, we aligned different 21 SaV GI, GII, GIII, GIV, and GV sequences, and included: Arg39, AY289803; Bristol, AJ249939; C12, AY603425; Cruise ship/00, AY289804; PEC, AF182760; Dresden, AY694184; Hou-7, AF435814; Houston/86/US, U95643; Houston/27/90/US, U95644; London/29845/92/UK, U95645; Lyon/598/97/F, AJ271056; Manchester, X86560; Mc2, AY237419; Mc10, AY237420; Mex340/1990, AF435812; Mex14917/00, AF435813; NK24, AY646856; Parkville, U73124; Potsdam, AAG01042; Plymouth, X86559; Sapporo/82/Japan, U65427; and Sakaeo-15, AY646855.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
GH carried out the study and wrote the manuscript. KK, TO, KN, and NT participated in the design of the study and helped to draft the manuscript.
Acknowledgements
This work was supported by Grants-in-aid from The Ministry of Education, Culture, Sports, Science and Technology, Japan and a Grant for Research on Re-emerging Infectious Diseases from The Ministry of Health, Labour, and Welfare, Japan. We are grateful to the Japanese Monbusho for the PhD scholarship provided to Grant Hansman.
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| 15727685 | PMC553994 | CC BY | 2021-01-04 16:39:00 | no | Virol J. 2005 Feb 23; 2:13 | utf-8 | Virol J | 2,005 | 10.1186/1743-422X-2-13 | oa_comm |
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J NeuroinflammationJournal of Neuroinflammation1742-2094BioMed Central London 1742-2094-2-61572535910.1186/1742-2094-2-6ResearchDoes angiotensin-1 converting enzyme genotype influence motor or cognitive development after pre-term birth? Harding David R [email protected] Sukhbir [email protected] David [email protected] Steve E [email protected] Andrew [email protected] Neil [email protected] Hugh E [email protected] Neonatal Intensive Care Unit, St. Michael's Hospital, Bristol, UK2 Division of Cardiovascular Genetics, University College London, London, UK3 University of Bristol Medical School, Southmead Hospital, Bristol, UK4 School of Human Development, University of Nottingham, Nottingham, UK2005 22 2 2005 2 6 6 22 11 2004 22 2 2005 Copyright © 2005 Harding et al; licensee BioMed Central Ltd.2005Harding et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Raised activity of the renin-angiotensin system (RAS) may both amplify inflammatory and free radical responses and decrease tissue metabolic efficiency and thus enhance cerebral injury in the preterm infant. The angiotensin-converting enzyme (ACE) DD genotype is associated with raised ACE and RAS activity as well as potentially adverse stimuli such as inflammation. The DD genotype has been associated with neurological impairments in the elderly, and thus may be also associated with poorer motor or cognitive development amongst children born preterm prematurely.
Methods
The association of DD genotype with developmental progress amongst 176 Caucasian children born at less than 33 weeks gestation (median birthweight 1475 g, range 645–2480 g; gestation 30 weeks, range 22–32; 108 male) was examined at 2 and 5 1/2 years of age. Measured neuro-cognitive outcomes were cranial ultrasound abnormalities, cerebral palsy, disability, Griffiths Developmental Quotient [DQ] at 2 yrs, and General Cognitive Ability [British Ability Scales-11] and motor performance [ABC Movement], both performed at 5 1/2 yrs. All outcomes were correlated with ACE genotype.
Results
The DD genotype was not associated with lower developmental quotients even after accounting for important social variables.
Conclusion
These data do not support either a role for ACE in the development of cognitive or motor function in surviving infants born preterm or inhibition of ACE as a neuroprotective therapy.
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Background
Delight over recent survival gains for the very premature infant has been tempered by the frequent presence of cerebral injury and developmental impairment. One quarter of those born before 26 weeks postmenstrual age (at least 11 weeks premature) show evidence of severe cerebral injury including cognitive dysfunction by 30 months of age [1]. Preterm children without any disability remain at risk of a range of motor, cognitive, behavioural and psychological deficits during childhood even if not born so close to the margin of viability [2]. To date, the pathophysiological processes leading to such impairment remain largely occult. In particular, cerebral imaging has failed to identify structural correlates of impaired higher function [3] although imaging can predict many cases of motor abnormality (such as cerebral palsy) due to the presence of periventricular white matter injury [4].
Three factors seem to play important roles in the aetiology of preterm cerebral injury. Firstly, exposure to inflammatory stimuli is associated with white matter injury and cerebral palsy in the preterm [5]. Secondly, reduced glucose and oxygen delivery to the developing brain (hypoxia-ischaemia: local cerebral or systemic) may cause excito-toxic neurotransmitter release followed by neuronal death [6]. Thirdly, free-radicals may damage the oligodendrocytes of white matter of the preterm brain [6]. Damage to the primitive white matter prevents the normal formation of grey matter connections which may influence cognitive development in childhood [7].
Candidate systems that might influence motor or cognitive outcome after premature birth are likely to be those which affect these responses. The human renin-angiotensin systems may be such a system. Angiotensin converting enzyme (ACE), a key component of the circulating (or endocrine) renin-angiotensin system (RAS), cleaves angiotensin I to yield the potent vasoconstrictor angiotensin II. In addition, ACE degrades vasodilator kinins. In these ways, endocrine RAS plays an important role in circulatory homeostasis. However, local RAS also exist in diverse human tissues including lung, myocardium, vasculature, lymphocyte and brain tissue. These are powerful regulators of mitochondrial respiration and whole-cell metabolism [8] and exert profound effects on whole-human metabolism and metabolic efficiency: elevated ACE may impair cellular aerobic metabolism [9]. RAS also plays a key role in the regulation of tissue inflammatory responses; ACE, through generation of angiotensin II, stimulates the synthesis of pro-inflammatory cytokines, including IL-6 which itself is thought to exert major neurocytotoxic effects with the genesis of functionally significant lesions in the developing preterm brain [5]. It has also been noted that the inhibition of RAS may reduce the effects of excitotoxic neurotransmitters and free radicals [10]. It is possible therefore that enhanced ACE activity may adversely influence the development of the child born prematurely.
A common variant of the human ACE gene provides a tool to determine if ACE activity does influence developmental progress after preterm birth. The presence (insertion, or 'I' allele) rather than the absence (deletion, or 'D' allele) of a 284-base-pair fragment in the human ACE gene is associated with lower ACE activity in organs including both circulating inflammatory cells [11] and the circulation itself [12]. Given the likely causal association of pro-inflammatory responses, ischaemic-hypoxia, excitotoxic neurotransmitters, and free radical attack with impaired neuro-outcome; and given the potential role of increased RAS activity in amplifying these effects, we might expect the DD genotype (encoding raised ACE activity) to be associated with poorer neuro-developmental progress after pretem birth. Comparable findings have been described with respect to the deterioration of cognitive function in the elderly by some authors [13-15]. We have tested this hypothesis by studying the association of the ACE I/D polymorphism with measures of neuro-developmental progress at 2 and 5 1/2 years of age in children who had participated in a neuro-developmental outcome study (The Avon Premature Infant Project, APIP [16]). All the patients were born at less than 33 weeks postmenstrual age (normal gestation is 37–40 weeks).
Methods
Patients
The study was approved by the ethical committees of Southmead Hospital and United Bristol Health Care Trust. Parental consent was obtained for participation in neurodevelopmental follow-up [16] (see below). Consent was not required for the genetic component of this study as all personal information was held separately from the genetic information and patients were identified only by study codes.
All children were born at 32 weeks gestation or less, between December 1990 and July 1993 at Southmead Hospital or St. Michael's Hospital, Bristol. All had participated in the Avon Premature Infant Project (APIP) [16]. Briefly, this was a randomised controlled trial in which developmental support (Portage) or supportive counselling (parental adviser), each started at discharge and continued for up to 2 years, were found to confer some measurable (3–4 DQ points (below)) but clinically insignificant benefit to development at 2 years of age, when given in addition to appropriate primary care and community support, after adjusting for social variables.
Neuro-developmental outcome
The Griffiths Mental Development Scales, used to assess motor and cognitive performance, was performed at 2 years corrected age [17]. The Griffiths scales comprise five subscales, including personal and social, hearing and speech, locomotor, eye hand co-ordination and performance domains, from which is derived an overall developmental quotient (DQ). A lower Griffiths DQ reflects a poorer neuro-developmental performance, with a difference DQ of five points being clinically apparent. DQ was standardised originally to a mean of 100, with a standard deviation of 15, but secular drifts in population scores have resulted in a higher population mean. Thus for severe disability a score of 70 (-2 standard deviations (sd)) would indicate severe disability. Cognitive developmental progress at 5.5 years of age was assessed using the British Ability Scales [18]. The BAS-II was standardised in the early 1990s and was used to compute general cognitive ability (GCA) together with visuospatial, verbal and non-verbal subscales. The GCA is a developmental quotient, equivalent to an IQ estimate, normalised at 100 (sd +/- 15) in which a lower score again indicates poorer conceptual ability. The Movement ABC scales were used to assess manual dexterity, ball skills, and balance over ten tests at 5 1/2 years of age. Scores of each component are summed to produce a composite score ranging from 0–40, with high scores indicating a more impaired motor skills and 0 indicating normal skills.
A psychologist performed the Griffiths Scales of Mental Development and a second psychologist performed the British Ability Scales (second edition) (BAS). The ABC Movement tests were performed by a trained research nurse. All assessments were blind to the child's neonatal course and subsequent progress.
ACE genotyping
DNA was extracted from the Guthrie card blood spots (newborn metabolic screening cards). ACE genotype was determined using 3-primer PCR amplification [9]. Primer ratios corresponded to 50 pmol of an I-specific oligonucleotide in a 20-υl reaction volume. The PCR was performed using Taq polymeraase yielding amplification products of 84 bp for the D allele, and 65 bp for the I allele. Amplification products were visualised using a 7.5% polyacrylamide gel stained with ethidium bromide. Genotyping was performed by staff blind to all clinical data.
Study Size
An estimate of sample size suggested that 144 patients would be needed for this study. The assumptions made for this calculation were that DD genotype infants had a mean DQ of 92.5 (1/2 SD below the norm) compared to a mean DQ of 100 in the ID+II group, assumed typical genotype distributions, and a significance of 0.05 with 80% power.
Statistical analysis
Data were stored in SPPS v9.0 for Windows. Lymphocyte [11] and tissue ACE [12] activity is primarily raised in DD genotype when compared to either ID or II genotype, and so data for those of DD genotype were compared to those from I-allele carriers. Categorical data were analysed by Chi square and continuous data by Student's T Test if normally distributed or Mann-Whitney U test as appropriate.
Results
Guthrie cards were located for 230 of 308 children. After exclusion of non-Caucasians and, at random, 1 child of any identical twin pairs (based on genotypes and gender) 176 babies with ACE genotype formed the study population (median birthweight 1475 g, range 645–2480 g; gestation 30 weeks, range 22–32) with follow-up data at 2 years. 122 of these also had follow-up at 5 1/2 years. The ACE genotype distribution was 49 [27.8%] DD, 73 [41.5%] ID, 54 [30.7%] II, demonstrated Hardy-Weinberg equilibrium, and was similar to that observed in the newborn term population from the same region of the UK (203 [24.1%] DD, 433 [51.5%] ID, 205 [24.4%) II). Baseline characteristics were independent of genotype, except that fewer individuals of DD genotype were from twin births (p = 0.047) (table 1). There was no association between markers of neonatal cerebral injury: severe intraventricular haemorrrhage or white matter injury (table 1). There was no association with the presence of any disability at 2 years of age (DD 17% vs ID/II 15%, p = 0.65).
Table 1 Perinatal and social factors
DD Genotype (n = 49) ID/II Genotype (n = 127)
No maternal antenatal corticosteroids 44 (80%) 112 (81%)
No. of children from twin pregnancy* 4 (8%) 27 (21%)
Male 32 (65%) 76 (60%)
Gestation, weeks (± SEM) 29.7 (± 0.3) 30.0 (± 0.2)
Birth weight, g (± SEM) 1453 (± 56) 1461 (± 34)
Portage, parent adviser 17 (31%), 19 (35%) 46 (33%), 42 (30%)
Severe intraventricular haemorrhage 5 (11%) 7 (6%)
White matter injury 7 (14%) 14 (11%)
Maternal age (± SEM) 27.2 (± 0.8) 27.4 (± 0.8)
Manual occupation 28 (57%) 76 (60%)
Maternal car use 30 (61%) 75 (60%)
Mother educated beyond 16 yrs. 17 (35%) 48 (38%)
*p = 0.047 (Fisher's Exact Probability Test)
Continuous data is shown as mean (± standard error of mean).
Measures of developmental cognitive and motor outcome were entirely independent of genotype (table 2). The findings were unchanged after post hoc subgroup analysis of singletons, infants with normal cranial scans, amongst children without disability and after adjusting for potential influential variables (including twin birth) using multiple regression (data not shown).
Table 2 ACE genotype and developmental performance at 2 and 5 1/2 years of age. Data shown is mean (± SEM).
Developmental tests DQ for DD DQ for ID/II p
Griffith DQ at 2 years 96.2 (3.1) 96.3 (1.3) 0.95
Locomotor subscale 92.7 (2.7) 92.4 (1.3) 0.92
Personal & social subscale 101.9 (3.0) 101.0 (1.6) 0.80
Hearing and speech subscale 92.9 (4.1) 94.0 (2.1) 0.80
Eye hand co-ordination subscale 90.8 (3.1) 92.8 (1.2) 0.46
Performance subscale 102.2 (4.3) 101.3 (1.6) 0.79
Griffith DQ at 2 years (adjusted for social variables) 100.0 (0.9) 99.3 (0.6) 0.43
ABC Movement summative score 8.1 (1.8) 8.0 (0.9) 0.97
GCA at 5 1/2 years 99.2 (3.4) 100.2 (2.0) 0.80
Verbal ability subscale 98.0 (4.0) 103.2 (1.7) 0.22
Pictoral ability subscale 99.9 (3.3) 98.7 (1.7) 0.99
Spatial ability subscale 98.4 (3.3) 97.3 (1.9) 0.67
Discussion
After a search of Embase and Medline we believe that this study is the first to attempt to dissect out the contribution of genetic variation in the ACE gene to developmental progress after pre-term delivery. Despite much physiological and biochemical evidence to support our hypothesis, we found that ACE DD genotype was not associated with adverse long term developmental outcome in infants of < 33 weeks gestation in this study.
These data are perhaps at variance with previous studies of Alzheimer's disease, age-associated memory impairment and vascular dementia, all of which have implicated the ACE D allele in having a role in mental decline [13-15]. However this is not a universal finding. Furthermore although ACE inhibitors appear to reduce inflammatory responses, ischaemic effects, and excitotoxic and free radical induced injury [10], angiotensin II does not (indeed angiotensin II may actually enhance ischaemic and excitotoxic neural injury via the AT2 receptor). In addition, both captopril and losartan (RAS inhibitors) appear to improve cognitive performance in mice [19] and humans [20]. It should be noted however that little is known about the ontogeny of the RAS in the human foetus. Certainly RAS (and angiotensin II receptors in particular) play a role in blood-brain barrier and central nervous system development in mice, and alterations in RAS receptor expression over foetal and neonatal life are recognised. It is thus possible that developmentally regulated patterns of AT1 receptor expression might offer some level of protection against the potentially detrimental effects of ACE-mediated angiotensin II synthesis.
Although there may be similar molecular pathways that effect cerebral injury in the preterm infant and the elderly, ontological differences in the expression of genes involved in predisposition to neural injury are well described. In particular reactive production of nitric oxide may be enhanced in the elderly and the ability to protect the brain from oxidants may be reduced in the elderly (22). Thus the effect of any one polymorphism, with a relatively minor effect, may be swamped in the newborn infant by other protective mechanisms.
The lack of any association between ACE genotype and scores of developmental progress was also surprising because we have demonstrated an association between DD genotype and markers of poor cardio-respiratory instability in the perinatal period in this patient group [21]. This association (between genotype and worse early cardio-respiratory status) could predispose to death, which would in turn weaken any association (if it exists) between DD genotype and worse developmental quotients. It is of course possible that our sample size was insufficient to demonstrate any association with ACE genotype and developmental progress. However, similar-sized studies have been sufficient to demonstrate an association between ACE D allele and cognitive decline in the elderly [13-15], and power calculations suggested we had enough patients to demonstrate at least a trend. If an undetected genotype-association does exist such an effect is weak.
Conclusion
We cannot support an association of ACE genotype with cognitive or motor development in survivors born preterm or, thus, the use of RAS inhibition as a neuroprotective agent in the preterm. Given the current lack of understanding of the mechanisms leading to cerebral injury and subsequent impairment – particularly of higher function – in such patients, further genetic association studies of other candidate genes are warranted.
List of abbreviations used
ACE, angiotensin-1 converting enzyme; DQ developmental quotient, BAS, British ability scales (second edition); GCA, general cognitive ability; RAS, renin angiotensin system; PCR, polymerase chain rection.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
DH, HM, AW, NM conceived the study and its design and wrote the manuscript. DH, DD and SD performed data collection, DNA extraction and PCR and participated in analysis of the data with SH and HM. NM reviewed all cranial imaging. All authors participated in the writing of the manuscript and approved the final manuscript.
Acknowledgements
The developmental assessments were performed by Dr Margaret Robinson (Griffiths Assessments), Pat Anderson (BAS-II) and Wendy Ring (Movement ABC). This research was supported by awards from The Southmead Hospital Research Foundation to AW and DH. The British Heart Foundation (grant numbers RG200015, SP98003, FS01XXX) SHE, HM, and SD. The original APIP study was supported by Action Research (Grant to NM).
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| 15725359 | PMC553995 | CC BY | 2021-01-04 16:38:21 | no | J Neuroinflammation. 2005 Feb 22; 2:6 | utf-8 | J Neuroinflammation | 2,005 | 10.1186/1742-2094-2-6 | oa_comm |
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Malar JMalaria Journal1475-2875BioMed Central London 1475-2875-4-131572370610.1186/1475-2875-4-13ResearchIllness-related practices for the management of childhood malaria among the Bwatiye people of north-eastern Nigeria Akogun Oladele B [email protected] Kauna K [email protected] The Health Programme, Common Heritage Foundation, No. 1 Bishop St., Box 5124, Yola, Nigeria2005 21 2 2005 4 13 13 3 11 2004 21 2 2005 Copyright © 2005 Akogun and John; licensee BioMed Central Ltd.2005Akogun and John; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
A wide range of childhood illnesses are accompanied by fever,, including malaria. Child mortality due to malaria has been attributed to poor health service delivery system and ignorance. An assessment of a mother's ability to recognize malaria in children under-five was carried out among the Bwatiye, a poorly-served minority ethnic group in north-eastern Nigeria.
Methods
A three-stage research design involving interviews, participatory observation and laboratory tests was used to seek information from 186 Bwatiye mothers about their illness-related experiences with childhood fevers.
Results
Mothers classified malaria into male (fever that persists for longer than three days) and female (fever that goes away within three days) and had a system of determining when febrile illness would not be regarded as malaria. Most often, malaria would be ignored in the first 2 days before seeking active treatment. Self-medication was the preferred option. Treatment practices and sources of help were influenced by local beliefs, the parity of the mother and previous experience with child mortality.
Conclusion
The need to educate mothers to suspect malaria in every case of febrile illness and take appropriate action in order to expose the underlying "evil" will be more acceptable than an insistence on replacing local knowledge with biological epidemiology of malaria. The challenge facing health workers is to identify and exploit local beliefs about aetiology in effecting management procedures among culturally different peoples, who may not accept the concept of biological epidemiology.
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Background
Malaria is a major cause of death among children in many parts of the world, despite the availability of simple and effective treatments [1] and is one of the main causes of morbidity and mortality in Nigeria [2,3]. Although, treatment often begins early and at home, a mother's inability to correctly recognize malaria has contributed substantially to child morbidity and mortality due to malaria [4]. In Tanzania, as in western Nigeria, studies show that mothers were unable to recognize severe malaria despite perceiving the signs and symptoms of onset of childhood malaria as including high temperature and loss of appetite [5]. Malaria control depends on many factors, some of which have not been studied at the level of rural communities, and in different cultural settings. An understanding of the mother's ability to appropriately manage childhood malaria in the home is crucial. In order to provide a a community with the capacity for dealing with the management of malaria in the home, a study of mothers' illness-related experiences in the management of childhood malaria was carried out among the poorly-served Bwatiye ethnic group of north-eastern Nigeria, the results of which are presented in this article.
Methods
The study was carried out during the malaria season (May-September) of 1999 among the Bwatiye, a minority group living in six villages along the Benue valley in Adamawa State of north-eastern Nigeria. A multiple-stage approach consisting qualitative, quantitative and laboratory techniques was used.
Local knowledge and help-seeking practices
In order to orient the data collection process, informal conversations were held with women and lay people in markets and health facilities about malaria illness experience within communities, the method of recognition and help-seeking behaviour. On the basis of received information, key informants were identified and selected (medicine vendors, folk healers, health-workers) and formally interviewed about local knowledge and practices regarding malaria in children. The information was used for designing a cross-sectional household survey questionnaire. Households with children between 9 and 60 months in six Bwatiye villages were identified with the assistance of community health personnel. The study objectives and procedures were explained to household heads and mothers of children that were eligible participants in the study. It was emphasized that anyone was at liberty to decline participation and that those participating were also free to withdraw at any stage of the study. The pre-tested cross-sectional survey questionnaire was administered in the Hausa language to 186 mothers in their homes. Information on the mother's demographic, educational and parity status was collected. Mothers were also asked about symptoms used for recognizing and classifying malaria and to describe their help-seeking practices when a child has malaria. Previous history of child mortality was also recorded. The interviews were complemented with observations of fifteen mothers (with previous child mortality, first childbirth, and more than two children) identified during the interview process. The results were analysed and used for developing a group discussion guide.
Six group discussions, each consisting of eight carefully selected mothers of different ages. family settings (polygamous or monogamous) and religions were held.
Culturally appropriate explanations were further obtained from four key informants when the data had been analysed and a draft report written.
Results
Presumptive diagnosis
Most mothers (88%) refer to malaria as zazzabi, meaning "hot body" with intermittent episodes of cold shivers ("when the child wants to stay in the sun") accompanied by headache as the main symptom of malaria in their children. Diarrhoea and teething were also important (44.6%) besides dehydration, sweating and vomiting (33.3%), loss of appetite (30.6%) and persistent crying (29.6%). Only very few mothers (6.5%) use faint spells, restlessness, moodiness and withdrawal as symptoms. Only 9.7% mentioned convulsion as an important symptom of malaria in children. Several symptoms were combined, although "child feels cold but has hot body" is an omnipresent clue to presuming that a child has zazzabi (Table 1).
Table 1 Symptoms1 used by Bwatiye mothers for the recognition of childhood malaria
Symptom Age (years) Parity (childbirths) Previous child mortality Education Total
<25 26–35 >35 1st 2nd-3rd 4th or more Yes No Literate Illiterate
No in sample (%) 54(29) 59(32) 72(39) 42(23) 63(34) 81(44) 36(19) 150(81) 139(75) 47(25) 186(100)
Hot body (>38C) 77.9 89.3 93.2 100 92.1 79.0 100 85.3 87.8 89.4 88.2
Diarrhoea/ enteric complaints 38.9 47.5 46.6 61.9 81.0 28.4 80.6 36.0 49.6 29.8 44.6
Vomiting 29.6 32.2 37.0 33.3 30.2 35.8 11.1 38.7 37.4 21.3 33.3
Loss of appetite 31.5 30.5 27.4 38.1 28.6 28.4 58.3 24.0 28.1 38.3 30.6
Persistent crying 38.9 32.2 20.5 47.6 15.9 30.9 19.4 32.0 20.9 55.3 29.6
Dehydration /sweating 37.0 33.9 32.9 35.7 31.2 35.8 66.7 26.7 38.1 23.4 34.4
Convulsions 9.3 10.2 9.6 9.5 9.5 9.9 5.6 10.7 8.6 12.8 9.7
Other 3 9.3 5.1 5.5 11.9 4.8 4.9 8.3 6.0 6.5 6.4 6.5
1Multiple responses allowed
2Vertical comparison only (based on number of responses)
3Faint spells, insomnia, Restlessness, moodiness, withdrawal, colds
While the number of childbirths and previous experience with child mortality were important factors in the recognition of malaria (Chisquare test, P > 0.05), age and education (Chisquare test, P < 0.05) were not. Association between age and number of childbirths on the one hand and age and child mortality on the other were significant (Chisquare test, P < 0.05). Young mothers had fewer children and fewer child mortality rates than the older mothers. Among first time mothers, 47.6% identified fever with persistent crying, an association unique to this group and used zazzabi as the major symptom (100%), while 79.0% of those with more than three child-births were content to presume malaria on noticing "hot-body with cold shivers" without any additional symptom. Mothers with more than three children (28.4%) and mothers with two or three children (81.0%) differed widely with respect to enteric symptoms.
A history of child death in the family plays an important role in a mother's approach to recognition of malaria. Surprisingly, education played a minor role in the recognition of childhood malaria. For mothers who had experienced a child dying, emphasis was placed on diarrhoea.
Classification
Malaria was classified into two categories; the female and the male. The female form goes away after a couple of days without any particular treatment other than that provided by the household or in some cases the mother. When illness defies home remedies after several days, or deteriorates, it is considered to be the male form. However, should extra-community consultants become necessary, the illness is no longer regarded as zazzabi and "evil doers" are suspected to be using zazzabi to hide a more severe ailment.
Convulsions or any form of complications including hallucinations were not regarded as symptoms of malaria but manifestations of other problems that the local healer (boka) rather than the health facility could handle.
Help-seeking behaviour
The sequence of action in progressive malaria infection among the Bwatiye is described in Figure 1.
Figure 1 Flowchart of the sequence of help-seeking habits.
Self-medication with herbs and herbal potions was the preferred option (60.2%) for most mothers (Table 2). However, young mothers were less inclined to use self-medication with herbs (53.6%) than older women (65.7%). They either waited for their husbands to take action (42.9%) or consulted an older woman or a mother-in-law (25.0%). On the other hand, those who had previously experienced child death (69.4%) preferred self-medication. Most mothers went to the health-care centre as the third line of action. Only few women without history of child death (11.3%) took their children to the health-care centre for treatment. Mothers often give traditional treatments for childhood convulsions and wait until fits cease before the next action. First-time mothers tended to consult their husband before anyone else. Multigravid women would first go to friends with previous experience with malaria in children to collect leftover tablets before informing the child's father or other male relatives.
Table 2 Help-seeking behaviour to malaria in children
Young women (< = 30) Older women (>30) Previous mortality No history of child births Total (%)
Sample (N = 186) 84 102 36 150 186
Behaviour*
Self-medication (with herbs, herbal potions): 45(53.6) 67(65.7) 25(69.4) 87(58.0) 60.2
Self-medication with antipyretics from market/shops 9(10.7) 15(14.7) 2(5.6) 22(14.7) 12.9
Treatment with drugs from health facilities 12(14..3) 8(7.8) 4(11.1) 16(10.7) 10.7
Treatment with herbs and drugs from market/shops 14(16.7) 16(15.7) 5(13.9) 25(16.7) 16.1
If symptoms do not go away/get worse
Visit the herbalist 6(7.1) 48(47.1) 23(63.9) 31(20.7) 29.0
Visit the health facility/staff 8(9.5) 15(14.7) 6(16.7) 17(11.3) 12.4
Wait for husband before any action 36(42.9) 9(8.8) 2(5.6) 43(28.7) 24.2
Consult mother-in-law 21(25.0) 19(18.6) 2(5.6) 38(25.3) 21.5
Consult a more experienced friend or neighbour 13(15.5) 11(10.8) 3(8.3) 21(14.0) 21.9
* Simultaneous treatment with herbs and modern medicine is very common
Extra-communal effort at getting the child back to good health would then be made. Treatment which hitherto was confined to consulting immediate family members would now be extended to the wider community. Local herbalists and other neighbours then contribute to the management of the ailment on the basis of previous experience with similar or other illness. This is often done more as an attempt to show concern and solidarity rather than an understanding of the illness at hand. However if the child remains unwell, evil doers were regarded as blocking the effort to heal the child. The men would gather around the child to administer any medicine that may be brought by other community members. Women are excluded from the room at this stage. The "male form" of malaria is treated with both modern and traditional medicine of all sorts as precautions against spiritual dimensions to the illness. When convulsions set in, the child was immediately assumed to be severely ill and the treatment in any orthodox setting was most often ruled out. A furore of activities would begin to get the child out of the village to some other village, away from the reach of local enemies.
The most common anti-malarial drug used by the few mothers, who bought over-the-counter drugs, was chloroquine that was often administered at inappropriate dosage.
The 3-day course of chloroquine was never used in most cases mainly due to access and ignorance of the importance of full dose. Children with convulsions were taken first to the traditional healer and if it did not seem to be making any improvement the health-worker was consulted. Some health personnel thought convulsion was a different illness caused by cold and was best handled by a boka.
Discussion
Aetiological perspective and natural action
In Bwatiye, the term zazzabi, a Hausa word, implies an ordinary illness that does not kill. If death eventually results, it could not have been malaria, but some other cause.
As in other cultures, an attempt to differentiate between the severe and the mild forms of malaria has led to classification in order to determine at which stage a particular remedy would be required. While the Yoruba and Igbo are reported to have three classifications for malaria or fever episodes [6,7] the Bwatiye classified malaria into two as did the Kenyan tribe described by Munguti [8]. It has been demonstrated in many studies and for many illnesses, including river blindness and malaria [9-11], that many African communities are unable to perceive illness as a continuum of symptoms with one mild one giving way to a more severe one. Unlike the observations made by Linder [12] that the onset of fever in children often prompts mothers to seek immediate treatment, the natural primary action among the Bwatiye is to ignore the illness and hope it will go away. Persistent illness only elicits home remedy using available drugs or herbs within the household. Help is sought from the wider community (or from the local healer) only when the home remedy fails. Since there is a strong belief in the spiritual causes of illness, confidence and reliance in the local healer (the boka) is strong, especially among the older mothers. The local belief is that convulsions are not curable in health facilities and a sick child taken to another village, out of reach of the local enemies, is likely to survive. Most child deaths occur at this stage. In some cases, the child is taken to the health facility, often too late to be helped. This experience, which seems common, has created a strain between the health personnel and mothers, one accusing the other of negligence. The delay action of first-time mothers is interpreted as stemming from fear of rebuke by their husband, while third-time mothers being more confident, would rather wait to be sure it was not an ordinary illness. Third-time mothers are often older, more experienced, but also more entrenched in the local beliefs about malaria aetiology than the young mothers. Although they have more responsibilities, they are economically more independent and are able to take vital decisions with respect to their children with little reference to the husbands, not the case with the younger, inexperienced mothers. Unlike the younger ones, older and multigravid mothers are assisted by their older children who serve as child-minders and help with domestic and economic tasks. When drugs are used, the habit of sharing health resources in times of illness signifying solidarity contributes to non-compliance with management regulations. A mother stops administering drug as soon as the child seems to have improved in health and the remaining drugs are kept for another episode. The act of sharing has been exploited for the development of community management of childhood malaria in Uganda. The Bwatiye and other similar societies could benefit from this management approach.
The logical categorization of malaria as female (mild malaria) and male (severe), the interpretation of the class of ailment and its consequences, provide useful clues in the search for healing within the local milieu. Local consultants are useful allies in appropriately managing malaria or other febrile illnesses among the Bwatiye and perhaps other ethnic groups in north-eastern Nigeria.
Implications for health education
The Bwatiye have an all-pervading belief that enemies often use zazzabi to cover up their evil deeds until it is too late for the affected person to seek a potent cure. This belief may not be ignored when drafting health education material. During post-analysis discussions, the women saw the logic of promptly administering antimalarial drugs at the onset of any febrile illness as a means of eliminating malaria and exposing any underlying evil that may not be malaria. The belief that malaria is incapable of killing a child without enemy intervention is a challenge to health educationists in this and other parts of Africa, where such beliefs prevail. These findings raise a number of points about what is appropriate in health education provision and empowerment for community health-care delivery. Should the development of educational materials be based on promoting biomedical aetiological theory of disease and should we insist that others accept it first, or should we work within the traditional understanding of disease transmission, which includes concepts of evil and ill intent?
The health authorities need to consider working within cultural epidemiological parameters if the programme objective is to reduce morbidity and mortality due to malaria. Insistence on acceptance of biomedical models of disease transmission belongs to another goal that may not be directly linked to reduction in morbidity and mortality.
Conclusion
Until mothers are empowered with the capability to recognize and treat malaria, its impact on child mortality will continue unabated. The need is to educate mothers to suspect malaria first in every case of febrile illness and take appropriate action to rid the child of malaria to expose the underlying "evil". This is a more acceptable form of education than an insistence on replacing local knowledge with biological epidemiology of malaria. The challenge before health personnel is to identify and exploit local beliefs about aetiology in effecting management procedures among culturally different peoples irrespective of their acceptance of biological models of disease transmission.
Authors' contributions
OBA conceived, designed, coordinated the implementation of the protocol, analysed and developed the manuscript while KKJ led the data collection team.
Acknowledgements
We are grateful to all those who participated in the study especially the mothers and their children who enthusiastically shared their knowledge and skills with the team. We appreciate the cooperation of the community leaders and health workers. We are grateful to Dr Niyi Oriolowo of Peace Hospital for the use their laboratories. This study was supported by Common Heritage Foundation for community development and education.
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| 15723706 | PMC553996 | CC BY | 2021-01-04 16:37:31 | no | Malar J. 2005 Feb 21; 4:13 | utf-8 | Malar J | 2,005 | 10.1186/1475-2875-4-13 | oa_comm |
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Cardiovasc UltrasoundCardiovascular Ultrasound1476-7120BioMed Central London 1476-7120-3-51573724210.1186/1476-7120-3-5Case ReportPreventing complicated transseptal puncture with intracardiac echocardiography: case report Shalganov Tchavdar Nikolov [email protected] Dora [email protected]ás Sarolta [email protected]ári András [email protected]örök Tamás [email protected] Gottsegen György Hungarian Institute of Cardiology Haller utca 29, H-1096, Budapest, Hungary2005 1 3 2005 3 5 5 31 1 2005 1 3 2005 Copyright © 2005 Shalganov et al; licensee BioMed Central Ltd.2005Shalganov et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Recently, intracardiac echocardiography emerged as a useful tool in the electrophysiology laboratories for guiding transseptal left heart catheterizations, for avoiding thromboembolic and mechanical complications and assessing the ablation lesions characteristics. Although the value of ICE is well known, it is not a universal tool for achieving uncomplicated access to the left atrium. We present a case in which ICE led to interruption of a transseptal procedure because several risk factors for mechanical complications were revealed.
Case presentation
A case of a patient with paroxysmal atrial fibrillation and atrial flutter, and distorted intracardiac anatomy is presented. Intracardiac echocardiography showed a small oval fossa abouting to an enlarged aorta anteriorly. A very small distance from the interatrial septum to the left atrial free wall was seen. The latter two conditions were predisposing to a complicated transseptal puncture. According to fluoroscopy the transseptal needle had a correct position, but the intracardiac echo image showed that it was actually pointing towards the aortic root and most importantly, that it was virtually impossible to stabilize it in the fossa itself. Based on intracardiac echo findings a decision was made to limit the procedure only to ablation of the cavotricuspid isthmus and not to proceed further so as to avoid complications.
Conclusion
This case report illustrates the usefulness of the intracardiac echocardiography in preventing serious or even fatal complications in transseptal procedures when the cardiac anatomy is unusual or distorted. It also helps to understand the possible mechanisms of mechanical complications in cases where fluoroscopic images are apparently normal.
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Background
Since the advent of ICE in the electrophysiology practice it proved its value in guiding transseptal procedures with providing an extra safety margin for the patients. The possibility to visualize the oval fossa, the LA free wall and the aortic root helps in preventing mechanical complications. ICE can visualize also intracardiac thrombus and spontaneous echocontrast, which is helpful in avoiding thromboembolic complications. Although the value of ICE is well known, it is rather hard to admit that it is a universal tool for achieving uncomplicated access to the left atrium. The aim of this case presentation is to show that ICE can lead to interruption of a transseptal procedure due to the presence of risk factors for mechanical complications when the fluoroscopic image is seemingly satisfying.
Case presentation
A seventy-year-old male patient with paroxysmal atrial fibrillation and atrial flutter, and concomitant arterial hypertension was referred to our institution for LA circumferential ablation. The preprocedural TEE described an aneurysm of the IAS (See Additional file 1: TEE.mpeg for the TEE), however the left heart catheterization was deemed feasible. ICE performed at the electrophysiology laboratory (catheter Ultra ICE 9900, 9 MHz, EP Technologies, Boston Scientific Corp., San Jose, CA, USA connected to a console ClearView Ultra, Boston Scientific Corp., San Jose, CA, USA) as a routine part of the transseptal puncture showed more detailed picture of the intracardiac anatomy – dilated aortic root, enlarged right atrium displacing the IAS towards the LA free wall, and small oval fossa with flapping motion of the fossa ovalis membrane. The distance between the IAS and the LA free wall during the atrial diastole was small (Fig. 1). During the atrial contraction the LA cavity appeared almost obliterated and the fossa ovalis membrane virtually touched the LA free wall, thus making the transseptal puncture potentially dangerous (Fig. 2) (See Additional file 2: ICE1.mpeg for the unusual intracardiac anatomy). The fossa itself was abouting anteriorly to the non-coronary aortic sinus of Valsalva and although the fluoroscopic position of the transseptal needle was apparently correct, ICE showed that it was always sliding to the junction between the IAS and the aorta and that the needle tip was pointing towards the aortic root. Several unsuccessful attempts were made to change its direction towards the oval fossa. That was also deemed a condition predisposing to complicated transseptal puncture (Fig. 3) (See Additional file 3: ICE2.mpeg for the sliding of the transseptal needle towards the aorta). During the procedure two echocardiographers, including the one, which had performed the TEE, re-evaluated the videotaped TEE and agreement was achieved that the initial interpretation of the TEE needed correction. Although the patient was symptomatic and the arrhythmia episodes could not be suppressed effectively with antiarrhythmic drugs, after considering all pros and cons we decided not to perform the left atrial procedure and thus to avoid serious and potentially fatal complications. Only a cavotricuspid isthmus ablation was done.
Figure 1 ICE during atrial diastole before the jump of the transseptal needle into the oval fossa. In this patient the LA cavity has a crescent-like shape at this time point of the cardiac cycle. Ao – non-coronary sinus of the aorta; CT – terminal crest; FO – oval fossa; LA – left atrium; LAFW – left atrial free wall; RA – right atrium; TSN – transseptal needle.
Figure 2 ICE during atrial systole before the jump of the transseptal needle into the oval fossa. The LA cavity is virtually missing at this time point of the cardiac cycle. Ao – non-coronary sinus of the aorta; CT – terminal crest; FO – oval fossa; RA – right atrium; RV – right ventricle.
Figure 3 ICE after the jump of the transseptal needle into the oval fossa. The needle tip points towards the aorta. Ao – non-coronary sinus of the aorta; LA – left atrium; RA – right atrium; TSN – transseptal needle.
Discussion
In the last years ICE emerged as a useful adjunctive tool in the field of interventional electrophysiology. It serves not only scientific purposes but practical issues as well. Its value for achieving successful and uncomplicated transseptal access to the LA cavity is well known [1-4]. However, it has not been used consistently for elucidating the possible mechanisms of mechanical complications during transseptal left heart procedures.
ICE for transseptal puncture
In this case ICE showed that the aneurysm of the IAS observed during the TEE was actually the angulated continuity between the enlarged aortic root and the IAS. The echocardiographic orientations of ICE are sometimes clearly off axis in comparison to standard transesophageal echocardiographic views. Nevertheless, to our opinion ICE is superior in providing more detailed picture of the neighboring cardiac structures. Although useful in guiding transseptal catheterizations, TEE does not always provide complete avoidance of complications even in patients with normal hearts [5,6]. Furthermore, the oval fossa itself was small but its membrane nevertheless showed bidirectional flapping motion. This suggests that ICE and TEE images are indeed no equivalent. As the fossa was abouting the aortic root this flapping motion was located in the angle between the two structures and had probably given rise to the false impression of an aneurysm. Additionally, very small distance between the oval fossa and the LA free wall was also observed. In such circumstances the risk of puncturing the left atrial free wall is prominent even if the puncture of the oval fossa itself is uncomplicated [2,3]. Usually ICE-guided redirection of the transseptal needle is all that is needed for achieving uncomplicated access to the LA in such cases. In the case presented there was virtually no distance between the IAS and the LA free wall during the atrial systole, so such a maneuver would not be of help. Also it was not possible to achieve a stable position of the transseptal needle in the fossa itself. It always slided and pointed to the aorta. This means that even if the distance from the oval fossa to the LA free wall was large enough the puncture would be complicated. To our opinion this is one possible explanation for the rare instances of inadvertent puncturing of the aorta when the fluoroscopic images appear to be completely satisfying.
ICE for avoidance of other complications
EP procedures are relatively safe procedures and have low complication rate. One of the most frequent complications is related to cardiac wall perforation with consequent pericardial effusion and tamponade. ICE, especially the one with phased-array transducer (deeper penetration) allows continuous monitoring of the pericardial space during EP procedures. This permits prompt detection of a pericardial effusion and an immediate guidance of a therapeutic puncture. Phased-array transducers are equipped with Doppler capabilities allowing assessment of the pulmonary venous flow pattern after pulmonary vein ablation on top of diameter measurements to exclude pulmonary vein stenosis, which is the most important complication of this procedure. Monitoring microbubble formation during RF energy application allows prevention of pulmonary vein stenosis [7]. ICE also allows detection of intracardiac thrombi during the procedures, especially during left-sided ablations [8].
Conclusion
We strongly believe that this case is a good illustration of the usefulness of the ICE in the electrophysiology field and further enhances its value as a tool for avoiding complications when the intracardiac anatomy is unusual or distorted. As the number of transseptal procedures in the electrophysiology laboratories all over the world is steeply growing ICE definitely has the potential to become a routine at least in those institutions with large volume of left atrial procedures.
List of abbreviations
EP – electrophysiologic
IAS – interatrial septum
ICE – intracardiac echocardiography
LA – left atrium; left atrial
TEE – transesophageal echocardiography
Supplementary Material
Additional File 1
Preprocedural transesophageal echocardiography. The irregular oval-shaped structure at the center of the screen is the aortic root. At a certain moment one can see at its upper part the ostium and the most proximal part of the right coronary artery. Below is situated the left atrium and to the left – the right atrium. The oval fossa is in between.
Click here for file
Additional File 2
Intracardiac echocardiography, showing distorted intracardiac anatomy. The oval shape in the center of the screen is the non-coronary sinus of Valsalva. Below is the right atrium at the bottom of which the transseptal needle is clearly visible. The prominent muscular structure in the left-hand part of the image is the terminal crest. The membrane of the oval fossa, adjacent to the right-hand part of the non-coronary aortic sinus shows bidirectional flapping motion. During the atrial contraction the cavity of the left atrium virtually disappears.
Click here for file
Additional File 3
Intracardiac echocardiography showing sliding of the needle towards the aorta. The transseptal needle is already in the oval fossa with its tip pointing to the aorta. This is especially clearly visible after a premature beat.
Click here for file
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| 15737242 | PMC553997 | CC BY | 2021-01-04 16:38:31 | no | Cardiovasc Ultrasound. 2005 Mar 1; 3:5 | utf-8 | Cardiovasc Ultrasound | 2,005 | 10.1186/1476-7120-3-5 | oa_comm |
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Immun AgeingImmunity & ageing : I & A1742-4933BioMed Central London 1742-4933-2-41571591210.1186/1742-4933-2-4ResearchDirect analysis of thymic function in children with Down's syndrome Prada Nicole [email protected] Milena [email protected] Leonarda [email protected] Erika [email protected] Marcello [email protected] Elisa [email protected] Enrico [email protected] Roberta [email protected] Luigi [email protected] Davide [email protected] Ornella [email protected] Milena [email protected] Cristina [email protected] Liviana [email protected] Elisabetta [email protected] Mauro [email protected] Ugo [email protected] Fiorella [email protected] Andrea [email protected] Dipartimento di Biopatologia e Metodologie Biomediche, Università di Palermo, via Tukory 211, 90134 Palermo, Italy2 Cattedra di Immunologia, Dipartimento di Scienze Biomediche, Università di Modena e Reggio Emilia, via Campi 287, 41100 Modena, Italy3 Clinica Odontoiatrica, Università di Modena e Reggio Emilia, via del Pozzo 71, 41100 Modena, Italy4 Servizio di Neuropsichiatria Infantile, AUSL Modena, via Cardarelli 45, 41100 Modena, Italy5 Clinica Pediatrica, Università di Modena e Reggio Emilia, via del Pozzo 71, 41100 Modena, Italy2005 16 2 2005 2 4 4 5 2 2005 16 2 2005 Copyright © 2005 Prada et al; licensee BioMed Central Ltd.2005Prada et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Down's syndrome (DS) is characterized by several immunological defects, especially regarding T cell compartment. DS is considered the best example of accelerated ageing in humans. Direct observations of the thymus have shown that in DS this organ undergoes severe histological and morphological changes. However, no data on its capacity to generate T cells are present in the literature. Here, using a new technology based upon real time PCR, we have investigated the capacity of the thymus to produce and release newly generated T lymphocytes (the so called "recent thymic emigrants", RTE) in children with DS.
Methods
We studied 8 children affected by DS, aged 2–7 years, compared with 8 age- and sex-matched healthy controls. Flow cytometry was used to determine different lymphocytes subsets. Real time PCR with the Taqman system was used to quantify the amount of RTE, i.e. peripheral blood lymphocytes that express the T cell receptor rearrangement excision circles (TREC).
Results
In comparison with control children, those with DS had a significant lower number of TREC+ peripheral blood cells. Moreover, in DS children but not in controls, a strong negative correlation between age and the levels of TREC+ cells was found.
Conclusions
The direct measure of thymic output indicates that the impairment of the organ results in a reduced production of newly generated T cells. This observation could suggest that cytokines able to modulate thymic function, such as interleukins, could be useful to improve the functionality of the organ and to treat the immunodeficiency present in DS subjects.
Down's syndromethymusTRECT lymphocytes
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Introduction
Down's Syndrome (DS) is the most common chromosomal abnormality in humans, that occurs in 1 out of every 800–1,000 births. It is an autosomal disorder resulting by triplication of chromosome 21. Many characteristics are commonly seen in DS, including some degree of intellectual impairment, that varies widely from individual to individual, heart defects, hypotonia, hyperuricemia, and the development of Alzheimer disease-type neuropathology beginning at about 40 years of age [1].
DS subjects also present alterations of the immune system which are similar to those of aged people, including increased susceptibility to bacterial and viral infection and to the onset of different types of haematological malignancies, along with a high frequency of autoantibodies. Alterations of B lymphocytes, T cell subsets, and natural killer cells, defective phagocytosis and chemotaxis of polymorphonuclear leukocytes and interleukin-2 production by activated T cells were also reported [2-7]. Quantitative studies of peripheral blood T lymphocytes reveal a reduction, often quite small, in the percentage and/or absolute number of T lymphocytes, although normal proportions or numbers of T and B lymphocytes in DS children have also been reported [8].
Several studies have focused their attention on the role of the thymus, and have described a variety of structural and anatomic alterations present in DS [9]. Few data, if any, exist on the direct measure of thymic functionality in terms of production of newly generated T cells. Accordingly, the aim of this study was to measure the capability of the thymus of DS children to produce new T lymphocytes, and to analyze how such capability changes with age. For this reason, we have quantified the so called "recent thymic emigrants" (RTE), that are the main contributors to the naïve T-cell pool, and are characterized by the presence in the nucleus of a circular, episomal DNA molecule called TREC (T cell receptor Rearrangement Excision Circles), generated during the intrathymic rearrangement of the α-chain locus of the T cell receptor (TCR).
Genes for the δ-chain of the TCR are distributed within the genomic region that codifies for the α-chain, and are removed in two steps during the recombination of Vα with Jα. Thus, a thymocyte that starts to rearrange the α-chain produces the first TREC, called signal-joint (sj)-TREC, then proliferates three or four times, and finally completes the rearrangement of Vα with Jα, so producing the second TREC, called coding-joint (cj)-TREC. The removal of genes for the δ-chain from the α region does not imply their elimination, as such DNA remains into the nucleus as a circle that is not able to replicate. As a consequence, when a cell undergoes a division, TREC are passed only to one of the two daughter cells. During the following cell cycles, TREC are then diluted into the population that origins from the first cell. Several data indicated that the percentage of TREC+ cells is a marker of thymic activity, that TREC+ cells are almost present within the subset of virgin T lymphocytes, and that their number consistently declines with age [10].
Materials and Methods
Subjects
We could analyze blood samples from 8 children with DS (with a mean age of 4.62 years, range: 2–7 years), 7 males and 1 females. As control group, we studied 8 children with a mean age of 4.75 years (range, 2–8 years), 7 males and 1 female. All subjects were in good conditions, and had no acute or chronic disease affecting the immune system. Written informed consent was obtained by their parents, according to the Italian laws.
Isolation of PBMC from blood
Peripheral blood mononuclear cells (PBMC) were separated from a minimum of 8 mL freshly collected blood according to standard procedures.
Analysis of the phenotype of peripheral blood lymphocytes
Blood samples were stained with different monoclonal antibodies for the cytometric analysis, as described [11]. The quantification of the main subpopulations present among lymphocytes (electronically selected on the basis of an electronic gate put in the region of lymphocytes) was performed by flow cytometry using a CyFlow ML instrument (Partec, Münster, Germany), according to standard procedures [11].
DNA extraction
DNA was extracted from 5 × 106 PBMC using the QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer's protocols and stored in sterile water at -20°C until use.
Quantification of percentage of sjTREC positive PBMC by a Real Time PCR approach
The percentage of PBMC containing sjTREC was measured by an original method that has been recently developed (patent pending) using a Real Time PCR approach. This assay was performed by two parallel polymerase chain reactions (PCR), that quantify sjTREC or nuclear DNA (nDNA) in a given sample, carried out in two different reaction tubes, but in the same plate, in order to have similar reaction conditions.
In the first reaction, we quantified sjTREC using a mix that consisted of: Supermix Biorad 1X, primers for sjTREC 500 pmol and we added 3 μl of the sample in each tube. The sjTREC primers we used were: sjTREC Dir (5'-CAC ATC CCT TTC AAC CAT GCT-3') and sjTREC Rev (5'-GCC AGC TGC AGG GTT TAG G-3'). TaqMan probe for sjTREC (5'-FAM-ACA CCT CTG GTT TTT GTA AAG GTG CCC ACT – BLACK HOLE-3') was included in the reaction mixture at the concentration of 200 nM, as a real time detector for the amplified product. One cycle of denaturation (95°C for 6 min) was performed, followed by 50 cycles of amplification (95°C for 30 sec, 58°C for 1 min 30 sec). The same approach was used to quantify nDNA, which was required to obtain the number of cells present in the sample. In this case, primers GenDir (5'-GGC TCT GTG AGG GAT ATA AAG ACA-3') and GenRev (5'-CCA ACC ACC CGA GCA ACT AAT CT-3'), designed on FasL gene sequence, present in two copies in the human genome (one in each chromosome 1), were used at the concentration of 600 and 400 nM respectively, and the TaqMan probe GenProbe (5' TexasRed – CTG TTC CGT TTC CTG CCG GTG C – BlackHole Quencher2 3') was included in the reaction mixture at the concentration of 300 nM. One cycle of denaturation (95°C for 3 min 30 sec) was performed, followed by 45 cycles of amplification (95°C for 30 sec, 60°C for 35 sec).
All of the aforementioned primers and probes have been designed using the program "primer3", available on the internet address: . The length of amplified fragments is 104 pb for nDNA and 101 pb for sjTREC.
PCR was performed by using an iCycler Thermal cycler (BioRad, Hercules, CA, USA), that monitors changes in the fluorescence spectrum of each reaction tube during the annealing phase. The fluorescence signal was processed using the "Real-time detection system iCycler iQ" software, that calculates the threshold and the threshold cycles of each sample. All reactions were carried out in triplicate.
To optimize the precision of the assay, whose results derive from the comparison between the threshold cycles of two different real time PCR reactions, we have developed a new approach. Indeed, the regions used as template for the two amplifications (i.e., those of sjTREC and nDNA) were purified and cloned tail to tail in a vector (pGEM-11Z, from Promega), to have a ratio of 1:1 of the molecules used as reference.
The nDNA region used has been excised as a SacI-ApaI fragment and cloned in the pGEMT easy vector (Promega), obtaining the pGEM11Z-nDNA vector. The sjTREC region used has been excised as a PstI-SacI fragment and cloned in the same vector, obtaining the pSJ vector we used. The plasmid has been purified, quantified with the spetrofotometer and linearized using SacI. Then a series of reactions has been performed for nDNA and sjTREC with serial dilution of the standard. All the times the threshold cycles of nDNA and sjTREC were the same, proving the ratio 1:1 of the two fragments.
Then, serial known dilutions of this vector, amplified in triplicate, were included in each PCR run to generate a standard curve from which the relative copy number of either sjTREC or nDNA present in the unknown samples was determined. The measured values for sjTREC and nDNA were always within the range of the standard curve, whose correlation coefficient was always >0.990. The values of sjTREC and nDNA present in each sample were calculated using the mean of the threshold cycles of the three replicates.
Then, the percentage of PBMC containing sjTREC in each sample was simply obtained from the ratio between the relative values of sjTREC (obtained dividing for 3 the result given by the iCycler, as we used 3 μl of DNA of the sample and 1 μl of standard to quantify sjTREC) and nDNA obtained (obtained versus the same vector), multiplied by 2 (as two copies of the nuclear gene are present in a cell).
Results
Peripheral blood cell phenotype in DS
Down's syndrome is characterized by severe immunological alterations, which mainly affect the T cell compartment and are often regarded as signs of accelerated ageing. Numerous reports suggested that thymic retention of T cells or maturation defects might be the cause of the observed alterations in the T cell compartment. Accordingly, we first checked the presence of possible changes in the phenotype of peripheral blood lymphocytes from DS children. As shown in Figure 1, we found that, in comparison with karyotypically normal healthy controls, DS children had less T helper cells and more cytotoxic T lymphocytes or cells expressing markers related to NK activity. This indicates that the population we analyzed can well represent the immunological situation present in DS children.
Figure 1 Phenotypic analysis of peripheral blood lymphocytes in patients with Down's syndrome (DS) and healthy subjects. Data are referred to 8 individuals per group. Asterisk indicates a statistically significant difference (p < 0.05).
Quantitative analysis of cells expressing TREC
We then studied directly the capacity of the thymus to produce new T lymphocytes, by the analysis of TREC+ cells. Figure 2 shows a typical example of real time PCR assay for quantification of the amount of TREC per cell. It is to note that the threshold cycle, i.e. the cycle of PCR in which the fluorescent signal deriving from the amplification of the DNA becomes evident, is quite different in DS children and controls. Indeed, such cycle is much lower in control children, indicating the presence of a higher number of TREC.
Figure 2 Representative example of real time PCR for the quantification of TREC in a group of 3 children with Down's syndrome (DS) and in 4 healthy controls (each measure is performed in triplicate). Note how the threshold cycle is different in the two groups.
The percentage of TREC+ lymphocytes in children affected by Down's syndrome and in controls is reported in Figure 3. A statistically significant difference was present between the two groups (p = 0.007), indicating that DS is characterized by a lower thymic output.
Figure 3 DS children have less TREC+ lymphocytes than healthy controls, as shown in this box-and-whiskers graphics. The boxes extend from the 25th percentile (x[25]) to the 75th percentile (x[75]) [i.e., the interquartile range (IQ)]; lines inside boxes represent median values. Lines emerging from boxes (i.e., the whiskers) extend to the upper and lower adjacent values. The upper adjacent value is defined as the largest data point ≤x[75]+1.5xIQ, and the lower adjacent value is defined as the smallest data point ≥x[25]-1.5xIQ. Note that no outliers are present in the two groups.
Correlation between age and TREC
Finally, we have investigated the correlation between age and TREC levels in the two groups. As reported in Figure 4, it is noteworthy that while control children did not display any age-related change (in the age range 2–8 years), those with DS had a significant age-related decrease in the number of TREC+ cells. This indicates that in DS, in contrast with control children, major changes in thymic output occur in the very first years of life.
Figure 4 Correlation between age and TREC levels in DS (squares) and control (triangles) children. In the year range 2–8, the correlation was significant in DS but not in control children.
Discussion
Immunological ageing is part of a continuum of developmental processes, encompassing complex reorganizational events, compensatory mechanisms and qualitative alterations in the functionality of several systems and organs. Among those organs that undergo major changes with ageing, thymus plays a special role [12]. The thymus is a central lymphoid organ that is the primary site of T-cell maturation and development. Shortly after birth, the thymus undergoes a life long process of involution whereby the organ is replaced by adipose tissue. The result is a reduction in the number of constituent thymocytes with age, a consequent shrinking of the thymus and a decline in the output of newly generated T lymphocytes [13].
Aged peripheral T-cell pool is characterized by the accumulation of T-cell capable of limited replication [14]. Since an efficient immune response is based upon the expansion of antigen-specific clones, the consequence of a qualitative and quantitative impairment of the system is an increased susceptibility to infections or cancer. Several groups, including ours, have studied the immune system during human ageing, using different models, including the one that represents the best example of successful ageing, i.e. healthy centenarians, and conditions of accelerated ageing, such as DS [2-6,15].
In DS subjects, the proportion of T-helper cells is decreased, resulting in a decreased ratio of helper/cytotoxic cells, and we could observe this phenomenon also in the patients here described. Furthermore, peripheral blood T cells have a decreased number of cells expressing the T-cell receptor-αβ (TCRαβ) complex, elevated numbers expressing TCRγδ and a decreased proportion of CD4+, CD45RA+ naïve T cells, along with a high number of cells with NK phenotype, suggesting that the DS thymus is inefficient in the release of functionally mature T cells [16]. An age-dependence in the proliferative response to phytohemagglutinin (PHA) of DS lymphocytes, but not of lymphocytes from healthy individuals, was observed. Allogeneic mixed lymphocyte proliferative responses are decreased, as are PHA-induced interleukin-2 production and cytotoxic T-lymphocyte activity [7].
All of the above mentioned alterations have a common substrate, i.e. the dysfunction of thymus. Indeed, defects in the capacity to produce thymic hormones has been described several years ago [17,18]. Furthermore, anatomic studies gave further evidence that changes in the T-lymphocyte system derive from structural abnormalities of the thymus. In comparison to age-matched controls, thymuses from infants with DS from 1 day to 15 months of age have marked lymphoid depletion, with a thin cortex and poor corticomedullary demarcation. The Hassall corpuscles are increased in size and frequently cystic. The presence of lower proportions of cells bearing high levels of the TCR-αβ complex and of CD3, a signal-transducing complex for the TCR, in thymuses of children with DS and the increase in the proportions of cells with these markers with age are indicative of delayed maturation of T cells within the thymus [9]. In addition, DS thymuses contain elevated levels of IFN-γ and TNF-α mRNA expressing cells, and there is mast cell hyperplasia and overexpression of class I MHC, CD18 and ICAM-1 [19]. DS thymocytes also have a greater than normal sensitivity to inhibition of IL-4-induced proliferation by IFN-γ and TNF-α [20]. Taken together, these findings indicate an abnormal thymocyte maturation and cytokine dysregulation in the DS thymus, possibly initiated by gene dose-related increased sensitivity to IFN-γ and to overexpression of CD18 (LFA-1β).
In conclusion, in this paper we show that the aforementioned alterations of the thymus result in the reduced production and output of newly generated lymphocytes, that can be directly measured by the assay we have developed and used. The quantitative analysis of TREC+ cells in the periphery is a relatively new and sensitive marker of thymic functionality, which is able to provide information on the status of the organ in different pathological conditions, and on its capability to generate new T cells [21-25]. A markedly reduced capacity of the thymus to produce RTE is present in the DS children we have studied. Such observation, if confirmed in a higher number of cases, could be useful to develop novel strategies to treat the immunodeficiency typical of this syndrome, based for example on the use of cytokines such as interleukin-2 or interleukin-7, which is capable of maintaining or restoring an efficient thymic output [26].
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| 15715912 | PMC553998 | CC BY | 2021-01-04 16:36:31 | no | Immun Ageing. 2005 Feb 16; 2:4 | utf-8 | Immun Ageing | 2,005 | 10.1186/1742-4933-2-4 | oa_comm |
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J Neuroengineering RehabilJournal of NeuroEngineering and Rehabilitation1743-0003BioMed Central London 1743-0003-2-31573724110.1186/1743-0003-2-3ResearchReduction of motion artifact in pulse oximetry by smoothed pseudo Wigner-Ville distribution Yan Yong-sheng [email protected] Carmen CY [email protected] Yuan-ting [email protected] Joint Research Center for Biomedical Engineering, The Chinese University of Hong Kong, Shatin, Hong Kong2005 1 3 2005 2 3 3 20 1 2005 1 3 2005 Copyright © 2005 Yan et al; licensee BioMed Central Ltd.2005Yan et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The pulse oximeter, a medical device capable of measuring blood oxygen saturation (SpO2), has been shown to be a valuable device for monitoring patients in critical conditions. In order to incorporate the technique into a wearable device which can be used in ambulatory settings, the influence of motion artifacts on the estimated SpO2 must be reduced. This study investigates the use of the smoothed psuedo Wigner-Ville distribution (SPWVD) for the reduction of motion artifacts affecting pulse oximetry.
Methods
The SPWVD approach is compared with two techniques currently used in this field, i.e. the weighted moving average (WMA) and the fast Fourier transform (FFT) approaches. SpO2 and pulse rate were estimated from a photoplethysmographic (PPG) signal recorded when subject is in a resting position as well as in the act of performing four types of motions: horizontal and vertical movements of the hand, and bending and pressing motions of the finger. For each condition, 24 sets of PPG signals collected from 6 subjects, each of 30 seconds, were studied with reference to the PPG signal recorded simultaneously from the subject's other hand, which was stationary at all times.
Results and Discussion
The SPWVD approach shows significant improvement (p < 0.05), as compared to traditional approaches, when subjects bend their finger or press their finger against the sensor. In addition, the SPWVD approach also reduces the mean absolute pulse rate error significantly (p < 0.05) from 16.4 bpm and 11.2 bpm for the WMA and FFT approaches, respectively, to 5.62 bpm.
Conclusion
The results suggested that the SPWVD approach could potentially be used to reduce motion artifact on wearable pulse oximeters.
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Introduction
Wearable medical devices are capable of continuously monitoring an individual's vital signs in real time. These devices are particularly important to the world's increasingly aging population, whose health conditions have to be assessed regularly or monitored continuously. The devices can warn individuals of symptoms of deterioration, e.g. alerting them when their blood pressure is increasing to a level above a predetermined threshold. The devices can also automatically notify emergency services in critical situations. In order to make wearable devices practical, a series of technical problems have to be solved. For example, these devices need to be miniature in size, must possess a user-friendly interface and be efficient in power consumption. Most importantly, these devices need to have a low failure rate and must report minimal false alarms. In other words, these devices are required to provide an accurate estimate of the monitored vital sign under normal daily life situations. This leads to the important topic on the reduction of motion artifacts [1-4]. In this paper, the smoothed pseudo Wigner-Ville distribution (SPWVD) is investigated as a novel motion artifacts resistant approach for estimating one of the most important vital signs – the blood oxygen saturation level (SpO2).
The paper is organized as follows. Section 2 reviews the techniques commonly used for attenuating motion artifacts in pulse oximetry. Section 3 discusses the basic theory for SpO2 computation and the techniques used in this study for reducing motion artifacts. Section 4 compares the performance of two time-frequency techniques, i.e. the short-time Fourier transform (STFT) and the SPWVD. Section 5 presents the protocol and the results of an experiment to assess motion artifact reduction in real data. Section 6 discusses the performance of the SPWVD approach as compared to the traditional time domain and spectral methods. Lastly, the major findings of this paper are summarized in section 7.
Background
SpO2 is commonly monitored by a pulse oximeter, which has been widely adopted as a standard measure during anesthesia, neonatal care and post-operative recovery [5,6]. Pulse oximeters currently available on the market normally perform remarkably well when the monitored subject is in the resting position. However, their reliability is significantly reduced when the subject moves, even when movements are only involuntary, such as shivering [1-4,7]. Therefore, the reduction of motion artifacts is of particular concern in the development of pulse oximeters to be applied in ambulatory, pediatric and trauma settings, as well as for implementing them into wearable devices for personal home healthcare [8].
A number of attempts have been made in the past decade to improve the accuracy of pulse oximeters when subjects move. Typical methods can be generally classified into three categories: (1) based on an independent measure of motion; (2) based on a model of the ideal signal or the noise; and (3) based on features recognized from the corrupted signal. For techniques based on an independent measure of motion, one or more transducers (e.g. piezo or optical sensors) are employed to record the user's motion. By assuming that the artifact is a linear addition to the pulsatile photoplethysmographic (PPG) signal, the original signal can be reconstructed from the corrupted signal [9-11]. This hypothesis is however often doubted when inspecting PPG signals under typical artifact-producing forces [12]. This observation drives researchers to develop more realistic models for the PPG signal or the artifact.
A recently proposed PPG artifact reduction methodology was based on the inversion of a nonlinear physical artifact model and could significantly reduce the effect of changes of probe coupling [8,12]. However, model-based techniques suffer inherently from the specificity of the model design and are unable to cope with all aspects of real-life scenarios.
On the other hand, techniques based on feature recognition are free of the generic problem of model designs. Instead, these techniques often utilize some predetermined criteria to separate regions of corrupted and uncorrupted PPG signal and estimate the desired parameters from the uncorrupted portion of it. For example, Swedlow et al. calculated the derivative of a signal and identified a portion of it as a motion artifact whenever the ratio of adjacent positive and negative peaks of the derivative is below a threshold [13]. J.E. Scharf et al. evaluated the use of spectral analysis to separate the cardiac physiologic components from the recorded PPG signal that is contaminated by motion artifact for SpO2 estimation [14-16].
The above methodologies employ techniques in the time domain or frequency domain. However, due to the nonstationary nature of PPG signals, the use of time-frequency analysis appears to be extremely attractive. Dowla et al. proposed using a neural network together with a wavelet transform (WT) to estimate SpO2 in the presence of a motion artifact, and found out that this technique performs better than conventional algorithm that detects peaks and troughs of the PPG signal for estimating SpO2 levels [17]. In their method, a neural network was trained to identify the motion level, which was then fed into a second neural network together with the amplitude ratios at different scales of WT of the PPG signal to estimate SpO2 levels. It has been pointed out by another researcher [16] that using WT for SpO2 computation requires careful analysis and additional testing. WT does not result in a spectrum where the amplitude of a unique cardiac frequency can be directly obtained for SpO2 estimation. On the other hand, although such a unique component is available on the spectrum obtained from fast Fourier transform (FFT), the time-frequency resolution of FFT or STFT is relatively low when compared to other time-frequency techniques such as the Wigner-Ville distribution. The goal of this study is to investigate the use of SPWVD, a high resolution time-frequency transformation where the amplitude of a unique cardiac frequency is apparent, for the estimation of SpO2 levels.
Methods
Basic theory
The traditional algorithms for estimating SpO2 detect peaks and troughs of the PPG signal in the time domain. Based on the Beer-Lambert law, which relates the optical path length and effective absorbance to the intensity of transmitted light, the relationship between intensity of transmitted light and SpO2 is commonly described as:
I (λ, t) = I0 (λ) exp[(-sεHbO2 (λ) + (1 - s)εHb (λ))·c·d (t)], (1)
where, εHbO2 and εHb are the extinction coefficients of oxygenated and de-oxygenated hemoglobin, and s, c, and d represent SpO2, total concentration of hemoglobin and the optical path length respectively.
By using two light sources – red and infrared lights – and calculating a normalized ratio of the AC component to the DC component for each light source, SpO2 can be computed from the ratio of ratios R, i.e. the normalized ratio of the red to the infrared transmitted light intensity. That is,
In practice, SpO2 can be obtained from equation (3) directly or by an empirical equation that relates SpO2 and R. In this study, SpO2 is estimated directly from equation (3).
SpO2 computation by weighted moving average (WMA)
By calculating the ratio of the AC components and the ratio of the DC components of the two light sources, SpO2 can be obtained from every single pulse of a PPG signal. To stabilize the reading, the weighted moving average (WMA) is often used [18]. Typical averaging methods, e.g. the median averaging and standard arithmetic averaging, are applied to every several samples or samples in every several-second intervals. In this study, the SpO2 obtained by the WMA approach was the average of SpO2 samples in an 8-second period. Overlap processing was performed at 1-second interval. The 8-second period is selected in order to satisfy clinical requirements [15,16].
SpO2 computation by fast Fourier transform (FFT)
Based on the hypothesis that cardiac rate can be estimated more easily by spectral analysis than time domain analysis, techniques in the frequency domain have been widely investigated as alternatives in pulse oximetry. For example, the FFT and discrete cosine transform (DCT) were proposed for estimating SpO2 [14-16]. These techniques calculate the spectrogram of the PPG signal in a fixed time period and select the strongest spectral line in the cardiac frequency band as the AC component. The cardiac frequency band is usually predetermined by certain thresholds or obtained from an independent pulse rate estimator, e.g. by applying electrocardiography in parallel. In this study, FFT is applied to every 8-second PPG signal at 1-second interval. The cardiac frequency band is predetermined as 0.8–2 Hz, i.e. corresponding to 48–120 bpm.
SpO2 computation by SPWVD
The Wigner-Ville distribution (WVD) of a signal x(t) is given as:
where x(t) and x*(t) are the time series of the signal and its complex conjugate respectively.
The problem of the WVD is the so-called cross-term interference, which appears as frequencies that lie between the frequencies of any two strong components. In order to suppress cross-term interference, the smoothed pseudo WVD is often used:
The two windowing operations h and g are equivalent to smoothing the WVD in the frequency and time domain respectively. Selection of the window is a compromise between the joint time-frequency resolution and the level of cross-term interference. Common choices of window include the rectangular and Kaiser windows [19-21]. In our experiment, we chose the Hamming window as both the time and frequency smoothing windows, g(t) and h(τ).
The maximum magnitude within the cardiac frequency band of the SPWVD in each second was used for SpO2 computation. Since SPWVD represents energy distribution, the square root of the magnitude was used for calculating the ratio of ratios R and SpO2. Moreover, as the rate of change of SpO2 is relatively slow, SpO2 that changed by more than 2% per second was considered to be physiologically impossible, and was rejected from the calculation [22].
Simulation: STFT spectrogram versus SPWVD
The performance of SpO2 computation by the SPWVD approach was evaluated in a simulation using PPG signals collected from a volunteer under modest random motions. For comparison, SpO2 was also estimated from the spectrogram obtained by STFT.
The noise-mixing-composition (NMC) method was applied to mimic the clinical situation and to induce a range of signal-to-noise ratios (SNR) [23]. To synthesize a noise-contaminated signal, an artifact noise that has been verified to be similar to the real noise was added to an undisturbed basis signal. The synthesized signal and SNR are formulated as:
with the basis episode S and artifact episode N and a mix parameter ε, which was adjusted to achieve the desired SNR.
512 samples of the PPG signal were recorded at 20 Hz from a volunteer. The signal was contaminated with in-band noise but with the cardiac frequency recognizable. The artifact noise was extracted by a filtering technique in the frequency domain. The spectrogram of the PPG signal was calculated using an FFT-based algorithm. Coefficients of the spectrum within 0.5 Hz of the primary cardiac frequency or its harmonics were set to zeros. The inverse FFT of the modified spectrum allowed us to obtain a pure artifact noise. Typical spectra of the contaminated signal (solid) and the resulting pure artifact noise (starred) are shown in Figure 1.
Figure 1 Typical spectra of contaminated signal and extracted artifact. Spectrum of the contaminated signal (solid) is obtained by FFT. Coefficients of the spectrum within 0.5 Hz of the primary cardiac frequency or its harmonics were set to zeros to result in the spectrum of pure artifact noise (starred).
A set of synthesized signals with different SNR values was obtained by changing the value of the mix parameter ε in equation (6). The undisturbed signal was also estimated from equation (6) by setting ε = 0. SpO2 were estimated from the set of synthesized signals in an 8-second period at 1-second interval by using the STFT and SPWVD approaches. The mean SpO2 error during the complete 25.6 seconds is shown in Figure 2 as a function of SNR.
Figure 2 The mean SpO2 error obtained by the STFT and SPWVD approaches at different levels of SNR. The SPWVD approach outperforms the STFT-based technique for low SNR.
Figure 2 suggests that the two approaches lead to similar results for high SNR values (e.g. SNR>-5 dB). However, the SPWVD method outperforms the STFT-based technique for low SNR (e.g. SNR<-5 dB). Also, it is observed in this simulation that the errors are randomly positive or negative for high SNR values, but is mostly positive for low SNR values, i.e. the approaches consistently overestimate the SpO2 level. When the SNR value decreases, energy in the side-bands of the noise artifact that overlapped with the cardiac frequency components increases. And therefore, the resultant SpO2 level approaches a value that would have been estimated from the pure noise artifact, which differed by 7.2% from the actual SpO2 level for this specific trial.
It should be noted that the pulse rate was predetermined in the simulation, which helped both approaches to determine the cardiac frequency band more accurately. In practical situations, the electrocardiogram can be recorded simultaneously and used as a reliable pulse rate estimator. As for the computational cost, the SPWVD approach can be implemented efficiently by making use of its symmetry properties, and thus, it can reduce the computational cost to a quarter of that of the STFT technique [20].
Experiment and Results
Experimental protocol
The purpose of this experiment is to compare the performance of three different methods (WMA, FFT, and SPWVD) in estimating SpO2 on subjects when they are (a) in a resting position and (b) in motion.
Six healthy subjects participated in the study. Four kinds of motions have been investigated: horizontal movement (M1) and vertical movement (M2) of the hand, as well as the bending motion (M3) and pressing motion (M4) of the finger. These motions were selected because they are some of the common movements attributable to the motion artifact in pulse oximetry [7,24]. Subjects were asked to perform all four movements, 4 times each, and each time for a duration of 30 seconds. When performing each movement, subjects were asked to move their right hand, or the index finger of their right hand, for a magnitude of 2–5 cm at a frequency of 0.5–4 Hz, while keeping their left hand stationary. Signals were recorded simultaneously from the index fingers of both hands. Throughout the analysis, SpO2 or pulse rate estimated from the left hand, which was stationary at all times, was used as the reference. The reference estimates were obtained by the WMA method.
The collected signals were separated into an AC and a DC component. The AC component was filtered out by a 4th order Butterworth band-pass filter with cut-off frequencies at 0.5 Hz and 20 Hz. The ratio of the DC components was computed directly in the time domain and the same value was used for the three different approaches, i.e. the WMA, FFT and SPWVD approach. On the other hand, a different ratio of the AC components was computed using each of the three approaches.
To evaluate the performance of the different approaches, the SpO2 bias and precision, the pulse rate error, the dropout rate and the SpO2 performance index (PI) were calculated. The bias and precision are defined as the mean and standard deviation of the difference between reference and estimated SpO2 respectively. The pulse rate error is the difference between reference and estimated pulse rate. The dropout rate and SpO2 PI are evaluation parameters adopted from previous work by S.J. Barker [24]. The dropout rate is the percentage of time during which the technique fails to give a SpO2 reading, and SpO2 PI is the percentage of time during which the SpO2 level was within 7% of the reference reading.
Results
Table 1 shows the composite values from all the experiments when subjects were in a resting position and in motion. As indicated in Table 1, all three approaches can achieve 100% SpO2 PI, 0.0% dropout rate and less than 3 bpm mean absolute pulse rate error in this experiment with a limited dataset.
Table 1 Performance statistics of the different approaches. The bias, precision and performance index (PI) of SpO2, as well as the mean absolute pulse rate error and dropout rate, are used to evaluate the performance of the WMA, FFT and SPWVD approaches when subjects are in a resting position and in motion.
State Approach SpO2 bias (%) SpO2 precision (%) SpO2 PI (%) Mean absolute pulse rate error (bpm) Dropout rate (%)
Resting WMA 0.19 0.34 100 1.25 0.0
FFT 0.24 0.53 100 2.51 0.0
SPWVD 0.21 0.41 100 1.35 0.0
Motion WMA -1.31 3.58 81 16.4 4.6
FFT -1.42 3.18 83 11.2 0.0
SPWVD -1.07 2.42 91 5.62 0.0
However, the SPWVD approach shows significant improvement in both SpO2 and pulse rate estimation as compared to the WMA and FFT approaches when subjects were in motion. SpO2 estimated from the SPWVD, WMA and FFT approaches differed from the reference by -1.07 ± 2.42%, -1.31 ± 3.58% and -1.42 ± 3.18%, respectively. The mean absolute pulse rate error is reduced significantly (p < 0.05) from 16.4 bpm and 11.2 bpm for the WMA and FFT approaches, respectively, to 5.62 bpm for the SPWVD approach. The SpO2 PI also has the highest SpO2 PI (91%). Both the SPWVD and FFT approaches achieve 0.0% dropout rate. The WMA approach sometimes failed to give a reading during bending or pressing motions (dropout rate = 4.6%), which would lead to instrument "dropout" or "freeze" in clinical situations.
Figure 3 shows the distribution of SpO2 bias and pulse rate error of the three approaches. As shown in Figure 3(a), the SpO2 errors obtained by the SPWVD approach have a higher incidence (72%) in the main error band (-3%, 3%), which is the range of bias commonly accepted by most pulse oximeter manufacturers, as compared to that obtained by the WMA (55%) and FFT (56%) approaches.
Figure 3 The distributions of (a) SpO2 bias and (b) pulse rate error obtained by the WMA, FFT and SPWVD approaches
For the estimation of pulse rate, 90% of the pulse rate error falls in the error band (-10 bpm, 10 bpm) when the SPWVD approach is used (see Figure 3(b)). When compared to the WMA and FFT approaches, where only 36% and 40% of the error fall in this error band respectively, the SPWVD significantly outperforms the other two approaches.
Figure 4 shows the SpO2 output bias and precision under conditions with different kinds of motions: horizontal and vertical movements of the hand, as well as bending and pressing motions of the finger. It can be seen that the estimation of SpO2 by the SPWVD approach improved significantly (p < 0.05) as compared to the WMA and FFT approaches when subjects bend their finger or press their finger against the sensor. The three approaches show no significant differences (p > 0.05) when subjects move their hand horizontally or vertically.
Figure 4 SpO2 (a) bias and (b) precision when subjects performed different types of motions: horizontal movement and vertical movement of the hand, as well as the bending motion and pressing motion of the finger.
Figure 5 gives the error distribution of SpO2, obtained by the SPWVD approach, when subjects were in different types of motions. It is found that the bending (M3) and pressing motions (M4) of the finger have a relatively broader error distribution than the horizontal and vertical movements of the hand (M1 and M2). It can also be seen that the error distribution of M2 is slightly more concentrated than that of the M1.
Figure 5 The error distributions of SpO2, obtained by the SPWVD approach, when subjects performed different types of motions: horizontal movement and vertical movement of the hand, as well as the bending motion and pressing motion of the finger.
Discussion
Spectral analysis is useful for separating motion artifact and cardiac physiologic spectra [14-16]. However, these techniques will not be applicable to spectra that contain frequency bands close to each other. Moreover, since both the motion and cardiac frequency are nonstationary in nature, simply using techniques in the frequency domain would not be able to separate them when one of the spectra varies within the fixed time window (i.e. an 8-second period in this study). Therefore, a time-frequency representation of the corrupted signal would be useful. The SPWVD approach is proposed for the reduction of motion artifacts because it can suppress cross-term interference while maintaining a good time-frequency concentration [19]. In addition, the approach utilizes the fact that SPWVD is an energy distribution and directly calculates the magnitude of the AC component from the spectrum. The approach solves the problem of WT, where a unique value for the cardiac frequency may not always be available [14-16]. Moreover, the approach does not require a large amount of samples for training, as the back-propagation neural network approach proposed in [17].
Standard parameters used to evaluate the performance of the techniques in pulse oximetry have been adopted in this study. S.J. Barker [24] evaluated 20 commercial pulse oximeters on 70 subjects, where data were recorded on each subject for 6 minutes during normal situation and 3 minutes during a hypoxemic episode. A motorized motion table was used to induce rubbing or tapping motions of the finger, with amplitude of ± 2 cm and frequency either fixed at 3 Hz or randomly varied between 1–4 Hz. As compared to the performance of some of the commercial products evaluated in [24], which have SpO2 bias in the range of 0.4–12%, SpO2 precision in the range of 2–6%, and SpO2 PI in the range of 27–94%, the proposed SPWVD approach reports comparable performance.
The four motions investigated in this study are some of the common movements associated with motion artifacts affecting pulse oximetry [24]. By studying the effect of each component on the estimated SpO2, one would have a clearer picture of what kind of motion induces the largest error on SpO2 estimation. In future studies, it would be interesting to develop a model that specifically deals with one type of motion. As suggested by Figure 4 and Figure 5, bending the finger (M3) or pressing the finger against the sensor (M4) induces a larger error on SpO2 estimation than horizontal or vertical movements of the hand (M1 or M2). In fact, this is consistent with the clinical findings discussed in [7], which suggested that bending and/or pressing the finger may cause the irregular compression of the vascular bed between the emitter and detector of pulse oximeter sensor, and thus inducing higher errors in the estimated SpO2. A potential solution would be to place multiple sensors around or along the finger so that the ratio of the light intensity received or a pressure reading could be an indication of the degree of bending, pressure exerted or even the level of distortion made on the peripheral blood vascular bed.
Compared with the WMA and FFT approaches, the SPWVD approach showed a significant improvement (p < 0.05) in pulse rate estimation when subjects were in motion. Although such a significant improvement is not found in the estimation of SpO2, this is attributed to the fact that erroneous SpO2 estimates above the 100% upper bound were always rejected. It is hypothesized that when patients with SpO2 much lower than 100% are recruited as subjects for evaluating the different approaches, the performance of each approach will be more notably different from each other. However, this hypothesis remains to be proven in a clinical study involving a significantly large patient population.
Conclusion
Estimation of SpO2 by a time-frequency representation, the SPWVD, has been investigated in this study. The approach has been tested on four kinds of motions that are found in common movements associated with motion artifacts in pulse oximetry [7,24], i.e. the horizontal movement and vertical movement of the hand, as well as the bending motion and pressing motion of the finger. When compared with the WMA and FFT techniques, the SPWVD approach shows significant improvement (p < 0.05) when subjects bend their finger or press their finger against the sensor. When subjects were in motion, SpO2 levels estimated from the SPWVD, WMA and FFT approaches differed from the reference by -1.07 ± 2.42%, -1.31 ± 3.58% and -1.42 ± 3.18% respectively. The SPWVD approach achieves 0.0% dropout rate and 91% SpO2 PI when subjects were in motion. For the estimation of pulse rate, the SPWVD approach results in a mean absolute pulse rate error of 5.62 bpm, as compared to 16.4 bpm and 11.2 bpm by the WMA and FFT approaches respectively. The results of the study suggested that the SPWVD approach could potentially be used to improve the performance of wearable pulse oximeters by reducing the influence of motion artifacts, in particular when subjects bend their finger or press it against the sensor.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
YSY designed and carried out the experiment, analyzed and interpreted the data, and drafted the manuscript. CCYP helped to analyze and interpret the data, and assisted in drafting the manuscript. YTZ conceived of the study, and participated in its design and coordination and helped to finalize the manuscript. All authors read and approved the final manuscript.
Acknowledgements
We would like to acknowledge the support of Hong Kong Innovation and Technology Fund. We are also grateful to Standard Telecommunications Ltd., IDT Technology Ltd. and Jetfly Technology Ltd. for their support of the ITF project.
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| 15737241 | PMC553999 | CC BY | 2021-01-04 16:37:40 | no | J Neuroengineering Rehabil. 2005 Mar 1; 2:3 | utf-8 | J Neuroeng Rehabil | 2,005 | 10.1186/1743-0003-2-3 | oa_comm |
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J Neuroengineering RehabilJournal of NeuroEngineering and Rehabilitation1743-0003BioMed Central London 1743-0003-2-41574062310.1186/1743-0003-2-4ResearchInitial development and testing of a novel foam-based pressure sensor for wearable sensing Dunne Lucy E [email protected] Sarah [email protected] Barry [email protected] Dermot [email protected] Adaptive Information Cluster, Department of Computer Science, University College Dublin, Belfield, Dublin 4, Ireland2 Adaptive Information Cluster, National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland2005 1 3 2005 2 4 4 6 1 2005 1 3 2005 Copyright © 2005 Dunne et al; licensee BioMed Central Ltd.2005Dunne et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
This paper provides an overview of initial research conducted in the development of pressure-sensitive foam and its application in wearable sensing. The foam sensor is composed of polypyrrole-coated polyurethane foam, which exhibits a piezo-resistive reaction when exposed to electrical current. The use of this polymer-coated foam is attractive for wearable sensing due to the sensor's retention of desirable mechanical properties similar to those exhibited by textile structures.
Methods
The development of the foam sensor is described, as well as the development of a prototype sensing garment with sensors in several areas on the torso to measure breathing, shoulder movement, neck movement, and scapula pressure. Sensor properties were characterized, and data from pilot tests was examined visually.
Results
The foam exhibits a positive linear conductance response to increased pressure. Torso tests show that it responds in a predictable and measurable manner to breathing, shoulder movement, neck movement, and scapula pressure.
Conclusion
The polypyrrole foam shows considerable promise as a sensor for medical, wearable, and ubiquitous computing applications. Further investigation of the foam's consistency of response, durability over time, and specificity of response is necessary.
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Background
We live in a world of information, and emerging technologies compel us to look for new ways to collect, process, and distribute information. Today we are faced with a significant information overload problem as users struggle to locate the right information in the right way at the right time. In response, a number of researchers have suggested that adaptive information technologies may hold the key to the next generation of ubiquitous information systems, systems that automatically adapt to changes in their environment and usage in order to deliver users a more intelligent, proactive and personalized information service. In this paper we provide an overview of initial research conducted as part of the Adaptive Information Cluster a multi-disciplinary research cluster that brings together researchers in areas such as wearable computing, sensor technologies, information retrieval and artificial intelligence with a view to developing the next generation of intelligent, sensor-based wearable computing technologies.
Sensing in the wearable environment is crucial for many applications, but existing sensor technologies pose significant wearability problems when integrated into the user's peri-personal space. One of the most compelling needs for wearable technology is in the continuous monitoring of the human body, be that for medical monitoring or to inform the operation of a context-aware computerized application. While many technologies that are often made wearable (such as music players or telephones) function nearly as well (or sometimes better) as portable devices, almost all continuous body-sensing technologies must be worn to be effective. However, because of their ubiquitous, constant-wear nature, such technologies must prioritise the effects of the technology on the user's physical comfort as well as social comfort. Traditional sensing technologies are rarely designed for continuous, on-body use: those that require skin contact are generally designed to be used in a hospital or doctor's office, and those that do not are generally designed for use in stationary devices. Consequently, the achievement of certain design goals for existing sensors (such as durability) is ultimately detrimental to the user's comfort when applied to the wearable environment. For example, durability often equals stiffness, which results in a solid device that can cause discomfort by localizing pressure.
Textile-based sensors offer a compromise solution to this problem, by retaining the characteristics associated with comfort and wearability (properties of standard, non-electronic garments). Many textile-based sensors are actually sensing materials used to coat a textile [1] or sensing materials formed into fibres and woven or knitted into a textile structure [2]. The properties sought by textile-based sensors can include flexibility, surface area, washability, stretch, and hand (texture of textile). However, they must also include the properties required for the electronic device, including durability, power consumption, and ease of connection into a circuit. Metallic components, designed to function in rigid environments, often do not satisfy these needs. For instance, a metallic element in a high-flex environment (such as a garment) will soon break. However the recently discovered [3] conducting electroactive polymers (CEP), offer a potential solution to this problem. CEPs such as polypyrrole (PPy), polyaniline and polythiophene constitute a class of polymeric materials which are inherently able to conduct charge through their polymeric structure. They can be reversibly switched from the doped conducting state to the undoped insulating state upon chemical or electrochemical treatment. In particular, polypyrrole has attracted much interest because it is easily prepared as films, powders and composites, has a relatively high conductivity and is relatively stable in the conducting state. However, when the black precipitate of PPy has been formed it is insoluble to all known solvents and is non-processable. To overcome this PPy can be simultaneously polymerised and deposited onto the substrate [3]. The result is that the substrate is covered with a thin layer of PPy rendering the whole object conducting without compromising the mechanical properties of the substrate.
Methods
Sensor Development
In previous work [4], a novel polymer synthesis methodology was developed to create a textile-like structure capable of sensing changes in planar or perpendicular pressure, by coating an open-cell polyurethane (PU) foam with a CEP (polypyrrole). The method used for sensor fabrication is described in [4]. The method involved soaking the substrate, the PU foam in an aqueous monomer and dopant solution. An aqueous oxidant solution was then introduced into the reaction vessel to initiate polymerisation. This lead to the precipitation of doped PPy, which subsequently deposited onto the PU substrate.
Sensor Characterization
Characterisation for the PPy-coated PU foam was carried out using a number of methods as described in [4]. It was found that increasing the weight placed upon the PPy-PU foam or shortening the overall length of the foam resulted in a proportional decrease in the electrical resistance measured across the foam in a linear fashion. Results from tests carried out using the Instron™ tensile testing instrument, courtesy of the University of Bath, England, showed that the stress-strain profile of the unadulterated PU foam sample and that of the PPy-coated PU foams sample were similar showing regions of elastic and inelastic responses to force. Problems such as repeatability and long-term aging of the foam were identified. The issue of repeatability was due to hysteresis effects observed during the tensile testing of the foam. These effects were observed for the coated and uncoated samples thus originating from the PU substrate. The effect of the PPy coating was to make the entire foam conducting without compromising the soft, compressible mechanical properties of the foam substrate.
Torso Garment
Once a predictable reaction was observed from the foam, it was applied to the wearable environment to explore its utility in garment systems. It was integrated into a torso garment in several ways to investigate the ability of the foam sensor to monitor specific body changes and physiological signals. The test garment contained foam sensors in 6 locations: the top outer edge of each shoulder, the back of the neck, the superior protrusion of each scapula, and the right side rib cage under the bust (Figure 1). Sensor positions were chosen to test the foam reaction to 4 different actions: breathing, shoulder movement, neck movement, and shoulder-blade pressure.
Figure 1 Garment Structure and Sensor Layout
The test garment was a sleeveless, collared shirt, closely fitted and nonextensile. The outer garment layer was a 100% polyester satin weave, and the inner layer was a 100% acrylic satin weave. The collar was 80% nylon, 20% elastine jersey knit. The structure of the garment was crucial to the quality of data obtained, as its textile composition, design, and fit moderated the amount of force present between the body and the sensors. In this study, the prototype garment was fitted to one test subject, to eliminate inter-subject anthropometric variation.
Sensors were sewn between the two garment layers, allowing them to be easily removed and interchanged. In each test two wire leads were attached to the foam sensors and to a constant current digital multi-meter, HP, Leixlip, Ireland. Data was collected at a rate of 3 points per second. The finished prototype garment is shown in Figure 2.
Figure 2 Prototype pressure-sensitive torso garment
Breathing
The breathing sensor was attached on the subject's left-side rib cage, under the bust. The sensor measured 2.75 × 1.5 × 0.5 cm. Data was gathered with the subject standing, and the subject was instructed to breathe deeply for a period of approximately one minute.
Shoulder Movement
Two shoulder movement sensors were attached at the outer edge of the garment at the apex of each shoulder (above the subject's axilla). The sensors measured 1.5 × 2.0 × 0.5 cm. Data was gathered with the subject seated, and the subject was instructed to raise one shoulder repeatedly to its maximum height.
Neck Movement
The neck motion sensor was attached vertically along the subject's spine, at the back of the neck extending from 4 cm below the top of the collar (approximately 2nd vertebra) to 2.5 cm below the neckline of the garment (approximately 4th vertebra). The sensor measured 1.5 × 5.5 × 0.5 cm. Data was gathered with the subject seated, and the subject was instructed to perform four full neck extensions (backwards movement) and three full neck flexions (forward movement).
Shoulder Blade Pressure
Two pressure pads were attached, one over the superior edge of each scapula. The sensors measured 8 × 4 × 0.5 cm. Data was collected with the subject alternately supine and seated, on a hard surface.
Results
Sensor Characteristics
The sequential coating of PU foam with conducting polymers resulted in an increase of the overall weight of the foam and the conductivity of the foam also from being an insulating material to a conductive material (ca. 1.41 mS/cm). The conductivity of the modified foam depends on the weight of conducting polymer deposited, which in turn depends on the number of coating layers deposited on to the foam substrate. It has been shown previously [4] that by coating the PU foam substrate a total of three times with PPy an electrical resistance of 1 kΩ/cm can be achieved. The PPy-PU foam was rubbed vigorously and rinsed with cold Milli-Q water to remove any loosely bound PPy. The stability of the bound PPy onto the PU substrate was excellent and resistance of the foam did not change with subsequent hand washings in cold Milli-Q water. The electrical conductivity is good remaining in the kΩ/cm region for up to 3 months.
Torso Garment
Integrating the foam sensors into the torso garment caused little alteration in the visual or tactile properties of the garment. The largest sensors, the scapula pressure pads, caused the only visible change to the appearance of the garment, as these were the only sensors that possessed enough volume to change the surface topology of the garment. Although comfort was not a measured variable, there appeared to be no change in the tactile comfort of the garment when the sensors were added. In demonstration, both the test subject and other viewers had difficulty locating the sensors within the garment without direction.
Breathing
As seen in Figure 3a, deep breathing resulted in a sinusoidal resistance curve, varying between approximately 2 kΩ and 4 kΩ. These are absolute values and a low total change compared to the other sensors. This is a result of the age of the foam: The breathing sensor was replaced with week-old foam prior to the test, while the other sensors were 2 months old. The sensor foams are composites of PPy and PU and so the absolute resistance of the foam will be affected by each of these components. Firstly the absolute resistance of the PPy may vary with time due to the gradual oxidation of the polymeric backbone. Also hysteresis in the PU foam substrate as observed during the tensile testing will cause problems to the measured absolute resistance. This hysteresis effect of the PU foam during use can be seen as the gradual and positive drift in the measured resistance that can be seen in Figure 3a. This drift was calculated as 26.67% change of resistance per minute. However if the foam sensor is allowed to relax, un-used, for 2 hours, then the resistance returns to the initial resistance value. However, the sensor output appears to be sufficiently robust, even in its unfiltered state, for a reliable determination of the wearer's respiratory rate, for example. In order to normalise the data so that the sensitivity of the sensor could be determined, the relative resistance of the foam sensor was plotted as in Figure 3b. This was calculated by dividing the absolute resistance at a given time t, Rt, by the initial baseline resistance, R0. It can be seen in Figure 3b that there was an approximate 20% change in the relative resistance of the foam sensor between inhalation and exhalation.
Figure 3 a) Absolute resistance response to Deep Breathing, b) relative resistance response (Rt/R0) to Deep Breathing
Shoulder Movement
The response of the foam to shoulder movements was an approximate 100% decrease in relative resistance as seen in Figure 4. Once again the data appears sufficiently robust to reliable detect each shoulder movement; however no test was performed to detect the foam reaction to shoulder movements of varying magnitudes.
Figure 4 Resistance Response to Shoulder Lift
Neck Movement
The foam responded to full neck extensions, section A in Figure 5, with an 80% decrease in the relative resistance. Full flexion of the neck, section B in Figure 5 involved a smaller body movement, which was detected as a smaller decrease (30%) in the relative resistance of the sensor. This data indicates that the dorsal neck sensor placement exhibits a response of greater magnitude for extension than for flexion. Since the sensor provides no additional qualitative information, it is hypothesized that a second sensor would be required to determine the difference between a small extension and a large flexion.
Figure 5 Resistance Response to Neck Movement
Shoulder-Blade Pressure
The foam responded with a 60% increase in the relative resistance when the subject moved from supine (applying pressure to the scapula area) to a seated position (no pressure), as seen in Figure 6. The response time of this sensor, that is, the time taken for the resistance to stabilise after the subject moved to a seated position, was approximately 8 seconds. The response time was shown previously [4] to be inversely related to the force applied to it and is also influenced by the size of the sensor. The foam sensor in this position measured 32 cm2 versus 2–12 cm2 for the other sensors and so the response time for the shoulder-blade foam sensor would be slightly slower than that for the other sensor positions, e.g. 4 seconds for shoulder lift foam sensor.
Figure 6 Resistance Response to Constant Scapula Pressure
Discussion
As demonstrated, pressure sensing in the wearable environment can provide useful descriptive information about the physical state of the user. Conducting electroactive polymers are attractive for sensing in a garment-integrated context because of their ability to retain the tactile and mechanical properties of a textile-based structure. In the garment integration, the foam sensors had little effect on the comfort or wearability of a standard garment. However, more investigation is necessary to determine the accuracy of the foam sensor responses, particularly the repeatability of response.
As seen in the torso sensor evaluation, the age of the sensor had a significant impact on the absolute resistance of the sensors. It has been shown previously that if PPy is left to open to atmosphere then there is a gradual increase in the electrical resistance due to oxidation of the polymeric backbone [5]. However, the coating itself did not delaminate from the foam substrate, even during hand-washing of the foam sensors. This indicates that if the oxidation were prevented, the sensor would be durable and washable over an indefinite period of time. In a garment-integrated context, washability of components is important to the preservation of normal user patterns of care and maintenance of clothing.
In the torso integration, the raw pilot test data indicates that foam sensors can provide detectable responses to all of the body signals investigated, although careful sensor placement is important to the quality of data gathered. In this study, inter-subject anthropometric variation was controlled by limiting the number of subjects to one, and by custom-engineering the garment to fit that subject precisely. However, in a real-world scenario such control would not be possible, and sensor locations across a broad variety of body shapes and sizes would be hard to predict. Similar issues would arise with sizing, fit, and sensor locations on the foot. Because of the increased number of sensors and precision of locations, this variable would become even more difficult to control, however were the number and locations of sensors increased still more to create a uniform grid of pressure sensors, the fit issue could be avoided.
An additional problem of hysteresis caused by the PU foam substrate results in the gradual and positive increase in the resistance of the foam sensor. Since the position and the relative resistance of the PPy-coated PU sensors are crucial to their sensitivity, calibration of the sensors would be required on a regular basis. This calibration would involve setting the baseline resistance and range of the measured resistance of the sensors as determined through a series of standard repeatable exercises by the subject. Once these parameters are set subject monitoring could be commenced.
There are many applications of wearable sensing for which this type of sensor is particularly well suited. For example, in the monitoring of high-pressure body areas for individuals with reduced tactile sensation (such as diabetics suffering from neuropathy) the foam sensor would allow pressure points to be monitored without introducing a solid sensor element into a pressurized area close to the skin that could create more irritation. Rigid sensors in such an area could easily create more irritation and exacerbate the problem, but a foam sensor not only would not create irritation, it could actually protect the body from irritants by providing an additional layer of cushioning on key pressure points.
Outside of medical applications, knowledge of the state of the body is essential in many wearable, mobile, and ubiquitous computing applications. It is common in wearable and ubiquitous computing applications for a system to make decisions based on its perception of the needs and wants of the user. A subtle, comfortable sensor that demands no attention or adaptation from the user can allow the application to function invisibly, reducing the cognitive load on the user.
Conclusion
Based on these preliminary data, polypyrrole-coated conductive foam shows considerable promise as a basic sensing technology, and for use in detecting body movements, physiological functions, and body state from body-garment interactions. Importantly, the sensor maintains the attractive structural properties of foam, consistent with the objectives of wearability and comfort in a smart garment.
Further study is necessary to fully understand the ability of the foam to serve as a reliable sensor over time and under the hostile conditions that garments must usually face. For instance, further work is required to understand and determine the effects of oxidation on baseline drift, the influence of variable conductance responses, calibrations of these responses and the optimal locations for sensors. In addition, processing algorithms for extraction of patterns from gathered data are required, as well as wearable and wireless hardware to allow the data to be used in real-time.
Future work includes in-depth analysis of foam responses in controlled environments, and evaluation of optimal sensor location for monitoring of specific activities and conditions.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
LED created the garment prototypes, participated in the prototype pilot evaluations, and drafted the manuscript. SB created the foam sensors, participated in the prototype pilot evaluations, and drafted the manuscript. BS participated in the project organization and supervised the research. DD participated in the project organization and supervised the research. All authors read and approved the final manuscript.
Acknowledgements
This material is based on works supported by Science Foundation Ireland under Grant No. 03/IN.3/I361 and IRCSET under Grant No. RS/2002/765-1. We would also like to acknowledge W. Megill, his research team and the University of Bath for kindly allowing SB to use their facilities for experimental work.
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Hertleer C Grabowska M Van Langenhove L Catrysse M Hermans B Puers R Kalmar A van Egmond H Matthys D Towards a Smart Suit Proceedings of Wearable Electronic and Smart Textiles: Leeds, UK 11 June 2004
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Brady S Diamond D Lau KT Inherently conducting polymer modified polyurethane smart foam for pressure sensing, Sens & Actuat A Article in Press
Thieblemon JC Planche MF Petrescu C Bouvier JM Bidan G Stability of Chemically Synthesized Polypyrrole films, Synth Met 1993 59 81 96 10.1016/0379-6779(93)91159-Y
| 15740623 | PMC554000 | CC BY | 2021-01-04 16:37:40 | no | J Neuroengineering Rehabil. 2005 Mar 1; 2:4 | utf-8 | J Neuroeng Rehabil | 2,005 | 10.1186/1743-0003-2-4 | oa_comm |
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Proteome SciProteome Science1477-5956BioMed Central London 1477-5956-3-21573056610.1186/1477-5956-3-2ResearchHuman neuroglobin protein in cerebrospinal fluid Casado Begona [email protected] Lewis K [email protected] Gail [email protected] Daniel J [email protected] James N [email protected] Division of Rheumatology, Immunology & Allergy, Georgetown University, Washington DC 20007-2197, USA2 Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0508, USA3 Department of Medicine, Division of Rheumatology, University of Michigan, Ann Arbor 48109, MI, USA4 Dipartimento di Biochimica A. Castellani, Universita di Pavia, Pavia 27100, Italy2005 25 2 2005 3 2 2 5 3 2004 25 2 2005 Copyright © 2005 Casado et al; licensee BioMed Central Ltd.2005Casado et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Neuroglobin is a hexacoordinated member of the globin family of proteins. It is predominantly localized to various brain regions and retina where it may play a role in protection against ischemia and nitric oxide-induced neural injury. Cerebrospinal fluid was collected from 12 chronic regional or systemic pain and 5 control subjects. Proteins were precipitated by addition of 50% 0.2 N acetic acid, 50% ethanol, 0.02% sodium bisulfite. The pellet was extensively digested with trypsin. Peptides were separated by capillary liquid chromatography using a gradient from 95% water to 95% acetonitrile in 0.2% formic acid, and eluted through a nanoelectrospray ionization interface into a quadrapole – time-of-flight dual mass spectrometer (QToF2, Waters, Milford, MA). Peptides were sequenced (PepSeq, MassLynx v3.5) and proteins identified using MASCOT ®.
Results
Six different neuroglobin peptides were identified in various combinations in 3 of 9 female pain subjects, but none in male pain, or female or male control subjects.
Conclusion
This is the first description of neuroglobin in cerebrospinal fluid. The mechanism(s) leading to its release in chronic pain states remain to be defined.
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Background
The protein constituents (proteome) of cerebrospinal fluid (CSF) are altered in disease states such as meningitis, but may also be more subtly altered in many other neural conditions. CSF has been difficult to investigate because of the need for invasive lumbar punctures and the small volumes of CSF available for analysis. This situation is now rapidly changing as methods requiring microliter volumes and sophisticated analysis tools such as proteomics become available [1,2]. Proteomics has made it possible to identify scores of proteins that have not been previously discovered in this fluid.
One such protein is neuroglobin. Neuroglobin is a recently identified member of the globin family. It binds oxygen with an affinity between that of myoglobin and hemoglobin [3,4]. Neuroglobin is 151 amino-acids long with a molecular mass of ≈ 17 kDa. The mouse and human genes are 94% identical. Neuroglobin is an ancient protein (estimated < 550 Myr old) that is more related to the annelid Aphrodite aculeate intracellular globin (30% identify) [5] than to vertebrate myoglobin (<21% identity) and hemoglobin (<25% identity) [3]. Human neuroglobin mRNA is predominantly expressed in brain with high signal in the frontal lobe, subthalamic nucleus and thalamus. The concentration is estimated to be less of 0.01% of the total brain protein content [3]. Neuroglobin protein has not been previously detected in cerebrospinal fluids.
Results
Neuroglobin Peptides
Five peptides derived from neuroglobin (NCBInr ID accession 10864065) were identified using both the MASCOT software with NCBInr database and ProteinLynx Global Server with SwissProt database (Table 1). Two precursor ions with mass/charge (M/Z) ratios of 423.97 and 435.82 were identified in CSF sample #3 with the SwissProt, but not NCBInr, searches. Table 1 shows the mass-over-charge (m/z), charge state, elution time, position, sequence, molecular weight (Mr) for each peptide. The numbers of matching peptides were 5 in sample #1, 2 in sample #2, and 3 in sample #3. The peptides mapped 31.1%, 13.9% and 17.2%, respectively, of the total neuroglobin protein sequence. They matched amino acids 1–10 and 15–30 of the N-terminal and 131–151 of the C-terminal. Trypsin digestion missed cleavage sites at amino acids 18 and 146. Reproducibility was demonstrated by the consistent retention times for the same peptides from different subjects. No low abundance neuroglobin peptides were found in other samples. BLAST sequence analysis of all six peptides identified only one protein: hypothetical 16.9 kDa protein (neuroglobin: NREF and iProClass NF00135839; SwissProt/TrEMBL Q9NPG2).
Table 1 Amino acid sequence of neuroglobin peptides identified from human CSF using CapLC nanoESI Q-TOF tandem mass spectrometry.
#a Amino Acids M/z b Zc Molecular Weight Δd Amino Acid Sequences Timee (min)
Calc. Exp.
1 1–10 423.98 3 1268.65 1268.91 0.25 (-)MERPEPELIR(Q) 37.02
1 15–30 580.73 3 1738.95 1739.18 0.23 (R)AVSRSPLEHGTVLFAR(L) 42.94
1 19–30 442.99 3 1325.71 1325.95 0.25 (R)SPLEHGTVLFAR(L) 42.68
1 131–146 580.04 3 1736.87 1737.10 0.24 (R)AAWSQLYGAVVQAMSR(G) 68.23
1 131–151 761.42 3 2281.06 2281.23 0.17 (R)AAWSQLYGAVVQAMSRGWDGE(-) 74.49
2 131–146 580.02 3 1736.87 1737.03 0.16 (R)AAWSQLYGAVVQAMSR(G) 68.24
2 131–151 761.37 3 2281.06 2281.09 0.03 (R)AAWSQLYGAVVQAMSRGWDGE(-) 74.54
3 1–10 423.97 f 3 1268.65 1268.88 0.23 (-)MERPEPELIR(Q) 37.31
3 15–30 435.82 f 4 1738.95 1739.27 0.32 (R)AVSRSPLEHGTVLFAR(L) 43.27
3 19–30 442.99 3 1325.71 1325.96 0.25 (R)SPLEHGTVLFAR(L) 43.01
a. Subject Number
b. Mass / charge ratio
c. Charge
d. Difference (error) between the experimental (Exp.) and calculated (Calc.) molecular weights
e. Elution time expressed in minutes.
f. Peptides in Subject 3 identified only with ProteinLynx Global Server using the SwissProt database.
Mass Spectrometry
Figures 1 through 5 show the tandem MS data for the 5 [M+H+] precursor ions. The mass of each b- and y-fragment is listed. The amino acid sequence is shown at the top of each spectrum using the Roepstorff nomenclature [11]. The amino acid sequences were determined from both the N- and C-terminal directions. Figures 1 and 4 show two spectra from subject #1. Figures 3 and 5 show two spectra from subject #2. Figure 2 shows one spectrum from subject #3. Neuroglobin peptides were detected in 2 of the 3 CapLC runs for subjects #1 and #2, but only in 1 run for subject #3.
Figure 1 The tandem mass spectrum is shown for the neuroglobin amino acid 1 to 10 peptide. In this and the following figures, the top line represents the b-series, and the 2nd line the y-series. The x-axis presents M/z and the y-axis signal intensity. The numbers are the M/z values for each daughter ion (vertical lines).
Figure 2 The tandem mass spectrum is shown for the neuroglobin amino acid 15 to 30 peptide.
Figure 3 The tandem mass spectrum is shown for the neuroglobin amino acid 19 to 30 peptide.
Figure 4 The tandem mass spectrum is shown for the neuroglobin amino acid 131 to 146 peptide.
Figure 5 The tandem mass spectrum is shown for the neuroglobin amino acid 131 to 151 peptide.
The peptides with M/z of 580 and 761 (Table 1) overlapped with the 761 ion having a missed tryptic cleavage point at Arg146. Additional peptides were not identified, perhaps because we did not reduce disulfide bridges to reveal additional trypsin digestion sites. However, all the identified peptides were specific for neuroglobin, and so were appropriate markers for fast identification of this protein.
Neuroglobin and Pain Subjects
Neuroglobin-derived peptides were found in 3 of 9 female pain subjects, but none of the 3 male pain subjects; it was not detected in the 3 female or 2 male control subjects. Within the chronic pain group there was no association between the presence of neuroglobin and clinical factors such as age, extent or duration of pain, or tenderness to pressure. No hemoglobin or cytoglobin [12,13] were detected.
Discussion
This is the first description of neuroglobin protein in the CSF of any species. Neuroglobin joins cytoglobin (histoglobin) in a new globin subfamily that forms hexacoordinated heme iron complexes [12,13]. These are distinct from the pentacoordinated hemoglobin and myoglobin.
The source of neuroglobin in the CSF is likely to be brain regions such as the subthalamic nuclei (60% of total brain neuroglobin mRNA expression), frontal lobe, thalamus, occipital pole, pituitary gland, and medulla oblongata [3,14]. Immunohistochemistry confirmed this distribution with strong staining in the hippocampus, thalamus, hypothalamus (especially the paraventricular nucleus) and brainstem nuclei of cranial nerves [15]. Expression was often patchy within these regions indicating that only select neurons expressed neuroglobin. Regions with high sensitivity to hypoxia such as the cerebral cortex had constitutive expression [15]. Spinal cord was a less likely source since its neuroglobin mRNA expression was less than 10% of that from the subthalamic nuclei. Neuroglobin mRNA was expressed in the retina [16] and in peripheral nerves suggesting that the mRNA was axonally transported and translated to distal neurons [17]. The protein has a cytoplasmic distribution [18]. Neuroglobin could provide oxygen for high energy consuming processes such as synaptic activity, neural plasticity, or efferent transmitter release as in nociceptive nerve axon responses.
Neuroglobin mRNA was also present in adrenal cells and the β cells of the pancreatic islets of Langerhans [14]. Roles in diabetes or hypoxia-induced insulin secretion are unstudied. These studies of mRNA expression should not be extrapolated into relative levels of protein expression or neuroglobin turnover since concordance between microarray and proteomic studies can be as low at 13% [19].
Neuroglobin is likely to serve as an intracellular oxygen depot to facilitate oxygen diffusion to the mitochondria. A role in oxygen supply was supported by the very high expression of neuroglobin mRNA in retinal neurons but not the supporting ocular epithelium and other structures [16]. Retinal neuroglobin concentrations were estimated at > 100 μM, compared to > 1 μM for the whole brain. The retinal and muscle oxygen tensions, oxygen affinities and tissue concentrations of neuroglobin and myoglobin were comparable suggesting that the two play homologous roles in their respective tissues.
Neuroglobin might act in certain circumstances to limit neural cellular damage during hypoxia. Neuroglobin expression was inversely correlated to the sensitivity of the brain regions to ischemia [3]. For example, neuroglobin expression was 4 times higher in the cerebral cortex than the hippocampus, corresponding to the time for ischemia to cause half-maximal damage (19.1 and 12.7 min, respectively) in these tissues [20]. Neuroglobin-immunoreactive material was upregulated in the cytoplasm of neurons that were destined to survive acute cerebral ischemia, and was reduced in apoptotic neurons [21]. Hypoxic induction of neuroglobin was blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059 [22]. Like hemoglobin and myoglobin, hemin increased neuroglobin 4-fold through a separate signalling process mediated by protein kinase G and soluble guanylate cyclase. Hypoxia-inducible neuroprotective factor (HIF-1) that can induce β-globin production may play a role in neuroglobin induction. It is not clear if there are differential responses to intermittent, recurrent, or chronic cerebral ischemia.
Neuroglobin was also colocalized with nitric oxide synthase in the lateral tegmental nuclei, stria terminalis, habencule, nucleus of the tractus solitarius, periaqueductal grey matter, amygdala and subfornic organ [23]. The protein may act as a nitric oxide scavenger, a role that has also recently been proposed for myoglobin [24]. This function would protect against nitric oxide – induced damage that is part of hypoxia – ischemia related neuron injury. Nitric oxide appears to bind to the hexacoordinated deoxy ferrous form (F8His-Fe2+-E7His) and displace the protein from the globin [25]. This affinity may be a double-edged sword, since neuroglobin, hemoglobin and myoglobin may protect Plasmodium and Trypanosoma from the antiparasitic effects of nitric oxide [26]. Neuroglobin may also play a protective role in carbon monoxide poisoning [27].
In this study, neuroglobin was qualitatively identified in CSF from 3 female subjects with chronic pain conditions. Females have greater pain sensitivity to pressure and other stimuli (lower pain thresholds) [28], but pain is not thought to induce neural hypoxia or any of the known triggers of neuroglobin expression [21].
It is tempting to speculate that the source of neuroglobin in our samples was from nuclei involved in pain transmission or regulation such as the thalamus, prefrontal cortex, amygdala, or spinal cord dorsal horn somatic pain synaptic regions (e.g. layers 1 and 2 of Rexed). The fact that neuroglobin was not detected in any of the control females in our study makes it unlikely that the expression was related to gender. Examination of additional normal and chronic pain subjects is underway to determine the factors that may be responsible for neuroglobin expression. It is also possible that the proteomic detection of neuroglobin varies depending upon sample preparation, signal-to-noise ratio for relatively low abundance proteins compared to albumin and immunoglobulins that are present in high abundance, duration of storage, factors related to trypsin digestion, capillary liquid chromatography, mass spectrometry or bioinformatic neuroglobin peptide detection. These technical factors are unlikely to be significant since our samples were treated identically and were stored for approximately equal amounts of time.
In contrast to neuroglobin's localization, cytoglobin-immunoreactive material was localized to the cellular nucleus in all tissues examined [14]. Mammalian cytoglobin genes display an unique exon-intron pattern with an additional exon resulting in a C-terminal extension of the protein that is not present in lower species such as zebra fish [29,30]. Again, it is not clear if cytoglobin acts as an oxygen depot or sink, free radical scavenger, oxygen-sensor or transcription factor. No evidence for cytoglobin was found in cerebrospinal fluid suggesting that nuclear degeneration was not present in any of our subjects.
Conclusion
This is the first description of neuroglobin in cerebrospinal fluid and in humans. Neuroglobin was identified in 3 of 9 female pain subjects. The role(s) for this ancient oxygen and nitric oxide binding protein in humans, and potential links to pain, remain to be fully determined.
Methods
After obtaining informed consent, lumbar punctures were performed on 17 subjects as part of an evaluation of pain mechanisms. Twelve patients had musculoskeletal pain and five were healthy control subjects. Cerebrospinal fluid samples were aliquoted and frozen at -70°C. Lipids and peptides were extracted from 200 μl of thawed CSF by adding an equal volume of 50% ethanol, 50% 0.2 N acetic acid 0.02% sodium bisulfite ("acid-ethanol") [6]. Centrifuged pellets were reconstituted in 50 μl of 0.1 M ammonium bicarbonate buffer (pH 7.8) and digested with trypsin (protein-enzyme ratios of 20:1) at 37°C overnight. Digested peptides were separated by capillary liquid chromatography (CapLC, Waters, Milford, MA) over a Zorbax 18WSB reverse phase column (100 mm × 0.15 mm inner diameter) (Micro-Tech Scientific, Sunnyvale, CA) at room temperature for 100 min using a gradient starting at 95% solvent A (aqueous solution of 0.2% formic acid) and ending with 95% solvent B (acetonitrile with 0.2% formic acid). The elution was performed at a flow-rate of 1 μl/min.
The column eluate was pumped through a nanoelectrospray interface into a quadrapole – time of flight (Q-TOF-2, Waters, Milford, MA) mass spectrometer. MASSLYNX version 3.5 software was used to control the CapLC and Q-ToF-2, data acquisition, processing, and determination of peptide sequences. The protein identification was performed with the MASCOT MS/MS ion search software and NCBInr protein database [7,8], and with ProteinLynx Global Server Web (Waters) with SwissProt database. The BLAST algorithm was used to compare protein queries to database sequences (e.g. Protein Information Resource, PIR, ) [9,10], proteins derived from GenBank coding sequences, and PDB atomic coordinates.
Samples were assessed by CapLC-Q-ToF-2 in triplicate. At least 2 separate peptides from neuroglobin had to identify in each individual sample to ensure that this protein, and not a related protein, was present. In an attempt to detect low abundance expression of neuroglobin peptide ions that were not selected by MS-MS (false negative results), all MS data from the appropriate CapLC retention times were reassessed at high resolution. Positive results (MS data) were checked to see if ions in MS were present but not in MS-MS for other pieces. All putative neuroglobin peptide spectra were sequenced using PepSeq (Waters) and confirmed by visual inspection.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
Casado B 1,4, Sample preparation, chromatography, mass spectrometry and manuscript preparation
Pannell LK 2, Supervision and assistance with chromatography and mass spectrometry and manuscript preparation
Whalen G 1, Sample preparation
Clauw DJ 3, Clinical investigation of subjects
Baraniuk JN 1,*, Organization of study and selection of samples, preparation of manuscript
Acknowledgements
Supported by U.S. Department of Army Grant DAMD 17-002-0018 (DC) and U.S. Public Health Service Award RO1 AI42403 (JNB).
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| 15730566 | PMC554085 | CC BY | 2021-01-04 16:37:15 | no | Proteome Sci. 2005 Feb 25; 3:2 | utf-8 | Proteome Sci | 2,005 | 10.1186/1477-5956-3-2 | oa_comm |
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RetrovirologyRetrovirology1742-4690BioMed Central London 1742-4690-2-91571323410.1186/1742-4690-2-9ResearchKaposi's sarcoma associated herpes virus-encoded viral FLICE inhibitory protein activates transcription from HIV-1 Long Terminal Repeat via the classical NF-κB pathway and functionally cooperates with Tat Sun Qinmiao [email protected] Hittu [email protected] Preet M [email protected] Hamon Center for Therapeutic Oncology Research, UT Southwestern Medical Center, Dallas TX 75390-8593, USA2 Department of Medicine, Division of Hematology-Oncology and the Hillman Cancer Center, University of Pittsburgh, PA 15213, USA2005 15 2 2005 2 9 9 24 9 2004 15 2 2005 Copyright © 2005 Sun et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The nuclear transcription factor NF-κB binds to the HIV-1 long terminal repeat (LTR) and is a key regulator of HIV-1 gene expression in cells latently infected with this virus. In this report, we have analyzed the ability of Kaposi's sarcoma associate herpes virus (KSHV, also known as Human Herpes virus 8)-encoded viral FLIP (Fas-associated death domain-like IL-1 beta-converting enzyme inhibitory protein) K13 to activate the HIV-1 LTR.
Results
We present evidence that vFLIP K13 activates HIV-1 LTR via the activation of the classical NF-κB pathway involving c-Rel, p65 and p50 subunits. K13-induced HIV-1 LTR transcriptional activation requires the cooperative interaction of all three components of the IKK complex and can be effectively blocked by inhibitors of the classical NF-κB pathway. K13 mutants that lacked the ability to activate the NF-κB pathway also failed to activate the HIV-1 LTR. K13 could effectively activate a HIV-1 LTR reporter construct lacking the Tat binding site but failed to activate a construct lacking the NF-κB binding sites. However, coexpression of HIV-1 Tat with K13 led to synergistic activation of HIV-1 LTR. Finally, K13 differentially activated HIV-1 LTRs derived from different strains of HIV-1, which correlated with their responsiveness to NF-κB pathway.
Conclusions
Our results suggest that concomitant infection with KSHV/HHV8 may stimulate HIV-1 LTR via vFLIP K13-induced classical NF-κB pathway which cooperates with HIV-1 Tat protein.
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Background
The human immunodeficiency virus type 1 (HIV-1) establishes latent infection following integration into the host genome [1]. The expression of integrated HIV-1 provirus in cells latently infected with this virus is controlled at the level of transcription by an interplay between distinct cellular and viral transcription factors which bind to the HIV-1 long terminal repeat (LTR) [1-4]. The HIV-1 LTR is divided into three regions: U3, R and U5, which contain four functional elements: transactivation response element (TAR), a basal or core promoter, a core enhancer, and a modulatory element [1,4]. The viral transactivator Tat is a key activator of HIV-1 LTR via its binding to the TAR region, while the core region contains three binding sites for Sp1 transcription factor and a TATA box [1]. The enhancer region of HIV-1 LTR contains two highly conserved consecutive copies of κB elements at nucleotides -104 to -81 that are critical for HIV-1 replication in T cells [1]. Finally, the modulatory region harbors binding sites for numerous transcription factors, such as c-Myb, NF-AT, USF and AP1. Among the various signaling pathways known to activate HIV-1 LTR, the NF-κB pathway is particularly important as it is activated by several cytokines involved in immune and inflammatory response [1]. However, all pathways that stimulate NF-κB do not reactivate latent HIV and HIV-1 gene expression is also known to be regulated by NF-κB-independent mechanisms, for example via Tat [2,3].
There are five known members of the NF-κB family in mammalian cells including p50/p105 (NF-κB1), p52/p100 (NF-κB2), p65 (RelA), c-Rel, and RelB [5,6]. Although many dimeric forms of NF-κB have been described, the classical NF-κB complex is a heterodimer of the p65/RelA and p50 subunits. The activity of NF-κB is tightly regulated by their association with a family of inhibitory proteins, called IκBs [5-7]. The best characterized Rel-IκB interaction is between IκBα and p65-p50 dimer, which blocks the ability of NF-κB to enter the nucleus. Stimulation by a number of stimuli results in the activation of a multi-subunit IκB kinase (IKK) complex, which contains two catalytic subunits, IKK1/IKKα and IKK2/IKKβ, and a regulatory subunit, NEMO/IKKγ [7]. The IKK complex leads to the inducible phosphorylation of IκB proteins at two conserved serine residues located within their N-terminal region [5]. Phosphorylation of IκB proteins lead to their ubiquitination and subsequent proteasome-mediated degradation, thereby releasing NF-κB from their inhibitory influence [7]. Once released, NF-κB is free to migrate to the nucleus and bind to the promoter of specific genes possessing its cognate binding site. In addition to the above classical NF-κB pathway, an alternative (or noncanonical) pathway of NF-κB activation that involves proteasome-mediated processing of p100/NF-κB2 into p52 subunit, has been described recently [8]. Unlike the classical NF-κB pathway, which involves IKK2 and NEMO, activation of the alternative NF-κB pathway by TNF family receptors is critically dependent on NIK and IKK1 [9,10].
Kaposi's sarcoma associated herpes virus (KSHV), also known as Human herpes virus 8 (HHV8), is a γ-2 herpes virus which is frequently associated with malignancy among AIDS patients [11-13]. In addition to Kaposi's sarcoma (KS), KSHV genome has been consistently found in primary effusion lymphoma (PEL) or body cavity lymphoma and multicentric Castleman's disease. KSHV genome is known to encode for homologs of several cytokines, chemokines and their receptors [11-13]. However, none of the above proteins is expressed in cells latently-infected with KSHV [11]. KSHV also encodes for a protein called K13 (or orf71), which is one of the few viral proteins known to be expressed in cells latently infected with KSHV [11,14-16].
The K13 protein contains two homologous copies of a Death Effector Domain (DED) that is also present in the prodomains of caspase 8 (also known as FLICE), caspase 10 and cellular FLICE Inhibitory Protein (cFLIP, also known as MRIT) [17]. Proteins with two DEDs have been discovered in other viruses as well, including MC159L and MC160 from the molluscum contagiosum virus and E8 from the equine herpes virus 2 [18-20]. These virally encoded DED-containing proteins are collectively referred to as vFLIPs (viral FLICE Inhibitory Proteins) [18-20].
We recently demonstrated that KSHV vFLIP K13 possesses the unique ability to activate both the classical and the alternate NF-κB pathways [21-24]. Several recent studies suggest that binding of NF-κB to HIV-1 LTR may not be sufficient and interaction with additional viral and cellular factors may be required to induce its transcriptional activation [25,26]. As such, in this report we have carried out a detailed analysis of the ability of K13 to activate the HIV-1 LTR and analyzed the contribution of the canonical vs alternate NF-κB signaling pathways, various subunits of the IKK complex and the HIV-1 Tat to this process.
Results
vFLIP K13 activates the HIV-1 LTR
We used a luciferase reporter construct to test the effect of vFLIP K13 on HIV-1 LTR transcriptional activation. This reporter construct expresses the firefly luciferase gene downstream of the HIV-1 LTR. As shown in Fig. 1A–C transient transfection of vFLIP K13 in 293T and Cos7 cells led to significant (3 and 5 fold, respectively) activation of the HIV-1 LTR where as expression of the vFLIP E8 from the equine herpes virus 2 failed to do so. As HIV-1 LTR is known to be responsive to proinflammatory cytokines, we also carried out a comparative analysis of the HIV-1 LTR activation by K13, TNF-α and IL-1β in 293T cells. As shown in Fig 1C, while K13-induced approximately 3-fold increase in HIV-1 LTR transcriptional activation, treatment with TNF-α(50 ng/ml) and IL-1β (50 ng/ml) resulted in 5–6 fold increase. A possible explanation for this difference lies in the fact that unlike TNF-α and IL-1β, K13 lacks the ability to induce the transcription factor AP1, which is known to activate HIV-1 LTR. We also tested whether vFLIP K13 possesses the ability to activate the HIV-1 LTR in cells naturally infected with HIV-1. As shown in Fig. 1D, transient transfection of K13 in Jurkat cells (human T cell lymphoma cell line) led to modest (2-fold) activation of HIV-1 LTR transcription activity.
K13 mutants defective in NF-κB activation fail to activate HIV-1 LTR
We have recently generated point mutants of the vFLIP K13 which differ in their ability to activate the NF-κB pathway [27]. In order to test the hypothesis that vFLIP K13 activates the HIV-1 LTR via NF-κB pathway, we carried out a comparative analysis of the ability of wild-type and mutant K13 constructs to activate the HIV-1 LTR reporter construct. In a parallel experiment, we also tested the effect of different K13 constructs on an NF-κB luciferase reporter construct to serve as a positive control. The luciferase expression in the latter construct is driven by four copies of a consensus NF-κB binding-site [28]. Consistent with our published results [27], the triple mutant 58AAA demonstrated a complete lack of NF-κB reporter activation while the mutant 67AAA retained partial ability to do so (Fig 2A). Importantly, essentially a similar pattern of reporter activation was obtained when the wild-type and mutant K13 constructs were tested on the HIV-1 LTR reporter construct (Fig 2B). Collectively, the above results suggested the involvement of the NF-κB pathway in vFLIP K13-induced HIV-1 LTR activation.
vFLIP K13 induces binding of specific transcription factors to HIV-1 LTR
In order to test the hypothesis that vFLIP K13 activates HIV-1 LTR by inducing the binding of specific transcription factors to the NF-κB binding sites present in the HIV-1 LTR, we used an electrophoretic mobility shift assay (EMSA). As shown in Fig. 3A, nuclear extracts from Jurkat cells expressing vFLIP K13 demonstrated significant DNA-binding activity on radiolabelled oligonucleotides-derived from the NF-κB binding sites present in HIV-1 LTR. In contrast, no HIV-1 LTR DNA-binding activity was observed in nuclear extracts of empty vector-expressing cells (Fig. 3A, compare lanes 1 and 2). The specificity of the complex was demonstrated by its disappearance upon competition with excess cold HIV-1 LTR oligonucleotide duplex and lack of effect upon competition with a non-specific oligonucleotide duplex (Fig. 3A, lanes 3 and 4).
Nature and subunit composition of K13-induced transcription factors bound to HIV-1 LTR
In addition to the classical NF-κB pathway, an alternative (or non-canonical) pathway of NF-κB activation, which involves proteasome-mediated processing of p100/NF-κB2 into p52 subunit, has been described [8]. We have recently demonstrated that vFLIP K13 can activate the alternate NF-κB pathway via an IKK1-dependent and NIK- and IKK2-independent process [24]. In order to determine the contribution of the classical vs alternate NF-κB pathway to vFLIP K13-induced HIV-1 LTR activation, we used a supershift assay to analyze the nature of the protein complexes bound to HIV-1 LTR from nuclear extracts of vFLIP K13-expressing cells. This assay demonstrated that p50 and c-Rel subunits are the major components of the HIV-1 LTR-bound NF-κB complexes induced by vFLIP K13 with modest contribution from the p65 subunit (Fig. 3B). As the p50, c-Rel and p65 subunits are primarily activated by the classical NF-κB pathway, the above results support the hypothesis that K13 activates the HIV-1 LTR via the classical NF-κB pathway.
Role of classical NF-κB activation in K13-induced HIV-1 LTR reporter activity
We have previously demonstrated that vFLIP K13 activates the classical NF-κB pathway via phosphorylation of IκBα, which leads to its ubiquitination and subsequent degradation via proteasome [22]. We used a phosphorylation-resistant mutant of IκBα to test the involvement of the classical NF-κB pathway in vFLIP K13-induced HIV-1 LTR reporter activity. As shown in Fig. 4A, a phosphorylation-resistant mutant of IκBα (IκBαSS32/36AA), in which the two critical N-terminal serine residues have been mutated to alanine, completely blocked vFLIP K13-induced HIV-1 LTR reporter activity.
We used siRNA-mediated downregulation of key subunits of the classical and alternate NF-κB pathways to test their involvement in K13-induced HIV-1 LTR activation. As shown in Fig. 4B, we achieved effective silencing of c-Rel and RelA/p65 expression by siRNA-mediated silencing. Consistent with our supershift assay (Fig. 3B), siRNA-mediated silencing of c-Rel expression led to almost complete suppression of K13-induced HIV-1 LTR activation (Fig. 4C). Similarly, silencing of p65 expression led to significant suppression of HIV-1 LTR activity, although some residual activity was still evident (Fig. 4C). Although p100 acts as a precursor of p52, another important function of p100 is to retain the RelB/p50 and RelB/p52 complexes in the cytoplasm. As such, in order to shut-off the alternate NF-κB pathway, we chose to silence the expression of RelB. As shown in Fig. 4B–C, siRNA-mediated downregulation of RelB, had no significant effect on K13-induced HIV-1 LTR activity. We also failed to observe any effect of p100/p52 silencing on HIV-1 LTR activation (data not shown). Taken together, the above results demonstrate a key role of the c-Rel and p65 subunits of the classical NF-κB pathway in K13-induced HIV-1 LTR reporter activation.
Role of individual subunits of the IKK complex in K13-induced HIV-1 LTR activation
K13 is known to associate with a 700 kDa multi-subunit IKK complex, which consists of two catalytic subunits, IKK1/IKKα and IKK2/IKKβ and a regulatory subunit, NEMO/IKKγ [22]. We tested the involvement of the individual components of the IKK complex in vFLIP K13-induced HIV-1 LTR reporter activity by using mouse fibroblast (MEF) cells deficient in IKK1, IKK2 and NEMO, respectively. As shown in Fig. 5A, we observed significant HIV-1 LTR reporter activity by the expression of vFLIP K13 in the wild type MEF cells. In contrast, almost no HIV-1 LTR reporter activity was observed in NEMO-deficient cells. However, some residual HIV-1 LTR reporter activity was observed in IKK1- and IKK2-deficient MEF cells. Collectively, the above results suggest that synergistic action of IKK1, IKK2 and NEMO is required for maximal activation of HIV-1 LTR by K13.
Next we sought to determine whether pharmacological inhibitors of the NF-κB pathway may be used to block vFLIP K13-induced HIV-1 LTR reporter activation. Lactacystin and MG132 are inhibitors of proteasome and block the NF-κB pathway by preventing the degradation of IκB. On the other hand, arsenic acid is believed to block the NF-κB pathway by inhibiting the IKK complex [29]. As shown in Fig. 5B, vFLIP K13-induced HIV-1 LTR reporter activation was effectively blocked by MG132, lactacystin and arsenic acid. These results suggest that inhibitors of the NF-κB pathway might have a role in preventing K13-induced HIV-1 LTR reporter activation.
Effect of Murr1 on K13-induced HIV-1 LTR activation
Murr1 is a gene product that has been previously implicated in copper regulation [30,31]. A recent study demonstrated that Murr1 is highly expressed in CD4+ T cells and serve as a genetic inhibitor factor for HIV-1 replication in the resting lymphocytes [32]. Murr1 was shown to block HIV-1 LTR activation and HIV-1 replication by inhibiting the proteasomal degradation of IκB and blocking basal and cytokine-stimulated NF-κB activation [32]. Based on the above study demonstrating the importance of Murr1 as an endogenous regulator of HIV-1 LTR activation, we tested its effect on K13-induced HIV-1 LTR activation. As shown in Figure 5C, co-expression of Murr1 led to significant block in K13-induced HIV-1 LTR reporter activity, thereby suggesting that K13-induced activation of HIV-1 replication in resting lymphoid cells may be regulated by Murr1 and K13 may selectively activate HIV-1 replication in activated cells in which expression of Murr1 is known to be down-regulated [32].
Synergistic activation of HIV-1 LTR by vFLIP K13 and HIV Tat protein
HIV-1 Tat is a viral nuclear protein that plays an essential role in HIV-1 gene expression at the transcriptional level [2,3]. Tat has been shown to associate with p300/CBP and P/CAF histone acetyltransferases (HAT) and efficient activation of the integrated HIV-1 LTR is largely dependent on Tat-dependent rearrangement of the nucleosome positioned at the transcription start site [2]. HIV-1 LTR is known to bind and respond to HIV Tat protein via a specific Tat-binding site [2]. We used deletion mutagenesis of the HIV-1 LTR to test whether vFLIP induced transcriptional activation is dependent on this Tat-binding site. As shown in Figures 6A and 6B, a bulge mutant (containing deletion of nucleotides +23/+25) of HIV-1 LTR, which is defective in Tat activation [33], had no significant effect on vFLIP K13-induced reporter activity. In contrast, vFLIP K13 failed to activate a luciferase report construct containing an HIV-1 LTR in which the NF-κB binding sites had been mutated (Fig. 6C). The above results confirm that vFLIP activates the HIV-1 LTR via the NF-κB binding sites and can do so independent of the Tat-binding site.
Transcriptional activation of genes is usually regulated by multiple transcription factors acting in concert. Thus, while NF-κB has been shown to play a major role in the activation of the HIV-1 LTR, it fails to do so when acting alone [25,34-36]. Along the same lines, the transactivating function of Tat protein requires the presence of NF-κB sites in the HIV-1 LTR and Tat protein is known to cooperate with NF-κB to activate the HIV-1 LTR [1,34,36]. We hypothesized that a functional interaction between K13-induced NF-κB and Tat may be particularly important in the early stages of HIV-1 infection when the amount of Tat is limited. To test this hypothesis, we began by performing a dose-response analysis of Tat and selected a dose of Tat (20 ng/ml) which led to sub-maximal activation of HIV-1 LTR activation in 293T cells (Fig. 6D). Next, we analyzed the effect of co-expression of Tat on K13-induced HIV-1 LTR activation. As shown in Figure 6E, while transfection of K13 (250–500 ng/well) led to approximately 2.5–3.5 fold increase in HIV-1 LTR activation, transfection of Tat (20 ng/well) induced 4-fold increase in HIV-1 LTR activity. However, co-expression of K13 with Tat led to a synergistic 12-fold activation of the HIV-1 LTR. These results suggest that K13-induced NF-κB functions synergistically with the Tat protein to activate the HIV-1 LTR.
Effect of vFLIP K13 on LTRs-Derived from different strains of HIV
There is considerable sequence diversity among the HIV-1 isolates that comprise the current global pandemic and these can be grouped into several distinct subtypes or clades [37]. In particular, the LTRs of different subtypes show distinct enhancer-promoter configuration and vary in the sequence and number of binding sites for different transcription factor, including NF-κB [38,39]. Although different HIV-1 LTRs are transcriptionally active, they differ in the level of basal reporter activity [38,39]. In addition, different HIV-1 LTRs are known to show differential response to TNF-α treatment, which correlates with the number of NF-κB binding sites [38,39]. Therefore, we sought to determine whether vFLIP K13 will differentially activate luciferase reporter constructs driven by LTRs derived from different HIV strains. Consistent with the published studies [38,39], we observed considerable difference in the basal activities of different HIV-1 LTRs promoters when transfected into 293T cells along with an empty vector (Fig. 7A). More importantly, coexpression of vFLIP K13 led to differential activation of luciferase reporter constructs containing LTRs from different subtypes of HIV-1 (Fig. 7A). Thus, subtype C, which possesses three NF-κB binding sites showed the maximum increase in vFLIP-induced HIV-1 LTR reporter activity while subtype E, which possesses only one NF-κB binding site showed the lowest level of basal and vFLIP-induced HIV-1 LTR transcriptional activation (Fig. 7A,B). These results demonstrate that, similar to situation with TNFα, K13 may differentially activate LTRs derived from different strains of HIV-1, which correlate with their NF-κB binding sites.
Discussion
Although co-infection with HHV-8 and HIV-1 is known to synergistically increase the incidence of KS, until recently intracellular interaction between HHV8 and HIV-1 has not received adequate attention under the assumption that these viruses infect distinct cell types. Thus, HHV8 is typically believed to infect B lymphocytes, epithelial cells, keratinocytes, KS tumor cells, and endothelial cells [40,41], while the predominant host cells for HIV-1 are CD4+ T lymphocytes, dendritic cells, and mononuclear phagocytes [41,42]. However, as recently pointed out by Huang et al, several lines of evidence suggest that the above assumption may not be completely true and HHV8 and HIV-1 may, in fact, interact in vivo [41]. First, both HHV8 and HIV-1 can efficiently infect cells of monocyte/macrophage lineage, including dendritic cells [43,44]. Second, Moir et al have shown that induction of CD4 and CXCR4 on B cells by CD40 stimulation leads to an increased susceptibility of these cells to T-trophic HIV infection [45]. Third, HHV8-infected B cells can be infected by HIV-1 via a cell-cell pathway and such infected B cells can support productive HIV-1 replication [46]. Finally, the range of HHV8-susceptible cells in vivo is unclear at the present. Therefore, it stands to reason that HHV8 and HIV-1 genomes may co-exist in the same cells in vivo and reciprocally regulate the gene expression of each other. Support for the above hypothesis is provided by a recent study which demonstrated that co-culture of HIV-1-infected CD4+ T cells with HHV8-infected B cell lines resulted in increased HIV-1 replication [47].
With the goal of elucidating intracellular signaling interactions which could be potentially involved in the induction of HIV-1 replication by HHV-8, we carried out a detailed analysis of the effect of HHV8 vFLIP on HIV-1 LTR activation. Consistent with an earlier report, we observed that HHV8 vFLIP strongly activates HIV-1 LTR in an NF-κB-dependent fashion [48]. We further demonstrate that vFLIP K13 could activate HIV-1 LTR in both epithelial and human lymphoma cell lines, although the magnitude of stimulatory effect was more pronounced in the epithelial cells. A possible explanation for this difference may lie in the differential expression of proteins that could modulate the effect of K13 on NF-κB and/or HIV-1 LTR activation. As an example, we demonstrate that K13-induced HIV-1 LTR activation can be effectively blocked by Murr1, a recently identified inhibitor of the NF-κB pathway which is highly expressed in T cells [32]. However, alternative explanation, including difference in the transfection efficiency between different cell lines, could apply as well.
We have recently reported that vFLIP K13 can activate both the classical and alternate NF-κB pathways and, as such, we were interested in determining the relative contribution of these pathways to K13-induced HIV-1 LTR activation. Based on the following data, we believe that activation of HIV-1 LTR is mainly through the classical pathway. First, our gel super-shift assay demonstrated that NF-κB complexes formed by vFLIP expression were primarily composed of c-Rel, p50 and p65 subunits. Second, siRNA-mediated downregulation of c-Rel and p65 led to near complete inhibition of K13-induced HIV-1 LTR activation whereas silencing of RelB expression was without significant effect. Third, K13-induced HIV-1 LTR activation was completely inhibited by super-repressor form of IκBα, which primarily blocks the classical NF-κB pathway. Finally, while K13 activates the alternate NF-κB pathway independent of IKK2, it failed to activate the HIV-1 LTR in IKK2-deficient MEFs.
Based on some early gene-knockout studies, IKK1 was believed to be not involved in cytokine-induced activation of the classical NF-κB pathway [49-51]. In the present study, we have observed that, in addition to IKK2- and NEMO-deficient MEFs, K13-induced HIV-1 LTR activation was markedly reduced in IKK1-deficient MEFs as well. We believe that the above results with IKK1-deficient cells do not necessarily support the involvement of the alternate NF-κB pathway in K13-induced HIV-1 LTR activation for the following reasons. First, we have recently reported that K13-induced p65/50 DNA binding and NF-κB transcriptional activation is markedly reduced in IKK1-deficient MEFs [23] Thus, the reduced HIV-1 LTR activation in the IKK1-deficient cells observed in the current study is consistent with requirement for IKK1 in K13-induced classical NF-κB activation. Second, recent studies suggest that IKK1 may be involved in transcriptional activation of classical NF-κB responsive genes through its ability to phosphorylate histones and p65 [52-54]. Thus, taken together, our results demonstrate that K13 activates HIV-1 LTR through the activation of the classical NF-κB pathway, in which IKK1 plays a major role. Thus, selective inhibitors of IKK1 may have a role in blocking K13-induced HIV-1 LTR transcriptional activation. However, it is important to point out that while IKK1 may be uniquely important for K13-induced classical NF-κB activation pathway, maximal activation of this pathway via K13 relies on cooperative interaction between IKK1, IKK2 and NEMO.
The transcription of cellular and viral genes is regulated by structural and functional interactions among a number of transcriptional factors that act in concert. This is also known to be the case with HIV-1 LTR. Thus, while NF-κB plays a major role in the transcriptional activation of HIV-1, it requires synergistic interaction with a number of cellular and viral proteins for maximal stimulation of this activity [1]. Although NF-κB is known to interact with Sp1, Ets and NF-AT to activate HIV-1 LTR, cooperative interaction between NF-κB and Tat has received the most interaction in the literature [1,55]. Tat has been shown to act synergistically with PMA, PHA and Tax-induced NF-κB to activate the HIV-1 LTR [1,34,36,55]. Consistent with these previous studies, we demonstrate that although K13 can activate the HIV-1 LTR by itself, it functionally cooperates with Tat to synergistically activate transcription from HIV-1 LTR. HIV-1 infection itself is known to induce persistent NF-κB activation, which is probably mediated via Tat and Nef [56,57], and interacts in a positive-feedback manner with Tat to enhance HIV-1 replication. However, in the immediate post-integration period of the HIV-1 life-cycle, Tat is expressed at very low levels which may not be enough to effectively stimulate HIV-1 LTR activation. Therefore, it is conceivable that vFLIP K13 could amplify the activity of Tat via NF-κB activation and thus support enhanced HIV-1 replication during the early stages of HIV-1 infection or in cells which express Tat at suboptimal levels.
The human immunodeficiency virus has considerably diversified during its worldwide spread in the current pandemic and can be classified into several distinct subtypes [37]. Subtype B is predominant in North America and Europe, subtype E in Southeast Asia and subtype C in sub-Saharan Africa, respectively [58]. Previous studies have demonstrated that LTRs from HIV-1 subtypes B, C and E vary in number and binding sites for NF-κB in their enhancer elements [59]. Thus, subtype C isolates are known to contain three functional NF-κB binding sites, as compared to two such sites in the enhancer of the more commonly studied subtype B [59]. On the other hand, in the subtype E, one of the NF-κB-binding sites has been switched to a GABP site, resulting only one functional NF-κB site and gain of a new specificity [60]. Consistent with the above results, in the present study we demonstrate that vFLIP-induced HIV-1 LTR activation is strongest in subtype C and weakest in subtype E. Thus, the differential response of different HIV-1 LTRs to K13-induced transcriptional activation may be explained on the basis of number of functional NF-κB sites in their enhancer elements. Future studies should address the question whether co-infection with HHV8 has a differential effect on the replication and natural history of different HIV-1 subtypes.
Methods
Plasmids, cell lines and reagents
Plasmids containing pcDNA3-K13-Flag and pcDNA3-E8-Flag, pRSV/LacZ and 293T cells have been described previously [21]. An expression construct encoding Murr-1 was generated by RT-PCR using cDNA prepared from H460 cells as a template and subsequently cloned in pcDNA3 vector with a C-terminal HA tag. Luciferase reporter constructs containing LTRs derived from different strains of HIV-1 (pBlue3'LTR-Luc-A-F) were obtained from AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH from Drs. Reink Jeeninga and Ben Berkhout. Wild-type and mutant HIV-1 LTR reporter constructs [61] and expression constructs for HIV Tat were obtained from Dr. Richard Gaynor. The IKKα-/- and IKKβ-/- mouse embryonic fibroblast cells were generated in Dr. Inder Verma's laboratory [62,63] and IKKγ/NEMO-/- cells were generated in Dr. Michael Karin's laboratory [64]. These cells were kindly provided by Dr. Richard Gaynor and were maintained in DMEM supplemented with 10% FBS. Jurkat cells were cultured in RPMI medium supplemented with 10% FBS and selected in the presence of 1500 μg/ml of G418 (Invitrogen). 293T cells were grown in DMEM with 10% FBS. Arsenic Acid was purchased from Sigma. MG132 and lactacystin were purchased from Calbiochem and Biomol, respectively. Retrovirus constructs containing C-terminal Flag epitope tagged HHV8 vFLIP (K13-Flag) was generated in MSCV neo-based retroviral vector and amphotropic viruses generated and used for infection as described previously [22].
Electrophoretic mobility shift assay
Electrophoretic mobility shift assay was performed essentially as described previously [22], except an HIV LTR oligonucleotide duplex (sense strand, 5' TGC TAC AAG GGA CTT TCC GCT GGG GAC TTT CCA GG 3') was used instead of κB binding oligonucleotide. Nuclear extracts were prepared from Jurkat cells stably expressing an empty vector or vFLIP K13, which have been described previously [23]. Antibodies against p50, p65, RelB and c-Rel were purchased from Santa Cruz Biotechnology. An antibody against p52 was purchased from Upstate biotechnology
Luciferase reporter assay
293 T cells were transfected in duplicate in a 24-well plate with the various test plasmids along with an HIV LTR/luciferase reporter construction (10 ng/well) and a pRSV/LacZ (β-galactosidase) reporter construct (75 ng/well) using calcium phosphate transfection protocol as described previously [21]. Cells were lysed 36–48 hours later and extracts were used for the measurement of firefly luciferase and galactosidase activity. Luciferase activity was normalized relative to the galactosidase activity to control for the difference in the transfection efficiency. Cos-7, Jurkat and MEF cells were transiently transfected with empty vector (pCDNA3) or K13 (500 ng/well) along with a HIV/luciferase reporter construct (100 ng/well) and a synthetic Renilla luciferase (phRL-TK; Promega) reporter vector (75 ng/well) by using LIPOFECTAMINE 2000 Reagent (Invitrogen, Carlsbad, CA) according to manufacturer's instruction. Thirty-six hours after transfection, cells lysates used for reporter assays. Luciferase activity was normalized relative to the Renilla luciferase activity to control for the difference in the transfection efficiency. The values shown are averages (Mean ± S.E.) of one representative experiment out of three in which each transfection was performed in duplicate.
siRNA Oligonucleotides
siRNA oligonucleotides with two thymidine residues (dTdT) at the 3'-end of the sequence were designed to p65 (sense, 5'-GCCCUAUCCCUUUACGUCAdTdT-3'), c-Rel (sense, 5'-CAACCGUGCUCCAAAUACU dTdT-3'), RelB (sense, AGAUCAUCGACGAGUACAUdTdT-3') and control (sense, 5' GCGCGCUUUGUAGGAUUCGdTdT-3'), along with their corresponding antisense oligonucleotides. The RNA oligonucleotides were synthesized at RNA Oligonucleotide Synthesis Core facility, UT Southwestern Medical center. siRNA oligonucleotides (80 nM) were transfected using calcium phosphate as described previously [65].
Western Blot
Western blot analysis was performed essentially as described previously [22]. Primary antibodies used in these experiments were: p65, c-Rel, Rel-B (rabbit polyclonal, Santa Cruz biotechnology) and actin (mouse monoclonal, Sigma).
List of Abbreviations Used
DED, death effector domain; EMSA, electrophoretic mobility shift assay; FLICE, Fas-associated death domain-like IL-1 beta-converting enzyme; FLIP, FLICE inhibitory protein; HHV8, Human herpes virus 8; HIV-1, human immunodeficiency virus 1; KS, Kaposi's sarcoma; NEMO, NF-κB essential modulator; NIK, NF-κB-inducing kinase; NF-κB, Nuclear factor kappa B; MEF, murine embryonic fibroblast; PEL, primary effusion lymphoma, TNFR, Tumor necrosis factor receptor; IκB, inhibitor of NF-κB, IKK, IκB kinase; vFLIP, viral FLICE inhibitory protein; LTR, long terminal repeat.
Competing Interests
The author(s) declare that they have no competing interests.
Authors' contributions
QS and HM carried out most of the experiments described in this manuscript. PMC conceived of the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
Acknowledgement
We will like to thank Drs. Inder Verma, Michael Karin and Richard Gaynor for MEF cells and various expression and reporter plasmids. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: pBlue3'LTR-Luc-A-F from Drs. Reink Jeeninga and Ben Berkhout. This work was supported by a grant from the National Institutes of Health (CA85177).
Figures and Tables
Figure 1 K13 activates HIV-1 LTR promoter. A. 293T cells were transfected with an empty vector or the indicated constructs (100 ng/well) along with an HIV-1 LTR/luciferase reporter construct (10 ng/well) and a pRSV/LacZ (β-galactosidase) reporter construct (75 ng/well), and the experiment was performed as described under "Materials and Methods." The values shown are averages (Mean ± S.E.) of one representative experiment out of three in which each transfection was performed in duplicate. B. A dose-response analysis of HIV-1 LTR activation by K13 and pro-inflammatory cytokines. 293T cells were transfected with the indicated amounts of a K13 expression plasmid and luciferase assay performed 36 h post-transfection as described for (A). The total amount of transfected DNA was kept constant by adding an empty vector. For experiments involving TNF-α and IL-1β, cells were treated with the indicated concentration of cytokines 12 h after transfection of the reporter plasmids and assayed for reporter activity after 24 h of stimulation. C. K13 activates HIV-1 LTR in Cos-7 cells. The experiment was performed as described in 1A except LIPOFECTAMINE 2000 Reagent (Invitrogen, Carlsbad, CA) was used for transfection and Renilla luciferase was used for normalization. D. K13 activates HIV-1 LTR in Jurkat cells. The experiment was performed as described for 1C by using LIPOFECTAMINE 2000 Reagent (Invitrogen, Carlsbad, CA).
Figure 2 Activation of HIV- LTR by K13 mutants correlates with their ability to activate the NF-κB pathway. A. NF-κB activation by mutants of K13. 293T cells were transfected with an empty vector (pCDNA3) or the indicated K13 expression constructs (100 ng/well) along with an NF-κB/luciferase reporter construct (75 ng/well) and an pRSV/LacZ (β-galactosidase) reporter construct (75 ng/well) and luciferase reporter assay performed as described in Fig. 1A. The values shown are averages (mean ± SEM) of one representative experiment out of three in which each transfection was performed in duplicate. B. HIV-1 LTR activation by wild-type and mutant K13 constructs. The experiment was performed as described for Fig. 1A.
Figure 3 Electrophoretic mobility shift assay. A. The nuclear extract from Jurkat cells stably expressing an empty vector (lane 1) and K13 (lanes 2–4) were used for EMSA. The position of the induced HIV-1 LTR complex is marked with an asterisk. The specificity of the complex is demonstrated by competition with excess cold HIV-1 LTR probe (lane 3) and a nonspecific (N.S.) probe (lane 4), respectively. B. A supershift assay showing the subunit composition of K13-induced NF-κB subunits bound to HIV-1 LTR. The supershift assay was performed using a control rabbit antisera (lane 3), control mouse antisera (lane 4), or antisera against p50 (lane 5), p65 (lane 6), p52 (lane 7), Rel B (lane 8) and c-Rel (lane 9) subunits of NF-κB, respectively. The position of the induced HIV-1 LTR complex is marked with an asterisk, while the super-shifted bands are marked by arrowheads.
Figure 4 K13 activates HIV LTR through the classical NF-κB pathway. A. 293T cells were transfected with an empty vector or K13 along with an HIV LTR/luciferase reporter construct and a β-galactosidase reporter construct as described in Fig. 1A. The amount of IκBαSS32/36AA inhibitor plasmid (500 ng/well) was five times the amount of vector (pcDNA3) or K13 (100 ng/well) plasmid. The values shown are averages (Mean ± S.E.) of one representative experiment out of three in which each transfection was performed in duplicate. B. Western blot analysis showing siRNA-mediated knock-down of p65, c-Rel and RelB expression. The blot was re-probed with a monoclonal antibody against actin (bottom panel) to show equal loading of all lanes and specificity of gene silencing. C. 293T cells were transfected with an empty vector or a K13 expression plasmid along with a control siRNA oligo-duplexes or siRNA duplexes against c-Rel, p65 and RelB, respectively. The luciferase reporter assay was performed as described in Fig 1A.
Figure 5 Mechanism of K13-induced HIV-1 LTR activation. A. Role of IKK complex in K13-induced HIV-1 LTR reporter activity. Wild-type and IKK-deficient cells were transiently transfected with an empty vector or K13 expression plasmid (500 ng/well) along with an HIV/luciferase reporter construct (100 ng/well) and a synthetic Renilla luciferase (phRL-TK) reporter vector (75 ng/well) by using LIPOFECTAMINE 2000 Reagent (Invitrogen, Carlsbad, CA) according to manufacturer's instruction. Thirty-six hours after transfection, cell lysates were used for reporter assays. Luciferase activity was normalized relative to the Renilla luciferase activity to control for the difference in the transfection efficiency. The values shown are averages (Mean ± S.E.) of one representative experiment out of three in which each transfection was performed in duplicate. B. Inhibitors of the NF-κB pathway significantly block K13-actived HIV LTR promoter. 293T cells were transfected with the empty vector or K13 along with an HIV LTR/luciferase reporter construct and a β-galactosidase reporter construct as described in Fig.1A. Eight hours after transfection, cells were treated with DMSO or different inhibitors for 24 h and then lysed for the reporter assay. The values shown are averages (Mean ± S.E.) of one representative experiment out of three in which each transfection was performed in duplicate. C. Effect of Murr1 on K13-induced HIV-1 LTR activation. 293T cells were transfected with an empty vector (pCDNA3) or K13 along with an HIV LTR/luciferase reporter construct and a β-galactosidase reporter construct as described in Fig. 1A. The amount of Murr1 plasmid (500ng/well) was five times the amount of vector or K13 (100ng/well). The values shown are averages (Mean ± S.E.) of one representative experiment out of three in which each transfection was performed in duplicate.
Figure 6 Effect of HIV Tat protein on K13-induced HIV-1 LTR activation. A-C. 293T cells were transfected with an empty vector (pCDNA3) or K13 along with different HIV LTR/luciferase reporter constructs and β-galactosidase as described in Fig. 1A. The values shown are averages (Mean ± S.E.) of one representative experiment out of three in which each transfection was performed in duplicate. A, Wild type HIV-1 LTR reporter; B, HIV-1 LTR reporter with deletion of Tat-binding site (nucleotides +23 to +25); C. HIV-1 LTR reporter lacking the NF-κB binding-sites. D. Dose-response analysis of Tat-induced HIV-1 LTR activation. E. K13 and Tat synergistically activate HIV-1 LTR. The experiment was performed as described for Fig. 6 A. The total amount of transfected DNA was kept constant by adding empty vector.
Figure 7 Differential activation of HIV-1 subtype LTRs by K13. A. 293T cells were transfected with an empty vector (pCDNA3) or K13 along with luciferase reporter constructs containing LTRs derived from the indicated strains of HIV and a β-galactosidase reporter construct as described in Fig. 1A. The values shown are averages (Mean ± S.E.) of one representative experiment out of three in which each transfection was performed in duplicate. B. Partial sequence of LTRs of HIV-1 subtypes A through F. The LTR region spanning positions -129 to -77 of subtype A is shown at the top. The NF-κB binding motifs are shaded gray.
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| 15713234 | PMC554086 | CC BY | 2021-01-04 16:05:48 | no | Retrovirology. 2005 Feb 15; 2:9 | utf-8 | Retrovirology | 2,005 | 10.1186/1742-4690-2-9 | oa_comm |
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Front ZoolFrontiers in Zoology1742-9994BioMed Central London 1742-9994-2-41574061810.1186/1742-9994-2-4HypothesisAsymmetry in host and parasitoid diffuse coevolution: when the red queen has to keep a finger in more than one pie Lapchin Laurent [email protected] Thomas [email protected] "Biologie des Populations en Interaction", UMR 1112 "Réponse des Organismes aux Stress Environnementaux", Inra/Unsa, 400 route des Chappes, BP167 06903 Sophia-Antipolis cedex, France2005 1 3 2005 2 4 4 10 12 2004 1 3 2005 Copyright © 2005 Lapchin and Guillemaud; licensee BioMed Central Ltd.2005Lapchin and Guillemaud; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Coevolution between pairs of antagonistic species is generally considered an endless "arms race" between attack and defense traits to counteract the adaptive responses of the other species.
Presentation of the hypothesis
When more than two species are involved, diffuse coevolution of hosts and parasitoids could be asymmetric because consumers can choose their prey whereas preys do not choose their predator. This asymmetry may lead to differences in the rate of evolution of the antagonistic species in response to selection. The more long-standing the coevolution of a given pair of antagonistic populations, the higher should be the fitness advantage for the consumer. Therefore, the main prediction of the hypothesis is that the consumer trophic level is more likely to win the coevolution race.
Testing the hypothesis
We propose testing the asymmetry hypothesis by focusing on the tritrophic system plant/aphid/aphid parasitoid. The analysis of the genetic variability in the virulence of several parasitoid populations and in the defenses of several aphid species or several clones of the same aphid species could be compared. Moreover, the analysis of the neutral population genetic structure of the parasitoid as a function of the aphid host, the plant host and geographic isolation may complement the detection of differences between host and parasitoid trophic specialization.
Implications of the hypothesis
Genetic structures induced by the arms race between antagonistic species may be disturbed by asymmetry in coevolution, producing neither rare genotype advantages nor coevolutionary hotspots. Thus this hypothesis profoundly changes our understanding of coevolution and may have important implications in terms of pest management.
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Background
Coevolution is the result of reciprocal selective pressures exerted by interacting species. Many studies have been devoted to the hypothesis of an endless "arms race" between antagonistic species, in which each species develops escalating attack and defense traits to counteract the adaptive responses of the other species. In reference to Lewis Carroll's book "Through the Looking Glass", Van Valen [1] named this model of coevolution "the Red Queen Hypothesis" (RQH) because, even in a constant physical environment, interacting species must evolve continuously to maintain their position. The RQH can be seen as an arms race between "resistance" and "virulence" where, following recent reviews on coevolution (e.g. [2]), "resistance" is the target's ability to survive attacks by the consumer, and "virulence" is the consumer's ability to defeat the target's defenses.
The RQH was initially developed in the context of multiple species interactions, to account for the constant probability of species extinction. However, as modeling the coevolution of many species involves a number of difficulties, later studies based on the RQH have mostly been limited to interactions between pairs of species. From this restricted situation, two main predictions can be made: first, arms races induce an advantage of rare genotypes of which resistance or virulence is more efficient and thus, frequency-dependent fluctuations of resistance and virulence may be predicted. Second, a geographic view of the coevolutionary process suggests a dynamic mosaic structure, with local and temporary "hot spots" of antagonistic species coevolution [3].
However, for plant-pathogen interactions [4] and for animal host-parasite interactions [5-7], empirical observations and experimental tests of RQH have not given entirely convincing results. For instance, there is considerable evidence of genetic variation for resistance, but parasite-driven genetic change in resistance has never been observed directly [8]. Conversely, in certain systems of interacting species, matching genetic diversity, which is expected under the RQH, is lacking [9]. Another study [2] has suggested that, for a given host-parasitoid association, the rank order of survival of different host strains exposed to different parasitoid strains remains constant. This lack of frequency dependence has been interpreted as evidence against the RQH.
Nevertheless, the RQH continues to lie at the heart of the debate concerning the coevolution of antagonistic species, probably because of the lack of plausible alternative hypotheses. Although metapopulation structures or time lags between the responses of antagonistic species to selective pressures have been evoked as reasons for the lack of clear experimental evidence in favor of the RQH [10], the additional hypothesis that is considered the most satisfactory relates to the cost of resistance [11]. Defense is thought to carry costs when it is not needed, i.e. the fitness of resistant individuals in the absence of enemies is reduced compared to susceptible individuals. Following this hypothesis, the arms race is constrained by the resistance cost and thus resistance and virulence of antagonistic species reach fixed levels that can be considered optimal. This hypothesis may result in a low level of genetic polymorphism of resistance and virulence in spatially restricted areas. In contrast, resistance and virulence are expected to be polymorphic on a larger spatial scale if, because of ecological factors, the densities of antagonistic species are spatially heterogeneous. However, although resistance costs have often been demonstrated, expected geographical patterns of resistance and virulence are not observed: antagonistic species densities are not clearly spatially associated with resistance and virulence variability levels [2]. Thus, although taking account the resistance cost hypothesis is a useful addition to Red Queen arms race models, this theoretical view of coevolutionary process does not provide predictions that fit well with most situations observed in the field [10,11].
Presentation of the hypothesis
Adaptive responses to selective pressures exerted by antagonistic organisms are not necessarily the same for the target species and the consumer species. There is a first level of potential evolutionary asymmetry between them because the failure of virulence for a consumer is a delay in fitness acquisition whereas the failure of defense for a prey is the loss of its entire fitness. Such a difference was underlined by Dawkins [12] noting Aesop's fable of the hare outrunning the fox, because the former runs for life whereas the latter runs for a meal. The opposite asymmetry has been suggested in the case of host-parasitoid relationships [2,13]: all consumers are obliged to overcome the defenses of their target whereas not all targets suffer attacks from consumers.
Extending the field of interest to interactions and coevolution between more than two species (i.e. diffuse coevolution) may offer new perspectives. Considering the reciprocal selective pressures exerted by many species leads us to take into account the specificity of virulence and resistance. Until now, few works have been devoted to this subject (but see [14]). However, when considering diffuse coevolution, there is a second level of potential asymmetry because a consumer may choose its targets, whereas targets cannot be certain which of its enemies will attack it.
A recent model described the evolution of resistance of one host subjected to the attacks of two types of parasitoids differing in their virulence and specificity (different genotypes of a species or different species) and the evolution of virulence of one parasitoid attacking two types of hosts differing in their resistance and specificity [15]. The results suggested that the level of specialization of resistance traits was not affected by the total probability of being attacked. They also suggested that, if the probabilities of encounters fluctuate and differ between trophic levels, generalist traits of resistance and partially specialist traits of virulence are favored. Finally, this model showed that fluctuating host encounter probabilities across or within generations will promote a partial specialization of the parasitoid virulence rather than a total one.
The asymmetry hypothesis (AH) may lead to differences in the rate of evolution of the antagonistic species in response to selection. For a specialist consumer capable of target choice, chosen targets constitute a more or less "constant environment". On the other hand, for a generalist defender, the diversified, facultative and fluctuating attacks by a set of enemies constitute a "variable environment". The constancy of the environment may lead to the faster adaptation of specialists (mostly consumers under AH) than of generalists (mostly targets under AH) [16]. Thus, under the asymmetry hypothesis, the evolution of host defense traits is likely to be slower than the evolution of parasitoid virulence traits. The more long-standing the coevolution of a given pair of antagonistic populations, the higher should be the fitness advantage for the consumer. Therefore, the main prediction of AH is that the consumer trophic level is more likely to win the coevolution race [17]. This may account for the paradox described by Holt & Hochberg [18] – the lack of clear published examples of an increase in host resistance after biological control using parasitoids, despite the potentially strong selective pressure associated with parasitism.
The asymmetry hypothesis seems to fit well with numerous data concerning geographic structures published in the literature (reviewed in [15] but see also [19]). In most of these studies, the interaction trait under consideration is the capability of Drosophila species to encapsulate parasitoid eggs. In this case, it can be also noted that the resistance trait is not specific (artificial eggs are encapsulated as well as parasitoid ones) when virulence traits (reviewed in [20]) are diversified and specialized (i.e., escaping the host's immune response, just swamping the host with virus-like particles, or mimicking host tissue on the egg surface).
Testing the hypothesis
We propose a dedicated test of the hypothesis of the specialization of the parasitoid virulence and the absence of specialization in the defense of the aphids, against the RQH. The study will focus on the tritrophic system plant/aphid/aphid parasitoid. Two complementary experimental approaches could be considered: A- The analysis of the genetic variability of virulence and defense of the parasitoid Lysiphlebus testaceipes and the aphid Aphis gossypii. To measure this variability, several populations of the parasitoid collected on different clones of the aphid will be confronted to different clones of this aphid. The different clones of the aphid will be confronted to the different parasitoid populations. This system that considers intra specific genetic variability for each trophic level will be used to evaluate the fitness of the consumers and of the targets in the different parasitoid /aphid combinations. An important point is that virulence will be estimated from the success rate of parasitism (i.e. the production of offspring) whereas defense will be estimated through the measure of the aphid fitness, i.e. the number of offspring produced by the aphid whatever the outcome of the parasitism (success or failure). Two key factors of the host fitness will be considered: 1) the host survival rate in case of parasitism failure (an aphid may die or not, even if the parasitism does not result in parasitoid offspring production); and 2) the number of offspring produced by the host after parasitism: after a parasitoid's sting, the host may produce offspring until mummification in case of parasitoid embryo development or until a variable date in the case of parasitism failure. B- The analysis of the neutral population genetic structure of the parasitoid as a function of the aphid host, the plant host and geographic isolation. The specialization of L. testaceipes on different aphid clones can lead to a neutral genetic differentiation of the parasitoid as a function of the host and the plant because the reproduction of the parasitoid occurs soon after the emergence of the adults from their host. The genetic differentiation between populations of the parasitoid sampled from different aphid clones and from different plant species could be evaluated and compared to a putative effect of isolation by distance.
This test may allow local verification or rejection of the predictions of the AH (specialization of the parasitoid and generalism of the host). When performed several times on animals from diverse geographic origins, this should eventually allow rejection of the classical interpretation of RQH and the host spots theory of coevolution [3].
Implications of the hypothesis
The consequences of the asymmetry hypothesis are important. Genetic structures induced by the arms race between antagonistic species may be disturbed by asymmetry in coevolution, producing neither rare genotype advantages nor coevolutionary hotspots [17]. However some consequences of the AH are compatible with the resistance cost or the Red Queen hypotheses. Under the AH, a low variability in generalist resistance level, as expected under the hypothesis of regulation by the cost of resistance, would be selected for locally by the global pressure exerted by all the species of the upper trophic level. Also, higher levels of local variability in consumer specialist virulence would be selected, as in situations for which the classical RQH holds true.
The AH cannot be applied to every situation. In the case of host-parasitoid associations, the high level of genetic variability in resistance observed within some local host populations [20,21] is better explained by RQH if one dominant parasitoid species is present rather than a complex of species. Moreover, as generalism can be seen as a bet-hedging response to unpredictable environmental variations [15], the advantages of specificity or generalism depend on the level of such fluctuations. One can predict that host defenses will be less generalist when facing more stable enemy communities. Another key point deals with the supposed difference of speed in evolutionary responses between generalist and specialist traits. It may depend on other important biological factors like the discrepancy between population sizes, generation times and the reproductive mode (e.g. a sexual parasitoid versus an asexual aphid) or the genetic make up (e.g. an haplo-diploid parasitoid versus a diploid aphid) of the considered species. The relative speed of evolutionary responses of antagonistic species has thus to be evaluated in each biological model studied.
Asymmetry may be suspected in cases involving successive trophic levels other than host-parasitoid associations, such as plant-insect or parasitoid-hyperparasitoid combinations, as soon as individuals of the upper trophic level can choose their target. However, random attacks, such as those due to plant pathogens, may favor more generalist traits of virulence, but the specificity of consumers, and therefore asymmetry, may be restored through indirect choices: pathogens transmitted by vectors may use or manipulate the specificity traits of the vector. Large sets of species interactions may thus lead to asymmetric coevolution.
The AH may have consequences in terms of pest management. For instance, it is generally thought that variations in parasitism outcome result from a variability of host resistance due to the selection for higher resistance [2,22]. However, under the AH, virulence of the parasitoid is expected to evolve faster than does the resistance of the host. Therefore, the asymmetry hypothesis implies that variations in parasitism outcome more probably result from variability in parasitoid virulence. Asymmetry could also help to understand some surprising resistance-virulence patterns issuing from biological control. For instance, in the U.S.A., the pea aphid Acyrthosiphon pisum is parasitized by the hymenopteran Aphidius ervi and is specialized on different plant species. Hufbauer & Via [23] observed that pea aphids that are specialized on alfalfa are successfully parasitized less often than are pea aphids specialized on clover. Hufbauer [24] also observed that parasitoids collected from alfalfa and clover fields do not differ in their ability to overcome pea aphid resistance. They concluded that alfalfa host population is more resistant to A. ervi than is the clover host population. If the association they studied dealt with two aphid and one parasitoid populations, under the AH, virulence specialization rather than resistance level variations can explain the observations: the parasitoid population is specialized on the clover host population, but keeps a partial virulence on the alfalfa aphid biotype. This suggests that short-term parasitoid specialization may be a key factor in biological control efficiency. For instance, consumers introduced to control a pest could rapidly specialize against non-target hosts.
The asymmetry hypothesis thus provides food for thought concerning diffuse coevolution and could be applied to domains beyond host-parasitoid coevolution. Similar thoughts may be applicable to the durability and efficiency of plant resistance or immunological responses to diseases transmitted by vectors. Its theoretical implications and its consequences in terms of population management are potentially important and remain unexplored.
List of abbreviations
RQH: Red Queen Hypothesis
AH: Asymmetry Hypothesis
Authors' contributions
Both authors have been involved in the elaboration of the hypothesis and the evaluation of its consequences, and in the drafting of the paper.
Acknowledgements
We thank Corinne Vacher and two anonymous reviewers for helpful comments on the manuscript and Nick Miller for editorial help. This work was founded by "Action Concertée Incitative Ecologie Quantitative" from French "Ministère Chargé de la Recherche" and by the Department "Santé des Plantes et Environnement" of the "Institut National de la Recherche Agronomique".
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| 15740618 | PMC554087 | CC BY | 2021-01-04 16:38:34 | no | Front Zool. 2005 Mar 1; 2:4 | utf-8 | Front Zool | 2,005 | 10.1186/1742-9994-2-4 | oa_comm |
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Nutr JNutrition Journal1475-2891BioMed Central London 1475-2891-4-81572768210.1186/1475-2891-4-8ResearchOne year soy protein supplementation has positive effects on bone formation markers but not bone density in postmenopausal women Arjmandi Bahram H [email protected] Edralin A [email protected] Dania A [email protected] Latha [email protected] Brenda J [email protected] Jennifer [email protected] Andrea B [email protected] Mark E [email protected] Claudia [email protected] Department of Nutritional Sciences, Oklahoma State University, Stillwater, Oklahoma 74078, USA2 Department of Statistics, Oklahoma State University, Stillwater, Oklahoma 74078, USA2005 23 2 2005 4 8 8 21 12 2004 23 2 2005 Copyright © 2005 Arjmandi et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Although soy protein and its isoflavones have been reported to reduce the risk of osteoporosis in peri- and post-menopausal women, most of these studies are of short duration (i.e. six months). The objective of this study was to examine if one year consumption of soy-containing foods (providing 25 g protein and 60 mg isoflavones) exerts beneficial effects on bone in postmenopausal women.
Methods
Eighty-seven eligible postmenopausal women were randomly assigned to consume soy or control foods daily for one year. Bone mineral density (BMD) and bone mineral content (BMC) of the whole body, lumbar (L1-L4), and total hip were measured using dual energy x-ray absorptiometry at baseline and after one year. Blood and urine markers of bone metabolism were also assessed.
Results and Discussion
Sixty-two subjects completed the one-year long study. Whole body and lumbar BMD and BMC were significantly decreased in both the soy and control groups. However, there were no significant changes in total hip BMD and BMC irrespective of treatment. Both treatments positively affected markers of bone formation as indicated by increased serum bone-specific alkaline phosphatase (BSAP) activity, insulin-like growth factor-I (IGF-I), and osteocalcin (BSAP: 27.8 and 25.8%, IGF-I: 12.8 and 26.3%, osteocalcin: 95.2 and 103.4% for control and soy groups, respectively). Neither of the protein supplements had any effect on urinary deoxypyridinoline excretion, a marker of bone resorption.
Conclusion
Our findings suggest that although one year supplementation of 25 g protein per se positively modulated markers of bone formation, this amount of protein was unable to prevent lumbar and whole body bone loss in postmenopausal women.
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Background
It is estimated by the year 2010, 35 million women in the United States either will have osteoporosis or be at risk of developing the disease if appropriate preventive measures are not taken [1]. Aside from existing drug therapies, certain lifestyle and nutritional factors are known to reduce the risk of osteoporosis [2-5]. Additionally, there are a considerable number of women that would prefer dietary supplements as an alternative/adjunctive to conventional therapeutic options [5]. Examples of these alternative therapies include the use of natural or plant-based substances such as soy isoflavones [6-13]. Soy isoflavones have received considerable attention due to their estrogen-like properties on certain tissues such as bone, leading some investigators [14,15] to refer to them as naturally occurring selective estrogen receptor modulators (SERMs).
Epidemiological data suggest that populations with high intakes of soy, i.e. Asians, have a lower incidence of osteoporotic fractures [16,17]. Asian women typically consume about 20 g of soy daily which provides approximately 40 mg isoflavones [18,19]. However, lower rates of fractures in these populations may not be fully attributed to soy consumption as there are a number of other confounding factors which can influence skeletal health.
From the research point of view, there are a number of animal studies which have shown that soy protein and/or its isoflavones positively influence bone mineral density (BMD) [8,20-26]. In terms of human studies, there are limited numbers of trials that have examined the effects of soy and its isoflavones on bone. Some of these clinical trials [27-29] are of short duration varying between 3 to 6 months, making the findings questionable since periods less than one year may not be sufficient to detect clinically relevant changes in bone mass. Nonetheless, even the findings of the few clinical studies of one to two year duration that have been conducted [30-34] are inconclusive. For instance, Vitolins et al. [30] reported that daily consumption of 25 g soy protein with 5, 42 or 58 mg isoflavones had no bone preserving effects in peri- and post-menopausal women in a two-year study. Recent findings from two groups [31,32] have shown similar effects of soy protein and its isoflavones on bone. In contrast, other studies have suggested that isoflavone-rich soy milk delivering 80 to 90 mg isoflavones [33] or soy products delivering 40 to 60 mg isoflavones on a daily basis [34] have some bone protective effects.
The purpose of the present study was to examine the effects of one-year supplementation of soy-based products containing 25 g protein and 60 mg isoflavones on BMD, bone mineral content (BMC), serum and urinary markers of bone turnover in postmenopausal women. The rationale for choosing this amount of soy protein was based on the average intake of Asians [18,19] and the recommended amount in the FDA approved health claim [35].
Methods
Subjects
Postmenopausal women younger than 65 years who were not on HRT or any prescription medications or herbal supplements, including soy isoflavones, known to positively influence bone were recruited. Women with cancer, liver disease, hypo- or hyperthyroidism, gastrointestinal disorders, insulin-dependent diabetes mellitus, pelvic inflammatory disease, and endometrial polyps were excluded from the study. The study protocol was approved by the Institutional Review Board at Oklahoma State University. Subjects signed a consent form after being provided with oral and written descriptions of the study. A complete medical history was obtained from all subjects before initiating the treatments. Subjects were also given routine physical and gynecological examinations. Subjects were independent living and were advised to maintain their usual physical activity.
Study Design
Eighty-seven eligible postmenopausal women were randomly assigned to one of two dietary treatments in a double-blind parallel study. The dietary treatments consisted of 25 g protein from soy products (donated by DrSoy Nutrition Irvine, CA) or comparative control. The test foods were in the form of a snack bar, drink mix or cereal and were consumed daily for a period of one year. The soy products were soy protein-based and delivered 60 mg isoflavones per day whereas the control regimen was devoid of soy protein and isoflavones.
To ensure double-blinding, the study participants were randomly assigned to one of the two treatments and the study supplies were provided to the study participants in unlabeled packages. Additionally, the identity of each treatment was revealed to the investigators and research personnel involved in the collection and analyses of the data only after all analyses were completed. For this study compliance was measured in two forms. First, study participants were provided with customized calendars for subjects to record how much of each of the cereal, the snack bar, or the drink mix they consumed, if any. Second, study participants were asked to return any unconsumed foods to the study site so they could be tallied. The study participants were advised by a registered dietitian to make appropriate adjustments in their daily food consumption to account for the additional energy and nutrients supplied by the treatment regimen.
Dietary assessment and anthropometric measurements
For each subject, medical and nutrition histories were obtained at the beginning of the study. One-week food frequency questionnaires were completed via interview by a registered dietitian at the beginning and at the end of the study. Nutrient analysis was performed using food analysis software (Food Processor version 7.50, ESHA Research, Salem, OR). Anthropometric data were collected at the beginning, six months, and at the end of the study by a single trained staff member, as described elsewhere [36]. Height and weight were used to calculate body mass index (BMI). Abdominal and hip circumferences were used to calculate waist-to-hip ratio.
Bone Density Assessments
Bone density was assessed at the beginning and at the end of treatment using dual energy x-ray absorptiometry (DXA; Hologic QDR-4500C, Waltham, MA) equipped with appropriate software for whole body BMD and BMC. Additionally, select regional sites, i.e. total hip and lumbar spine (L1-L4) were analyzed using high resolution software. The intra- and inter- assay coefficients of variations were 3.4% and 5.1% and 2.5% and 4.7% for BMC and BMD, respectively.
Gynecological exam and blood and urine collection
Study participants were provided with routine physical and gynecological exams including a pap smear at baseline and at the end of the study. A venous blood sample was obtained after an overnight fast from each subject at the beginning, six months, and at the end of the study for various analyses. Blood samples were centrifuged at 2500 × g for 15 min at 4°C, serum samples were separated and stored at -20°C until analyses. Each study participant collected a 24-h urine specimen, excluding the first void, at the beginning, after six months, and at the end of the study. Urine volume was recorded and aliquots were stored at -20°C for later analyses.
Analytical methods
To assess whether soy isoflavones modulate sex steroids and their availability, serum levels of 17β-estradiol (E2), estrone (E1), estrone sulfate, follicle stimulating hormone (FSH), and sex hormone-binding globulin (SHBG) were assessed as we have previously described [37] using radioimmunoassay kits from Diagnostic Systems Laboratories Inc. (Webster, TX). Serum bone-specific alkaline phosphatase (BSAP) activity, a specific marker of bone formation [38], was quantified by immunoassay in a microtiter format (Metra Biosystems, Mountain View, CA). Alkaline phosphatase (ALP), a nonspecific marker of bone formation [38], was determined colorimetrically using a commercially available kit (Roche Diagnostics; Branchburg, NJ) and analyzed with a Cobas-Fara II Clinical Analyzer (Montclair, NJ). Additional serum biomarkers of bone formation i.e. osteocalcin, insulin-like growth factor-I (IGF-I), and IGF-binding protein-3 (IGFBP-3), which usually increases parallel to IGF-I, were also measured using kits from Diagnostic Systems Laboratories Inc.
Urinary creatinine was measured colorimetrically with commercially available kits (Roche Diagnostics) using a Cobas Fara II clinical analyzer. Urinary deoxypyridinoline (Dpd), a specific marker of bone resorption [39], was measured by competitive enzyme immunoassay in a microassay stripwell format (Quidel Corporation, Mountain View, CA). The intra- and inter-assay CVs were 4.3% and 4.6 %, and 6.5% and 8.6%, for creatinine and Dpd, respectively.
Statistical Analyses
Data were analyzed using analysis of variance methods with PROC MIXED in PC SAS (Version 8.2, SAS Institute, Cary, NC) analyzing the main and interaction effects of the two factors, treatment (soy protein or control) and time (baseline or after treatment), using the SLICE option. Since each subject was measured at baseline and after treatment, a split plot (repeated measures) model was utilized. The mean changes in endpoints for the soy protein and control treatment groups were compared by analyzing interaction effects of the two factors, treatment and time, using the SLICE option. Data are reported as least square mean ± standard error (SE); unless otherwise indicated, P < 0.05 was regarded as significant.
Results
Baseline characteristics, anthropometric measurements, and dietary intake
Sixty-two of the 87 women completed the one year study resulting in an attrition rate of approximately 29%. Reasons for dropping from the study included medical conditions preventing continuation in the study (2 women in the soy group and 1 in the control group), starting HRT (1 woman in the soy group and 3 in the control group), noncompliance (2 women in the soy group), dislike of the volume or flavor of the food (3 women in the control group), gastrointestinal side effects (2 women in the control group), food was causing headaches (1 woman in the control group), and personal reasons (3 women in the soy group and 2 in the control group). Five additional women in the soy group decided to discontinue the study without citing a particular reason.
Women in both treatment groups had similar baseline characteristics (Table 1). Body weight and BMI significantly increased in both treatment groups after one year of supplementation. On average, women in the soy group experienced a 1.6% increase in body weight, while a 3.3% increase was observed in women in the control group. Nonetheless, there was no significant change in waist to hip ratio in either treatment group. In terms of dietary intake, protein levels significantly increased in both treatment groups, as expected, due to the study supplements. Total caloric and carbohydrate intake increased in the control group, but decreased in the soy group after one year of supplementation (Table 2). No change in fat intake was found in either group.
Bone mineral density and bone mineral content
Subjects in both the soy and control groups lost whole body and lumbar BMD and BMC after one year, but no change was observed in the total hip (Table 3). Whole body BMD decreased by approximately 1.3% in both treatment groups, while lumbar BMD decreased by 1.0% in the soy group and 0.9% in controls (Figure 1). As shown in Figure 2, whole body BMC decreased by 1.4% and 1.0%, and lumbar BMC was reduced by 1.5% and 1.1% in the control and soy groups, respectively. Although the change in whole body bone mineral area (BMA) of the soy-treated group was increased and the control group remained relatively unchanged, neither of these alterations reached the level of statistical significance (data not shown). The decrease in whole body BMD of subjects on soy may have resulted from the small increase in whole body area and a significant decrease in BMC, whereas the change in BMD in the control group was primarily a reflection of a decrease in BMC. Lumbar and total hip BMA was unaltered by either dietary treatment (data not shown).
Serum and urinary parameters of relevance to bone and calcium metabolism
Both dietary protein supplements significantly increased serum markers of bone formation, i.e. osteocalcin, BSAP, IGF-I and ALP (Table 4). However, IGFBP-3, which usually increases parallel to IGF-I, was only significantly increased in the women consuming the soy products. Neither proteins had any effect on bone resorption as indicated by urinary Dpd (Table 4).
Since there are some reports [40-42] indicating that soy isoflavones may modulate sex steroids, we assessed serum levels of FSH, E2, E1, and estrone sulfate. None of the treatment significantly influenced these sex hormones (Table 5). However, soy but not control supplementation significantly decreased SHBG concentrations by 14.5%.
Discussion
The role of soy protein and its isoflavones in the maintenance of health such as the prevention of cardiovascular disease, certain types of cancer, and menopausal symptoms is now widely recognized [43-50]. In terms of bone, there are animal [20-26] and human [27-34] studies that have explored the role of soy in maintaining or increasing bone mass. In general, animal studies have shown that isoflavones in the context of soy protein have positive effects on BMD [20-26]. The findings of clinical trials have ranged from no significant changes [27-32] or a slight increase [28,33,34] in BMD. Nonetheless, the bone protective effects of soy and/or its isoflavones are at best inconclusive.
In the present study, the daily consumption of 25 g protein for one year irrespective of the source resulted in no significant changes in hip BMD and BMC. Other investigators [51,52] have reported that diets high in protein were associated with higher BMD in femoral neck. We speculate that higher dietary protein may have a protective effect on hip BMD over the long term. This notion, however, seems somewhat paradoxical because high protein diets, especially proteins rich in sulfur-containing amino acids, are known to increase urinary calcium that may result in accelerated bone loss [53]. Nonetheless, a counter-argument has been made that protein-associated hypercalciuria is due to enhanced intestinal calcium absorption and not the breakdown of bone [54,55].
Our findings do not support a bone protective role for soy protein and its isoflavones at the level used in this study. Whether higher amount of soy protein and/or its isoflavones can reverse bone loss remains to be illustrated. Nonetheless, higher doses of soy protein with varying levels of isoflavones have not consistently shown to exert beneficial effects on bone. For instance, Gallagher et al. [32] supplemented the diets of postmenopausal women for nine months with 40 g soy protein delivering three levels of isoflavones (0, 52, and 96 mg) but all three groups experienced bone loss. On the other hand, six-month studies by Potter et al. [28] and Alekel and colleagues [29] reported positive effects of soy protein supplementation on BMD. Potter et al. [28] showed that 40 g of soy protein containing 90 mg isoflavones was able to attenuate lumbar spine (L1-L4) BMD, however, the same amount of protein with 56 mg isoflavones had no such an effect. Although Alekel et al. [29] suggested that 40 g soy protein supplementation with 80 mg isoflavones was able to attenuate bone loss from lumbar spine, women still lost 0.2% BMD in six months. Their data [29] imply that soy protein or its isoflavones are incapable of increasing bone mass in perimenopausal women.
As for an effect of soy isoflavones alone, Chen et al. [56] recently reported that supplementing postmenopausal women with soy isoflavones (40 and 80 mg/d) for one year resulted in favorable increases in BMC of the hip in women who are at least four years postmenopausal and are of low body weight or have low levels of dietary calcium. Similar to that study [56], the majority of the women in our present study were four or more years postmenopausal; however, our study participants had adequate calcium intakes and did not have low body weights. It is possible that this difference in the nutrition status of the study participants between the two studies may be responsible for the discrepancy in the observed effects on bone.
As far as which of the many isoflavones in soy is responsible for the effects on bone, to date, the most convincing data on the effect of a single isoflavone, genistein, on bone have been reported in a one-year study by Morabito and colleagues [57]. They demonstrated that both genistein at a dose of 54 mg/d and HRT increased BMD in early postmenopausal women. In that study [57], genistein significantly increased BMD of the femoral neck by 3.6% and lumbar spine by 3.0% while HRT increased femoral neck and lumbar spine BMD by 2.4 and 3.8%, respectively. These authors [57] suggested that genistein reduces bone resorption markers and enhances new bone formation parameters resulting in a net gain of bone mass. Isolated isoflavones derived from other sources such as red clover have also been found to positively affect bone. For example, Clifton-Bligh and colleagues [58] reported that clover-derived isoflavones at doses of 57 and 85 mg isoflavones/day were able to significantly increase BMD of the proximal radius and ulna by 4.1 and 3%, respectively after 6 months. Similarly, Atkinson et al. [59] showed that red-clover derived isoflavones (26 mg biochanin A, 16 mg formononetin, 1 mg genistein, and 0.5 mg daidzein) slowed the loss of lumbar spine BMC and BMD. These data suggest that isoflavones from sources other than soy, also have osteoprotective effects.
In the present study, biomarkers of bone formation, i.e. osteocalcein, IGF-I, and BSAP were all significantly elevated in both groups. However, the specific marker of bone resorption, urinary Dpd, was not altered. A number of clinical studies have evaluated the effects of soy protein with its isoflavones on bone biomarkers. Overall, the effects of soy and/or its isoflavones have produced no consistent effects on biomarkers of bone turnover. For instance, biomarkers of bone formation have been reported to either increase [60,61] or not change [29,62] as result of soy supplementation. Similarly, biomarkers of bone resorption have been reported to decrease [62-64], not change [61] or even increase [60]. We have previously reported that 40 g of soy protein providing 90 mg of isoflavones/day reduced Dpd in postmenopausal women not on HRT [62]. In the present study, participants were asked to consume a lower amount of soy protein (only 25 g providing 60 mg of isoflavones/day). It is possible that the reduced dose of soy in this study may have contributed to the lack of effect on bone resorption (as assessed by Dpd excretion) and that a dose-response study may be justified in order to achieve both increases in markers of bone formation and reductions in markers of bone resorption. Nevertheless, the positive changes in biomarkers of bone formation in the present study have not translated to increases in BMD and BMC. Whether the positive effects of protein supplementation on bone biomarkers would translate to better bones needs to be assessed in a longer term study.
Although in this study, soy supplementation for one year did not produce any estrogenic effects as assessed by circulating sex hormone levels, it did decrease SHBG concentrations Decreases in SHBG result in increases in the availability of circulating estrogens [65]. Thus, soy supplementation may have increased the availability of estrogens without affecting actual concentrations. However, this is speculative and measurement of bioavailable estradiol is necessary to confirm this statement.
From the findings of our study and the collective review of existing literature, it is too early to state whether soy protein or its isoflavones can be substituted for estrogen in preventing the bone loss induced by ovarian hormone deficiency. Future studies are needed to address numerous questions including but not limited to whether: 1) isoflavones independent of soy protein can prevent ovarian hormone deficiency-associated bone loss; 2) consumption of soy containing food or intake of isoflavones on a daily basis is necessary to observe the expected beneficial effects on bone or simply intermittent use will produce the same results; 3) the effect of soy protein or its isoflavones on bone is transitory; and 4) the combination of soy isoflavones and lower doses of antiresorptive agents can prevent postmenopausal bone mineral loss. As these and other questions are answered, the efficacy of soy protein and its isoflavones as alternative and/or adjunctive treatments for postmenopausal osteoporosis can be determined.
Competing interests
The author(s) declare that they have no competing interests.
Acknowledgements
Supported in part by grants from the Oklahoma Center for the Advancement of Science and Technology (#AR982-006) and DrSoy Nutrition (Irvine, CA).
Figures and Tables
Figure 1 Mean percent change from baseline values in bone mineral density (BMD) of the whole body, lumbar (L1-L4), and hip after one year of supplementation with control or soy foods. Bars represent least square means ± SE.
Figure 2 Mean percent change from baseline values in bone mineral content (BMC) of the whole body, lumbar (L1-L4), and hip after one year of supplementation with control or soy foods. Bars represent least square means ± SE.
Table 1 Subject characteristics at baseline and at the end of the study
Measures Control (n = 27) Soy (n = 35) Control vs Soy
Baseline Final %change Baseline Final %change p values
Trtmt Time Trtmt × Time
Age (yrs) 56 ± 5 53 ± 6
Years since menopause 6 ± 5 5 ± 5
Weight (kg) 71.7 ± 2.4 74.1 ± 2.4 +3.3 75.5 ± 2.2 76.7 ± 2.2 +1.6 0.334 <0.001 0.170
BMI (kg/m2) 27.3 ± 1.0 28.2 ± 1.0 +3.3 28.6 ± 0.9 29.0 ± 0.9 +1.4 0.436 <0.001 0.178
Waist to hip ratio 0.817 ± 0.014 0.815 ± 0.014 -0.2 0.787 ± 0.013 0.788 ± 0.013 +0.1 0.114 0.921 0.827
Values are least squares mean ± SE. BMI = body mass index.
No treatment effects were detected.
Table 2 Daily total energy and macronutrient intake at baseline and after one year supplementation of soy or control foods
Measures Control (n = 27) Soy (n = 35) Control vs Soy
Baseline Final Baseline Final p values
Trtmt Time Trtmt × Time
Total energy (kcal) 1577 ± 95b 1850 ± 96a 1827 ± 82a 1582 ± 84b 0.933 0.853 0.001
Protein (g) 64.2 ± 4.1c 87.8 ± 4.1a 75.8 ± 3.6b 87.3 ± 3.6a 0.241 <0.001 0.035
Carbohydrates (g) 207 ± 14bc 247 ± 14a 243 ± 12ab 202 ± 12c 0.752 0.938 <0.001
Total fat (g) 56.6 ± 4.8 59.0 ± 4.8 62.5 ± 4.1 57.0 ± 4.2 0.719 0.667 0.277
Calcium (mg) 796 ± 69 1168 ± 70 873 ± 60 1183 ± 62 0.459 0.003 0.777
Values are least squares mean ± SE.
Values were calculated from 7-day food frequency questionnaires and included the treatment regimen.
Within each row, values that do not share the same superscript letters are significantly (P < 0.05) different from each other. Soy food products on the average provided approximately 232 kcal, 25 g protein, 4.4 g fat, 25 g carbohydrates, 500 mg calcium and 60 mg isoflavones daily. Control food products on the average provided approximately 454 kcal, 25 g protein, 6 g fat, 77 g carbohydrates, 500 mg calcium and 0 mg isoflavones daily. Soy and control foods were donated by DrSoy Nutrition (Irvine, CA).
Table 3 Effects of control and soy foods on bone mineral density (BMD) and bone mineral content (BMC)
Measures Control (n = 27) Soy (n = 35) Control vs Soy
Baseline Final Baseline Final p values
Trtmt Time Trtmt × Time
BMD, (g/cm2)
Whole body 1.050 ± 0.020 1.036 ± 0.0201 1.050 ± 0.018 1.036 ± 0.018 0.986 < 0.001 0.936
L1-L4 0.941 ± 0.026 0.933 ± 0.026 0.944 ± 0.022 0.934 ± 0.022 0.958 0.039 0.825
Hip, total 0.871 ± 0.021 0.870 ± 0.021 0.853 ± 0.018 0.852 ± 0.018 0.512 0.904 0.988
BMC, (g)
Whole body 2022 ± 58 1994 ± 58 2023 ± 49 2003 ± 49 0.944 < 0.001 0.465
L1-L4 53.631 ± 2.137 52.806 ± 2.137 54.737 ± 1.816 54.152 ± 1.816 0.662 0.018 0.681
Hip, total 57.532 ± 2.051 57.140 ± 2.051 57.278 ± 1.743 57.182 ± 1.743 0.967 0.738 0.839
Values are least squares mean ± SE. Lumbar spine (L1-L4).
No treatment effects were detected.
Table 4 Effects of control and soy foods on serum and urinary markers of bone metabolism
Measures Control (n = 27) Soy (n = 35) Control vs Soy
Baseline 6 months Final Baseline 6 months Final P values
Trtmt Time Trtmt × Time
Serum
IGF-I (nmol/L) 17.9 ± 1.4b 20.5 ± 1.4a 20.2 ± 1.4a 13.3 ± 1.2c 16.8 ± 1.2ab 16.8 ± 1.2ab 0.023 < 0.001 0.693
IGFBP3 (ng/mL) 3833 ± 133 3934 ± 133 3904 ± 135 3442 ± 120b 3751 ± 120a 3622 ± 121a 0.087 0.005 0.252
Osteocalcin (ng/mL) 8.3 ± 2.1b 13.9 ± 2.1a 16.2 ± 2.2a 8.9 ± 1.9b 17.4 ± 2.0a 18.1 ± 2.0a 0.365 < 0.001 0.671
BSAP (U/L) 19.8 ± 1.2c 23.3 ± 1.2b 25.3 ± 1.2a 19.8 ± 1.1c 22.6 ± 1.1b 24.9 ± 1.1a 0.796 < 0.001 0.818
ALP (U/L) 57.4 ± 3.0c 65.9 ± 3.0b 74.0 ± 3.1a 56.6 ± 2.7c 69.1 ± 2.7b 74.3 ± 2.8a 0.798 < 0.001 0.499
Urine
Dpd (nmol/mmol creatinine) 5.3 ± 0.4 5.3 ± 0.4 4.9 ± 0.4 5.2 ± 0.3 5.4 ± 0.3 5.1 ± 0.3 0.888 0.234 0.744
Values are least squares mean ± SE. IGF-I = insulin-like growth factor-I; IGFBP-3 = insulin-like growth factor binding protein-3; BSAP = bone-specific alkaline phosphatase; ALP = alkaline phosphatase; Dpd = deoxypyridinoline.
Within each row, values that do not share the same superscript letters are significantly (P < 0.05) different from each other.
Table 5 Effects of control and soy foods on on sex hormones
Measures Control (n = 27) Soy (n = 35) Control vs Soy
Baseline 6 months Final Baseline 6 months Final P values
Trtmt Time Trtmt × Time
FSH (mIU/mL) 41.0 ± 3.8 37.5 ± 3.8 36.4 ± 3.8 44.8 ± 3.4 47.6 ± 3.4 43.2 ± 3.4 0.143 0.195 0.235
E2 (pg/mL) 9.9 ± 4.1 12.3 ± 4.1 8.8 ± 4.2 12.7 ± 3.7 19.5 ± 3.7 17.7 ± 3.8 0.120 0.314 0.690
Estrone (pg/mL) 14.3 ± 4.3 21.9 ± 4.3 13.9 ± 4.4 19.6 ± 3.8 22.0 ± 3.9 22.4 ± 3.9 0.283 0.290 0.448
Estrone sulfate (ng/mL) 1.2 ± 0.4 1.9 ± 0.4 1.1 ± 0.4 1.8 ± 0.4 2.0 ± 0.4 1.9 ± 0.4 0.264 0.309 0.552
SHBG (nmol/L) 87.1 ± 9.2 81.3 ± 9.2 76.3 ± 9.3 105.3 ± 8.2 88.2 ± 8.2 90.0 ± 8.3 0.264 0.002 0.355
Values are least squares mean ± SE. FSH = follicle stimulating hormone; E2 = 17β-estradiol; SHBG = sex hormone-binding globulin.
No treatment effects were detected.
==== Refs
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| 15727682 | PMC554088 | CC BY | 2021-01-04 16:05:47 | no | Nutr J. 2005 Feb 23; 4:8 | utf-8 | Nutr J | 2,005 | 10.1186/1475-2891-4-8 | oa_comm |
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World J Surg OncolWorld Journal of Surgical Oncology1477-7819BioMed Central London 1477-7819-3-151574062510.1186/1477-7819-3-15Case ReportLocally advanced duodenal gangliocytic paraganglioma treated with adjuvant radiation therapy: case report and review of the literature Wong Adrian [email protected] Alexander R [email protected] John [email protected] Charles R [email protected] Department of Radiation Oncology, School of Medicine, University of Texas Health Science Center @ San Antonio, San Antonio, TX, 78229, USA2 Division of Surgical Oncology, Cancer Therapy & Research Center, San Antonio, TX, 78229, USA3 Department of Pathology, Methodist Hospital, San Antonio, TX, 78229, USA2005 1 3 2005 3 15 15 17 12 2004 1 3 2005 Copyright © 2005 Wong et al; licensee BioMed Central Ltd.2005Wong et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Gangliocytic paraganglioma are rare neoplasms that predominantly arise in periampulary region. Though considered benign the disease can spread to regional lymphatics.
Case presentation
A 49 year old woman presented with melena and was found to have a periampullary mass. Endoscopic evaluation and biopsy demonstrated a periampullary paraganglioma. The tumor was resected with pylorus-preserving pancreaticoduodenectomy and was found to represent a gangliocytic paraganglioma associated with nodal metastases. In a controversial decision, the patient was treated with adjuvant external beam radiation therapy. She is alive and well one year following resection. The authors have reviewed the current literature pertaining to this entity and have discussed the biologic behavior of the tumor as well as the rationale for treatment strategies employed.
Conclusion
Paraganglioma is a rare tumor that typically resides in the gastrointestinal tract and demonstrates low malignant potential. Due to rarity of the disease there is no consensus on the adjuvant treatment even though nearly 5% of the lesions demonstrate the malignant potential.
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Background
Gangliocytic paragangliomas are unusual neoplasms that may be identified anywhere within the gastrointestinal tract, but predominate in the periampullary region. This entity was first discussed by Dahl et al in 1957, and subsequently reported by Taylor and Helwig in 1962 [1,2]. Kepes and Zacharias named the tumor and described its characteristics in 1971 [3]. The pathognomonic features of these neoplasm is the identification of three distinct cellular elements: spindle cells, epithelial cells, and ganglion cells. These tumors are considered benign, yet occasionally metastasize to regional lymph nodes, as well as to distant organs [4]. Long term survival is common with appropriate resection. We report a case of a 49-year-old female who presented with melena and was found to have a periampullary gangliocytic duodenal paraganglioma. The details of the clinical presentation, histopathological findings, and therapeutic choices are provided.
Case presentation
A 49-year-old woman presented with a 6-month history of melena. During the preliminary consultation, she also complained of right upper quadrant pain, which radiated to her right lower quadrant and upper back. She underwent upper gastrointestinal (GI) endoscopy which demonstrated a 3 cm, ulcerated, ampullary mass (figure 1). Endoscopic biopsies suggested the diagnosis of paraganglioma. The lesion did not obstruct the ampullary orifice. Both computed tomography (CT) and magnetic resonance imaging (MRI) of the abdomen failed to demonstrate this lesion or any additional abnormalities.
Figure 1 Endoscopic photograph of the paraganglioma demonstrating an exophytic tumor whose edges enfold around a central area of necrosis that is actively bleeding.
The patient underwent pylorus-preserving pancreaticoduodenectomy and lymph node dissection. The postoperative course was uneventful except for delayed gastric emptying diagnosed by frequent vomiting during postoperative days 7 to 10, and confirmed by gastrograffin swallow. These symptoms eventually resolved with conservative management and the patient was discharged from the hospital on 15th postoperative day.
Histopathological analysis
Gross pathological evaluation of the resected specimen included a portion of duodenum with ampulla, measuring 16 cm. in length, and a portion of pancreatic head measuring 5 cm. in length. There was a polypoid periampullary mass protruding into the duodenal lumen, measuring 1.4 × 1.2 × 0.7 cm. On cut section, the mass was moderately firm and pink-tan. It was circumscribed but unencapsulated, and appeared to be covered by normal appearing duodenal mucosa. There was no evidence of pancreatic invasion by the tumor. A total of seven lymph nodes were also removed, 5 peripancreatic (3.0 cm in greatest dimension) and 2 periduodenal (1.2 cm and 1.0 cm in greatest dimension, respectively).
Histologically, the tumor consisted of a complex neoplastic proliferation that included a component resembling carcinoid or islet cell tumor (figure 2), admixed with a proliferation of spindled neurofibrillary cells and larger polygonal cells demonstrating gangliocytic differentiation (figures 3 and 4). There were areas of stromal hyalinization resembling amyloid, with focal calcification (figure 5). Congo red and Thioflavin T stains were negative for amyloid. The tumor extended through the muscularis propria and along the common bile duct, but did not invade the pancreas. The resection margins were free of tumor. Metastatic paraganglioma was present in 6 of 7 periduodenal and peripancreatic lymph nodes. The metastatic lymph nodes showed the same mixed histologic features as the primary tumor.
Figure 2 Photomicrograph showing areas of the tumor that had an epithelial pattern resembling carcinoid tumor or islet cell tumor. These cells were cohesive, uniform in size and shape with small round uniform nuclei, and formed rosettes, cribriform structures, solid nests and trabecular cords (Hematoxylin and Eosin original magnification ×100).
Figure 3 Photomicrograph showing areas of the tumor that consisted of a proliferation of spindled neurofibrillary cells with admixed larger polygonal ganglionic cells (Hematoxylin and Eosin original magnification ×100).
Figure 4 Photomicrograph showing the ganglionic cells having large round nuclei with chromatin clearing and large central nucleoli. The cytoplasm is abundant and amphophilic staining. The ganglionic cells are admixed with the spindled cells (Hematoxylin and Eosin original magnification ×400).
Figure 5 Photomicrograph showing the focal areas of stromal hyalinization with calcification. Special stains for amyloid were negative (Hematoxylin and Eosin original magnification ×200).
Immunohistochemical analysis demonstrated that the tumor stained positively for S-100, chromogranin, synaptophysin, and cytokeratin AE1 and AE3. No reactivity was observed with MART-1 or HMB 45. Staining for c-kit (CD 117) was performed on sections of the primary tumor and of one of the lymph nodes with metastatic tumor. The carcinoid-like epithelial cells and the spindle-shaped neurofibrillar cells stained negatively for the cell marker c-Kit. The gangliocytic cells stained strongly positive for c-kit.
Adjuvant therapy
Due to the evidence of regional lymph node metastasis the patient was counseled regarding adjuvant therapeutic options. The treating physicians queried recognized experts in the field of radiation therapy for gastrointestinal malignancies via email and a consensus developed that external beam radiation therapy might be reasonable, although it was admitted that no data are available regarding the use of this modality for this disease entity. Ultimately, a decision was made to administer external beam radiotherapy to the abdomen in an effort to eradicate any possible residual disease not removed during surgery and to reduce the risk of locally recurrent disease. No chemotherapy was advised due to the rarity of distant metastases and the lack of response of these neoplasms to conventional systemic therapy.
The patient was treated with intensity-modulated radiotherapy in 28 fractions of 180 cGy/fraction over 37 elapsed days; 6 mv photon beam energy was used. The target was a postoperative tumor bed with a 5–10 mm margin. The total dose was 5,040 cGy. She tolerated treatment well and is now symptom free more than one year following resection. Surveillance CT scans and endoscopy have been performed both of which reveal no evidence of recurrent disease.
Discussion
Gangliocytic paraganglioma is a rare, typically benign tumor of the gastrointestinal tract most commonly located in the second portion of the duodenum, with a few cases having involved the jejunum and pylorus [5-7]. Burke et al reported that there seems to be a slight male predominance and an average age of 54 at presentation [5]. Other authors have denied that there is any gender preference [8]. This lesion usually presents with abdominal pain and gastrointestinal bleeding due to mucosal ulceration. Obstructive jaundice is less common [9].
Histologically, our patient's tumor demonstrated the characteristic tricellular pattern of gangliocytic paragangliomas. These tumors are typically composed of an admixture of ganglion cells, spindle cells and epithelial cells [5,10-13]. These tumors are submucosal, and rarely recur or metastasize [14-16]. In most reported cases regional lymph node involvement, the metastatic cells consist predominantly of epithelial cells [15]. A relatively unique element of the case we present is that six of 7 regional lymph node metastases contained all three characteristic cell types, and thus, demonstrated the possibility for each of these cell types to acquire a malignant potential.
Immunohistochemically these tumors stain positive for a variety of markers as was demonstrated in this report. Such markers include those mentioned above as well as neuron-specific enolase, pancreatic polypeptide, somatostatin, myelin basic protein and neurofilament proteins [6,12,13,17]. The origin of gangliocytic paragangliomas has been widely debated and includes hypotheses ranging from a hamartomatous derivation to cellular elements arising from pancreatic neuroendocrine tissue, or that of the retroperitoneal celiac sympathetic or parasympathetic plexuses [4,12].
There is no data in the literature to guide clinicians on the use of adjuvant therapy despite the fact that approximately 5% of cases demonstrate malignant features [4]. Since this patient had multiple positive lymph nodes and is relatively young, a trial of adjuvant radiotherapy to the operative bed was considered reasonable and was endorsed by radiation oncologists at high volume cancer centers queried via email.
Conclusion
Gangliocytic paraganglioma is a rare duodenal tumor that can present with non-specific symptoms. Positive diagnosis can be obtained histologically by observing three characteristic cell types. Although this tumor is considered benign, the possibility exists for regional lymph nodal spread. Due to the rarity of the disease, no clear adjuvant treatment strategy has been determined in cases that demonstrate regional or distant metastasis.
Competing interests
The authors(s) declare that they have no competing interests.
Authors' contributions
AW wrote the original manuscript.
AM performed surgical resection and prepared requested revisions of the manuscript.
JM performed histopathological evaluation of the lesion and prepared photomicrographs.
CT administered radiation therapy to the patient, made editorial suggestions, and supervised AW.
Acknowledgements
Written consent was obtained from the patient for publication of this study and the related photos.
==== Refs
Dahl EV Waugh JM Dahlin DC Gastrointestinal ganglioneuromas: brief review with report of a duodenal ganglioneuroma Am J Pathol 1957 33 953 966 13458330
Taylor HB Helwig EB Benign nonchromaffin paragangliomas of the duodenum Virchows Arch Pathol Anat Physiol Klin Med 1962 335 356 366 13919963 10.1007/BF00957029
Kepes JJ Zacharias DL Gangliocytic paragangliomas of the duodenum: a report of two cases with light and electron microscopic examination Cancer 1971 27 61 70 4099700
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Sundararajan V Robinson-Smith TM Lowy AM Duodenal gangliocytic paraganglioma with lymph node metastasis: a case report and review of the literature Arch Pathol Lab Med 2003 127 e139 e141 12653602
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| 15740625 | PMC554089 | CC BY | 2021-01-04 16:39:05 | no | World J Surg Oncol. 2005 Mar 1; 3:15 | utf-8 | World J Surg Oncol | 2,005 | 10.1186/1477-7819-3-15 | oa_comm |
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Nutr Metab (Lond)Nutrition & Metabolism1743-7075BioMed Central London 1743-7075-2-71573331910.1186/1743-7075-2-7Brief CommunicationCanavanine-induced longevity in mice may require diets with greater than 15.7% protein Brown Dan L [email protected] Animal Science Department, Cornell University, 320 Morrison Hall, Ithaca, NY 14853 USA2005 25 2 2005 2 7 7 9 12 2004 25 2 2005 Copyright © 2005 Brown; licensee BioMed Central Ltd.2005Brown; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Dietary administration of 1% canavanine had been shown to improve survival in female BALB/c mice consuming diets containing 23.4% protein (dry matter basis).
Methods
In order to determine if this effect also obtains at more moderate dietary protein concentrations, 30 female BALB/c mice were fed a basal diet with 14% protein (15.7% dry matter basis) and another 30 were fed the same diet plus 1% canavanine.
Results
Neither mean (Control 873.2 d, Canavanine 870.0 d; SEM = 34.2 d; P = 0.949 from ANOVA) nor median (Control 902 d, Canavanine 884.5 d; P = 0.9058 from Mann-Whitney) lifespans differed between groups.
Although mean antinuclear antibody (ANA) titers did not differ between control and canavanine-treated mice at 833 days of age (19.84 vs 20.39 respectively; SEM = 2.64; P = 0.889 from ANOVA), one canavanine-treated mouse displayed an outlying ANA value of 50 (next lower value = 30) denoting possible early sign of incipient autoimmune disease in that individual.
Conclusion
There may be an interaction between dietary protein level and canavanine with respect to lifespan in mice.
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Background
L-canavanine is a common non-protein amino acid found naturally in alfalfa sprouts, broad beans, jack beans, and a number of other legume foods and animal feed ingredients [1] at up to 2.4% of food dry matter. This analog of arginine (Figure 1.) can also block NO synthesis [2-5], interfere with normal ammonia disposal [6,7], charge tRNAarg, cause the synthesis of canavanyl proteins [8], as well as prevent normal reproduction in arthropods [9] and rodents [10].
Figure 1 Chemical structure of canavanine and arginine
Canavanine has also been reported to induce a condition that mimics systemic lupus erythematosus (SLE) in primates [11,12], to increase antibodies to nuclear components and promote SLE-like lesions in auto immune-susceptible (e.g., (NZB X NZW)F1) mice [13].
In our previous study [14], eighteen female BALB/c mice were fed a 23.4% protein diet containing 1.56% L-canavanine sulfate (equivalent to 1% L-canavanine base) and eighteen control mice received control diet (23.4% protein) from 84–477 days of age. More canavanine-fed mice (16 of 18) survived to age 477 days than those fed the basal diet (9 of 18) (X 2 with Yates correction = 24.8).
The death rate and median life span of control mice were consistent with previously reported survival studies of female BALB-C mice fed high protein diets [15]. Necropsy and histopathology from both experimental animals and co-housed cages of contemporary sentinel mice performed by the University of California Comparative Pathology Laboratory (Davis, Ca.) did not detect any significant gross or histopathological lesions, parasites or pathogenic bacteria, viruses or mycoplasma. Serology results were negative as well. There was no evidence of infectious disease and no evidence of the lung tumors often found in older BALB-C mice [15]. One sentinel, but no experimental animals, showed evidence of a lymphosarcoma.
Kristal and Yu [16] have suggested that aging "...results in deleterious changes in cellular, intercellular, tissue and organismic functions."
Apparently, canavanine postponed the type of physiological decline that results in earlier death due to the inability of these aging mice to maintain homeostasis in the face of undetectable disease or some other stress.
These were serendipitous findings during a project designed to explore the mechanism of diet-induced autoimmunity. Ironically, no positive ANAs or anti SSDNA or anti dsDNA titers were detected in these mice as a result of canavanine treatment.
Previous reports of life span extension by restricting the intake of energy [17], protein [18], tryptophan [19] and methionine [20] have been accompanied by severe reductions in growth and eventual body size relative to controls. The severe stunting usually associated with amino acid deficiency and extended life span was not observed in the BALBc mice [14].
Our earlier results did leave unanswered the question of whether canavanine was extending life or merely offsetting the life-shortening effects of a very high protein diet. Since the objectives of the original investigation were unrelated to lifespan determination and required the harvest of body tissues, full lifespan data were not available.
The purpose of the experiment reported here was to test the hypothesis that canavanine will reduce middle age mortality and extend the lifespan of female BALB/c mice consuming a diet containing moderate concentrations of protein (15.7% DMB).
Methods
Five female 100-day-old 20-gram BALB/cAnHsd mice (first generation offspring of mice obtained from Harlan Sprague-Dawley) were assigned to each of twelve cages and six cages were randomly assigned to each of two treatments: Control = 25 g of ground Agway 1000 lab animal feed or Canavanine = 25 g of a mixture containing 1.56% canavanine sulfate (prepared by the method of Rosenthal [21]) and 98.44% ground Agway 1000 lab animal feed resulting in a diet containing 1% L-canavanine base (Table 1). All animals were weighed monthly for the first 28 months of age and cause of death determined by symptoms and necropsy. As animals died, feed offerings were reduced, but 5 grams per head ratio was maintained.
Table 1 Experimental Diets
Nutrient Basal (Agway Prolab 1000) Basal Diet + canavanine
Dry Master % 89.88 89.78
All others on DMBasis
NX6.25 % 15.70 17.84
Lipids % 7.05 6.94
Ash % 6.54 6.44
Gross Energy kcal/g 4.5 4.5
Estimated Metabolizable Energy kcal/ 3.3 3.3
Calcium % 0.90 0.89
Phosphorus % 0.8 0.79
Arginine % 0.81 0.80
Canavanine % ----- 1.05
At 833 days of age, blood samples were taken from the supraorbital sinus of the 37 surviving mice (19 controls and 18 canavanine-fed) and analyzed for antinuclear antibodies (ANA) by the methods of Naiki, et al [22].
Variations in lifespan and ANA values were evaluated by analyses of variance and median values were contrasted by the Mann Whitney procedure [23].
Results
Neither mean (870.0 vs 873.2, SEM = 34.2) nor median (885 vs 902, W = 923.5) lifespan differed significantly between mice fed canavanine and those fed the same basal diet without canavanine, respectively. (Figure 2).
Figure 2 Survival of mice fed 15.7% dietary protein
Although mean (20.39 vs 19.84) indices of ANA titers at 833 days of age were also similar for canavanine and control treatments, one canavanine-fed mouse displayed an unusually high titer (10X SEM above mean). (Figure 3).
About half of the mice (48.3%) displayed mammary tumors, pulmonary adenocarcinomas or both at the time of death (17 canavanine mice and 12 controls; but the difference was not significant by Chi-square analysis, [23]) (Table 2). No infectious diseases were detected in either experimental or sentinel mice.
Figure 3 Antinuclear Antibodies
Table 2 Probable cause of death
Mammary Tumor Pulmonary Adenocarcinoma Both Types of Tumor Total With Any Tumor Other Undetermined
Canavanine 9 7 1 17 2 11
Control 6 4 2 12 3 15
X2(Yates) 0.44 0.37 0.56 0.35 0.65 0.43
P >0.50 >0.50 >0.30 >0.50 >0.30 >0.50
Discussion
The median longevity of control mice observed in this experiment (in which a 15.7% protein was fed) considerably exceeds that of the previous trial in which the basal diet contained 23.4% protein (902 d vs 449 d). Storer [24] fed BALB/c mice a diet of protein content between those two extremes (17%, but probably closer to 19% on a dry matter basis) and observed mean longevity for females of 575 d, a result falling between our two observations. That decreasing protein concentrations should extend rodent lifespan is not a unique finding, the phenomenon is well known. The apparent corrective effect of canavanine at high protein concentrations and lack of effect at more moderate concentrations does offer a new dimension to dietary modulation of longevity that bears further study.
A number of populations consume protein in amounts that far exceed known nutritional requirements (humans in some Western countries, alfalfa-fed horses in some Western States and pet dogs in most of the Northern Hemisphere). The impact of these protein intakes on human and domestic animal longevity and the possible corrective effects of non-protein amino acids on those effects should interest a variety of medical, veterinary and nutritional workers.
For the second time, we have failed to induce significant autoimmune disease signs with dietary canavanine in BALB/c mice. Since this can be done in autoimmune susceptible strains and hybrids and in the "normal" (DBA/2) strain of mice [13], genetic differences may play a role in an animal's response to this secondary plant compound.
These preliminary experiments were not contemporary comparisons, nor were they conducted with both sexes, with the same source of BALB/c mice, the same basal diets or at the same locations. Known autoimmune-susceptible lines were not challenged in those trials. Although the data from those trials did indicate a canavanine-protein concentration interaction, we propose a contemporary, comprehensive, integrated experiment including both autoimmune susceptible and more normal populations. The proposed work will be more conclusive and provide an opportunity to examine the generality of the canavanine longevity effect, suggest mechanisms for its occurrence and find out if positive effects on oxidation event-dependent aging might be offset by autoimmunity in susceptible individuals. Examination of such indices as tissue and urine nitrate/nitrite, 8-OH deoxyguanidine, canavanyl residues in specific and total proteins would all contribute to a better understanding of mechanisms of longevity modulation.
Conclusion
This and a previous study suggest that an interaction between dietary protein concentration and canavanine effects on longevity may exist in BALB/c mice. Contemporary trials that vary both protein and canavanine concentrations are needed to test this possibility more conclusively.
Competing interests
The author(s) declare that they have no competing interests.
Acknowledgements
The author acknowledges M. Eric Gershwin for analyses of ANA in these mice and Thomas Kuntz for purification of the canavanine and preparation of experimental rations.
==== Refs
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| 15733319 | PMC554090 | CC BY | 2021-01-04 16:37:46 | no | Nutr Metab (Lond). 2005 Feb 25; 2:7 | utf-8 | Nutr Metab (Lond) | 2,005 | 10.1186/1743-7075-2-7 | oa_comm |
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Nutr JNutrition Journal1475-2891BioMed Central London 1475-2891-4-101574544910.1186/1475-2891-4-10ReviewAssessment of obesity management in medical examination Treyzon Leo [email protected] Divisions of Digestive Diseases and Clinical Nutrition, Center for Human Nutrition, David Geffen School of Medicine at UCLA, 900 Veteran Avenue, Room 12–217, Los Angeles, CA 90095, USA2005 3 3 2005 4 10 10 24 1 2005 3 3 2005 Copyright © 2005 Treyzon; licensee BioMed Central Ltd.2005Treyzon; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Obesity is a growing international health problem that has already reached epidemic proportions, particularly within the United States where a majority of the population is overweight or obese. Effective methods of treatment are needed, and should be taught to physicians by efficient means. There exists a disconnect between the rising obesity prevalence with its high toll on medical resources, and the lack of obesity education provided to practitioners in the course of their training. One particular shortfall is the lack of representation of obesity on standardized medical examinations. Physician attitudes toward obesity are influenced by their lack of familiarity with the management of the disease. This may include dietary restriction, increasing physical activity, behavior modification, pharmacotherapy, and surgical interventions. Thus, curricular changes in the medical education of obesity could help reduce morbidity and mortality associated with this disease.
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Wegeners's granulomatosis, errlichiosis, and tetrology of Fallot are examples of some of the rare diseases that I have learned to identify and look for in patients for over seven years as a medical student and resident in Internal Medicine. As a physician in training, I was frequently challenged to consider broad differentials and to watch out for the zebra that masquerades as a common disease throughout my clinical encounters. I have had my antennas up in search of these diseases for quite a while now. I have not, however, encountered any patients with these diseases so far. What I have usually encountered instead are common diseases, like type 2 diabetes, hypertension, and arthritis. More than anything, I have been struck by the incidence of obesity in our population. I find it alarming that many clinicians fail to actively address this growing epidemic in a formal manner with their patients, but rather regard obesity as a lifestyle choice that is the patient's sole responsibility.
My personal experience with obesity, having trained at a quaternary referral center in the relatively thin state of California, is that over one third of the patients I cared for were obese (not just overweight). Consistent with national trends, patients in my region have been becoming more obese and accordingly have been suffering increasingly from the complications of the disease, such as hypertension, insulin resistance, fatty liver disease, dyslipidemia, coronary heart disease, osteoarthritis, obesity-related cancer, and premature death [1]. I have been reassured that national policy makers at agencies such as the NIH and Medicare have taken administrative steps to address this epidemic [2]. What has perplexed me is that medical educators have been slow to prioritize obesity as an important challenge in the medical education of new physicians.
Many physicians cite their unfamiliarity with obesity as an impediment to their ability to adequately address the problem with their patients [3]. In one study by Jelalian et al., the authors found that when addressing obesity, one fourth of physicians thought that they were not at all or only slightly competent, while 20% reported feeling not at all or only slightly comfortable [3]. My impression is that obesity is not well represented in examinations of medical knowledge. As the latest confirmation of this hypothesis, I found it alarming that there were no questions addressing management of obesity on the American Board of Internal Medicine training examination in August 2004. This is my based on my immediate recollection of the exam, and confirmed by an informal same-day survey of five co-examinees. I later wrote to the ABIM to confirm my suspicion of this omission. In their response, they could not deny that the topic of obesity management had been left out [4]. I pose to you that our medical education is based largely upon that which we are tested. If this is true, it is not surprising that many physicians do not feel comfortable managing obesity.
I recalled at least five examination questions addressing Wegener's granulomatosis, but none directly on obesity. The differences in prevalence are remarkable. In the United States, approximately 61% (110 million) of adults (age 20–74 years) are overweight or obese. [5]. Educators must realize that if we are to aggressively arrest this epidemic, we must be armed with the knowledge to do so. Awareness, in part comes from exposure. By that, I mean, that which we study is that which we will be questioned on. If we all agree that obesity education is a priority, it follows that examinations should reflect that prioritization.
Diabetes, coronary heart disease, dyslipidemias, hypertension, and cancer were appropriately well represented in this ABIM exam and previous standardized examinations I have taken. Obesity should be right up there with these "big players". There is a lot to understand within this field. It is a lot more complex than "eat less, make better nutritional choices, and exercise more". The complexities of nutritional decision making are very important to the patient, and they want to discuss these choices with physicians as well as dieticians. Pharmacotherapy, although limited in terms of number of options, does have a role. Bariatric surgery is an evolving field and there are complexities associated with the use of the procedure that surgeons and internists alike must understand. Also, such psychosocial aspects of obesity as diminished quality-of-life, self-esteem, and work performance must be stressed and understood. Fogelman reported that in one survey of family practitioners, 72% believed that they had limited efficacy in treating obesity and considered themselves not well prepared by medical school to treat overweight patients [6]. Some 60% reported feeling that they have insufficient knowledge regarding nutritional issues. Regarding pharmacotherapy for treating obesity, only 66% knew the drugs' prescription indications. [6].
Primary prevention is the way to go in obesity research expenditure. In my opinion, the greatest healthcare cost-savings' "bang-for-the-buck" comes from preventive care and education. Consider how many fewer cases of diabetes, hypertension, CHD, fatty liver disease, dyslipidemia, thromboembolic disease, obesity-hypoventilation, obstructive sleep apnea, gout, osteoarthritis, cancer, GERD, reproductive and urinary tract abnormalities, cataracts, psychosocial abnormalities we would have to battle if the inciting factor (obesity) was aggressively treated at the onset. Is this not one prudent management step toward curtailing an overtaxed health care system?
The war on obesity seems winnable. We just need the tools and awareness to address the disease. Obesity is not a silent disease to be tolerated: it is an aggressive yet insidious disease (much like type 2 diabetes) that must be arrested before its negative health consequences become irreversible. The first step is to teach practitioners what to look out for, and in turn, to teach patients what to look out for. Meanwhile, I will have my antennas up in search of Wegener's granulomatosis.
Competing interests
I hereby declare that I do not have a financial association or other conflict of interest with the subjects mentioned in this manuscript.
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Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults Accessed December 1, 2004.
NIH Strategic Plan for Obesity Research Accessed December 1, 2004.
Jelalian E Boergers J "Survey of physician attitudes and practices related to pediatric obesity." Clin Pediatr (Phila) 2003 42 235 45 12739922
Personal e-mail communication with ABIM
National Center for Health Statistics, Centers for Disease Control and Prevention Accessed December 1, 2004.
Fogelman Y Vinker S "Managing obesity: a survey of attitudes and practices among Israeli primary care physicians." Int J Obes Relat Metab Disord 2002 26 1393 7 12355337 10.1038/sj.ijo.0802063
| 15745449 | PMC554091 | CC BY | 2021-01-04 16:39:30 | no | Nutr J. 2005 Mar 3; 4:10 | utf-8 | Nutr J | 2,005 | 10.1186/1475-2891-4-10 | oa_comm |
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Front ZoolFrontiers in Zoology1742-9994BioMed Central London 1742-9994-2-31571591510.1186/1742-9994-2-3ResearchOpisthobranchia (Mollusca, Gastropoda) – more than just slimy slugs. Shell reduction and its implications on defence and foraging Wägele Heike [email protected]_bonn.deKlussmann-Kolb Annette [email protected] Museum König Bonn, Adenaueralle 160, 53113 Bonn, Germany2 Institut für Evolutionsbiologie, Rheinische Friedrich-Wilhelms-Universität, An der Immenburg 1, 53121 Bonn, Germany3 Zoologisches Institut, J.W. Goethe Universität, Siesmayerstrasse 70, 60054 Frankfurt, Germany2005 16 2 2005 2 3 3 19 10 2004 16 2 2005 Copyright © 2005 Wägele and Klussmann-Kolb; licensee BioMed Central Ltd.2005Wägele and Klussmann-Kolb; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
In general shell-less slugs are considered to be slimy animals with a rather dull appearance and a pest to garden plants. But marine slugs usually are beautifully coloured animals belonging to the less-known Opisthobranchia. They are characterized by a large array of interesting biological phenomena, usually related to foraging and/or defence. In this paper our knowledge of shell reduction, correlated with the evolution of different defensive and foraging strategies is reviewed, and new results on histology of different glandular systems are included.
Results
Based on a phylogeny obtained by morphological and histological data, the parallel reduction of the shell within the different groups is outlined. Major food sources are given and glandular structures are described as possible defensive structures in the external epithelia, and as internal glands.
Conclusion
According to phylogenetic analyses, the reduction of the shell correlates with the evolution of defensive strategies. Many different kinds of defence structures, like cleptocnides, mantle dermal formations (MDFs), and acid glands, are only present in shell-less slugs. In several cases, it is not clear whether the defensive devices were a prerequisite for the reduction of the shell, or reduction occurred before. Reduction of the shell and acquisition of different defensive structures had an implication on exploration of new food sources and therefore likely enhanced adaptive radiation of several groups.
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Background
Very often, non-shelled gastropods are considered to be slimy and non-attractive. This connotation usually refers to terrestrial species of the Stylommatophora belonging to the well-known Limacidae or Arionidae and in particular to garden snails, which do have negative effects on our horticulture. However, the Opisthobranchia are beautifully coloured "slimy" gastropods and exclusively occur in marine habitats. Non-scientists only meet these animals while diving for pleasure. One group of opisthobranchs, however, has become very famous throughout natural and even medical sciences: Aplysia californica Cooper, 1863, the sea hare, belonging to the subgroup Anaspidea (Fig. 1E). It is a classic example for neurobiological investigations, involving behaviour. It was E.R. Kandel, who performed many of his investigations on learning and memory on this animal [1]. He created the basic understanding of nerve functioning and learning in human beings and was awarded the noble prize of medicine in 2000 for his life time research on these animals.
Figure 1 Examples of opisthobranch species. A Micromelo undata (Bruguière, 1792) (Acteonoidea) – Queensland, Australia, B Scaphander lignarius (Linné, 1758) (Cephalaspidea) – Northern Sea, C Chelidonura pallida Risbec, 1951 (Cephalaspidea) – Queensland, Australia, D Elysiella pusilla Bergh, 1872 (Sacoglossa) from the Indo Pacific, feeding on the green alga Halimeda; due to incorporation of chloroplasts, Elysiella has the same colour as the algae, E Aplysia punctata (Cuvier, 1803) (Anaspidea) – Mediterranean Sea, F Tylodina perversa (Gmelin, 1791) (Tylodinoidea) – Mediterranean Sea, G Pleurobranchaea meckelii Meckel in Leue, 1813 Mediterranean Sea.
Only in very few species of Opisthobranchia, the shell is big enough so that the animal can withdraw completely. In most species the shell is reduced in size, internalised or lost completely. Opisthobranchs are much less diverse in species numbers (5000 to 6000) than the terrestrial Stylommatophora (about 30000 species), or the shelled marine gastropods, in former times named "Prosobranchia" (60000 species), but they show many biological features that are unique or rare in the animal kingdom and that are often related to foraging or defensive strategies [2]. These include incorporation and usage of intact chloroplasts from algal cells for feeding strategies (Sacoglossa, Fig. 1D) [3], or storage of intact cnidocysts from cnidarians for defence (Aeolidoidea, Fig. 2E, G, H) [4]. Some of them are able to synthesize toxic compounds or to uptake these secondary metabolites from their food in order to use them as repellents (Nudibranchia, Chromodorididae, Fig. 2C) [5]. Many of these biological phenomena are hardly understood because investigation of biological data is scarce. Evolution of different strategies is not known, because of the lack of well-supported phylogenetic analyses.
Figure 2 Examples of nudibranch species. A Armina neapolitana (Delle Chiaje, 1824) (Cladobranchia) – Mediterranean Sea, B Risbecia tryoni (Garrett, 1873) (Anthobranchia) – Queensland, Australia, C Glossodoris cruenta Rudman, 1986 (Anthobranchia) – Queensland, Australia, D Bornella stellifer (Adams & Reeve in Adams, 1848) (Cladobranchia) – Queensland, Australia, E Flabellina exoptata Gosliner & Willan, 1991 (Cladobranchia) – Queensland, Australia, F Ceratosoma magnifica (Van Hasselt, 1824) (Anthobranchia) – New South Wales, Australia, G Spurilla major (Eliot, 1903) (Cladobranchia) – Queensland, Australia, H Cuthona sibogae Bergh, 1905 (Cladobranchia) – Queensland, Australia
Knowledge on the different subgroups of the Opisthobranchia differs according to their availability and spectacular appearance. For example, many more data on feeding strategies and other biological features are available for the beautifully coloured Nudibranchia (Fig. 2), than for the tiny and inconspicuous Acochlidacea. The authors have worked on different aspects of the biology of the Opisthobranchia for many years, trying to promote our understanding of this peculiar group. Although the Opisthobranchia is a rather small taxon, this group is ideal for evolutionary studies. Recently, Wägele reviewed potential key characters that might have enhanced radiation within the Opisthobranchia [2]. She has used a working hypothesis on opisthobranch phylogeny and published data on different strategies to deduce her proposals. She discussed the gizzard in Cephalaspidea, kleptoplasty in Sacoglossa, kleptocnides in Aeolidoidea, symbiosis with unicellular algae in Phyllodesmium Ehrenberg, 1831 and mantle dermal formations in Chromodorididae. Cimino and Ghiselin [6] and Cimino et al. [7] discussed the loss of the shell and the acquisition of toxic substances as a driving force in the evolution of Sacoglossa. Glandular structures and acquisition of chemical defence is subject of several reviews [8-13]. In the present review, our knowledge on Opisthobranchia is briefly summarized with emphasis on reduction of the shell and its implications on life history, especially regarding foraging and defence. Additionally new results on several glandular structures are presented. Some glands are described here for the first time. We point to a new aspect in the evolution of defensive devices. Their primary function as excretory or detoxification organs should be taken into consideration. It is beyond the scope of this review to include all new data on morphology, histology and phylogeny, as well as the literature published on the Opisthobranchia. The intention is to draw attention to a fascinating group of animals with a species number of manageable size, and in which similar evolutionary traits occurring in different groups at the same time and their implications can be analysed.
Results
Figure 3 represents a preliminary phylogenetic tree of the Opisthobranchia, as well as a few members of the Pulmonata and basal Heterobranchia. This tree is based on data obtained by morphological and histological analyses. Characters are listed in table 1 (see additional file 2) and the data matrix in table 2 (see additional file 1). A complete discussion of characters and obtained trees is in preparation. In the tree presented here, all characters are treated as unweighted and unordered (see methods below). A similar comprehensive tree of the Opisthobranchia, based on 18S and 28S genes is published by Vonnemann et al. [14]. These authors did not include basal Heterobranchia, Pteropoda and enigmatic forms, e.g., the Rhodopidae (Fig. 4). Comparison of these two most recently obtained morphological and genetic trees shows that nearly all major opisthobranch subgroups are monophyletic, but the position of some of these groups differs between the cladograms. These differences mainly concern the position of the Acteonoidea, Tylodinoidea and Acochlidacea (highlighted in both Figs. 3 and 4 by bold bars). Nevertheless evolutionary traits concerning the fate of the shell can be detected within well-defined clades (see Figs. 3 and 4: shell internalisation is indicated by grey arrows, shell loss by black arrows). Several groups included in the trees will not be considered in this study, because they are not assigned to the Opisthobranchia. These groups are the basal Heterobranchia, Pyramidellidae and Pulmonata. Furthermore, this discussion focuses more closely on the morphology-based tree, because more taxa are included there.
Figure 3 Phylogeny of Opisthobranchia. Cladogram based on morphological data. Grey arrows indicate internalisation, black arrows the loss, of the shell. Positions of Acteonoidea, Tylodinoidea and Acochlidacea are marked by bold lines, because they differ from those on the gene-based tree (Fig. 4)
Figure 4 Phylogeny of Opisthobranchia. Cladogram based on 18S and 28S gene, after Vonnemann et al. (in press). Positions of Acteonoidea, Tylodinoidea and Acochlidacea are marked by bold lines, because they differ from those on the morphology-based tree (Fig. 3). Grey arrows indicate the internalisation, black arrows the loss, of the shell.
Description of monophyletic groups
Acteonoidea
The families Acteonidae and Hydatinidae form sister-groups, the position of the debatable monophylum is under discussion. All acteonoids have a shell that resembles that of many prosobranchs (Fig. 1A). Some of the members are able to withdraw completely into the shell and to close the shell with an operculum, e.g. Acteon tornatilis (Linné, 1758). Acteonidae and Hydatinidae are carnivorous and mainly feed on polychaetes. No defensive strategies are known from these animals although histological investigations show a highly glandular area in the mantle cavity and the mantle rim. The mantle rim glands, for example, are very conspicuous. These comprise large epithelial cells that are filled with a non-staining vacuole (Fig. 5A). The glandular area is highly folded. The cells appear to lie subepithelially due to their size. They alternate with small ciliated cells. The hypobranchial gland in the roof of the mantle cavity is small and consists of violet-staining epithelial cells indicating acid mucopolysaccharides (Fig. 5B).
Figure 5 Glandular structures in opisthobranch species. A Acteon tornatilis (Linné, 1758) (Acteonoidea), mantle rim glands. B Bullina lineata (Gray, 1825) (Acteonoidea), hypobranchial gland. Note the ciliated raphe (arrow). C Clione limacina (Phipps, 1774) (Pteropoda, Gymnosomata), large single cellular glands (arrows). D Haminoea antillarum (d'Orbigny, 1841) (Cephalaspidea), Blochmann glands. E Chelidonura tsurugensis Baba & Abe, 1959 (Cephalaspidea), hypobranchial gland with violet stained glandular cells and above (arrow), single bluish stained glandular cells. F Dolabrifera dolabrifera (Cuvier, 1817) (Anaspidea), gland of Bohadsch, or opaline gland. G Umbraculum umbraculum (Lightfoot, 1786) (Tylodinoidea), dorsal mantle gland. H Tylodina perversa (Gmelin, 1791) (Tylodinoidea), dorsal mantle glands in the free mantle rim; above lies the shell.
Pteropoda
This group living exclusively in pelagic waters comprises two major clades, the herbivorous Thecosomata and the carnivorous Gymnosomata. Many thecosomates still have a shell whereas the Gymnosomata have lost it. The latter feed on the former. Many morphological adaptations have occurred due to their life in pelagic waters. Defensive mechanisms are hardly known from pteropods. Whereas the Thecosomata do not show specialized defensive glandular structures in the outer epithelium, peculiar structures of rather unknown function can be found in the two species of Gymnosomata investigated here. Both species show single large glandular cells, with one vacuole and a larger nucleus. The contents of the vacuoles only stain in smaller, probably immature, cells. In larger cells they are translucent (Fig. 5C).
Cephalaspidea
Members of following families have been included in this analysis: Smaragdinellidae, Haminoeidae, Retusidae, Cylichnidae, Bullidae, Philinidae, Aglajidae, Gastropteridae and Diaphanidae. Monophyly on family level was not recovered for the Diaphanidae and Cylichnidae. Furthermore, the Cephalaspidea (Acteonoidea excluded) is not monophyletic due to the inclusion of the Anaspidea. In our analysis, the gizzard-bearing groups form one clade, representing the Anaspidea (Fig. 1E) and the Cephalaspidean families Smaragdinellidae, Haminoeidae, Retusidae, Cylichnidae (Fig. 1B), Bullidae and Philinidae. Runcina Forbes & Hanley, 1853 and a yet undescribed Philinoglossa Hertling, 1932, both with a gizzard, are not part of that monophyletic group. The gizzard is a muscular oesophagus with 3 to 10 large gizzard plates that function like a grinding mill (Fig. 6A). Feeding strategies are highly diverse within these different groups. Herbivory is known only from cephalaspids with gizzard plates, whereas carnivory is widely spread within all other cephalaspidean groups with or without a gizzard. Prey items are mainly polychaetes, bivalves and in a few cases congeners. Runcina and Philinoglossa feed on diatoms.
Figure 6 Anatomical features characteristic for some opisthobranch species. A Haminoea antillarum (d'Orbigny, 1841) (Cephalaspidea), gizzard. B Aeolidia papillosa (Linné, 1761) (Nudibranchia, Dexiarchia, Aeolidoidea), longitudinal section of ceras with cnidosac with cleptocnides and outleading duct. C Aeolidia papillosa, oral tube with subepithelial glandular tissue. D Tomthompsonia antarctica (Thiele, J., 1912) (Pleurobranchoidea), spicules in notal tissue. E Onchidoris bilamellata (Linnaeus, 1767) (Nudibranchia, Anthobranchia), spicules in notal tissue.
Whereas many herbivorous slugs still have an external shell, e.g. Bulla Linné, 1758, Haminoea Turton & Kingston, 1830, Retusa Brown, 1827, Cylichna Lovén, 1846, etc., only a few carnivorous cephalaspids have retained a large shell (e.g. Scaphander Montfort, 1810) (Fig. 1B). Many have an internalized shell (Fig. 1C, Chelidonura Adams, 1850, Gastropteron Meckel in Kosse, 1813) or have lost it all together (Siphopteron Gosliner, 1989). Cephalaspideans have several glandular structures, although their function is hardly understood. The hypobranchial gland is composed of epithelial cells staining violet (Fig. 5E). This gland can be very voluminous (e.g. in Haminoea callidegenita Gibson & Chia, 1989) or can be reduced (in many cephalaspids with small and reduced mantle cavity, e.g. Chelidonura tsurugensis Baba & Abe, 1959). In many Cephalaspidea single glandular cells can be observed that stain bluish and open to the outside by a small duct (Fig. 5E, glandular cells above hypobranchial gland). These glands usually are confined to the mantle cavity roof. A special type of single gland is present in very few members of the Cephalaspidea, namely the Blochmann's glands. One characteristic is the duct leading to the outside that is composed of a few small cuboidal cells. The contents of these glands do not stain (Fig. 5D).
Anaspidea
This group, which is mainly characterized by two pairs of head tentacles (Fig. 1E), is closely related to the gizzard bearing cephalaspids. Nearly all species have a reduced shell or no shell at all (none of the latter species are included in the analyses presented here). In general, Anaspidea feed on red, brown and green algae. The group is known for their defensive habits, by using an ink gland when disturbed. The glands are typical Blochmann's glands already described for Cephalaspidea. Another gland is also widespread in Anaspidea and assumed to be an additional defensive gland, the so-called gland of Bohadsch, or opaline gland (Fig. 5F). It is composed of large cells containing a large nucleus. In general they are considered to be special forms of the Blochmann's gland [8,15]. The glands open at the bottom of the visceral cavity and stain violet. In a few species single glandular cells are arranged around a single opening. Additionally, the single blue-stained subepithelial glandular cells already described in Cephalaspidea (see Fig. 5E) are present in the dorsal mantle cavity.
Tylodinoidea
This tiny group is characterized by a rather large foot, and the umbrella-like shell covering the viscera, but not the foot (Fig. 1F). Data on biology of this small group are scarce. Probably all of them feed on poriferans. The Mediterranean Tylodina perversa (Gmelin, 1791) fosters the secondary metabolites from its exclusive food, the sponge Aplysina aerophoba Schmidt, 1862 [16]. Becerro et al. [17] demonstrated that the slug actively selects for sponges with a high concentration of cyanobacteria, whereas sponges without these bacteria (e.g. A. aerophoba from deeper waters, or A. cavernicola) were neglected. Histological investigation show that the Tylodinoidea have several peculiar glands. In the dorsal mantle tissue of Umbraculum umbraculum (Lightfoot, 1786), many tubules of a highly ramifying gland (Fig. 5G) lead to one or two main ducts that open to the outside in the anterior mantle rim above the mouth. Tylodina perversa has glandular tissue in the same area as U. umbraculum, but it has several ducts leading to the outside, all lying at the anterior dorsal mantle rim (Fig. 5H).
Sacoglossa
This group is monophyletic. In the morphological tree presented here (Fig. 3), the non-shelled Elysia species appears as the most basal one, whereas shell-bearing sacoglossans, like Cylindrobulla, are more derived. External morphology of sacoglossan species shows a high diversity. A rather primitive large and coiled shell is present in Cylindrobulla Fischer, 1857 and Ascobulla Marcus Ev., 1972. Others, like Oxynoe Rafinesque, 1814 and Lobiger Krohn, 1847, have tiny shells, whereas the shell is lost in all Elysiidae (Fig. 1D). A peculiar bivalved shell is present in the family Juliidae. Many sacoglossans feed on green algae (Ulvophycea) by piercing the algal cells with their radular teeth and by sucking the contents into their digestive tract. Defensive glands are not so obvious, but cryptic appearance obtained by an uptake and storage of chloroplasts is evident for many species as can be seen for Elysiella pusilla Bergh, 1872 feeding on the alga Halimeda Lamouroux, 1816 (Fig. 1D). Investigated Sacoglossa are characterized by many subepithelial glands with violet-stained contents (Fig. 7A). But the quantity of these cells differs to a great extent among species. Placobranchus ocellatus van Hasselt, 1824, a shell-less slug, is unique in having many globular structures arranged along the edge of the mantle rim (Fig. 7B). These structures have a diameter of nearly 1 mm and are composed of many cells each with a large vacuole. The contents of the vacuole stain bluish.
Figure 7 Glandular structures in opisthobranch species. A Elysia crispata (Moerch, 1863) (Sacoglossa), subepithelial glandular cells in dorsal epithelium. B Plakobranchus ocellatus van Hasselt, 1824 (Sacoglossa), mantle dermal formation in the edge of the parapodia. C Berthellina citrina (Rüppell & Leuckart, 1828) (Pleurobranchoidea), median buccal gland in visceral cavity, producing sulphuric acid. D Berthella edwardsi (Vayssiére, 1896) (Pleurobranchoidea), acid glands lying in the notum tissue. E Thecacera pennigera (Montagu, 1815) (Nudibranchia, Anthobranchia), subepithelial glandular cells in dorsal epithelium. F Marionia blainvillea (Risso, 1818) (Nudibranchia, Dexiarchia, Dendronotoidea), epithelial glandular cells with unusual large vacuoles filled with homogenously stained contents. G Risbecia tryoni (Garrett, 1873) (Nudibranchia, Anthobranchia), mantle dermal formations (MDFs) along the posterior mantle rim.
Pleurobranchoidea
Shells, when present, are internalised (Fig. 1G). Pleurobranch species feed on different prey items, but they are all carnivorous, some even feed on congeners. Typical for the group is a huge acid gland lying in the visceral cavity and opening into the oral tube next to the mouth. The gland is composed of huge cells with non-staining vacuoles (Fig. 7C). Additionally, several species show a highly glandular notum epithelium with huge subepithelial glandular follicles composed of cells with non-staining vacuoles (Fig. 7D). Several members, like Tomthompsonia Wägele & Hain, 1991, have spicules in their notum (Fig. 6D).
Nudibranchia
All members of the monophyletic Nudibranchia have lost the shell completely (Fig. 2A–H). This taxon, with about 3000 species and a high diversity in shape and in biology, is the largest opisthobranch group and comprises about half of the known opisthobranch species. Two monophyletic clades can be recognized, the Anthobranchia (Fig. 2B, C, F) and the Dexiarchia (Fig. 2A, D, E, G, H). The former mainly feed on Porifera, Bryozoa and Tunicata, the latter on Cnidaria, mainly on octocorals. Defensive strategies are very diverse in the Nudibranchia and comprise different techniques. Many species of the Anthobranchia, especially those feeding on sponges, have spicules in the notum (Fig. 6E). Many species are characterized by a highly glandular epidermis (Fig. 7E). Nearly all members of the very species-rich family Chromodorididae (Fig. 2B) have so called mantle dermal formations (MDFs) lying in the mantle tissue (Fig. 7G). These are globular structures with a diameter of up to 1 mm. MDFs are composed of cells with huge non-staining vacuoles.
Many Dexiarchia species are also characterized by a glandular epidermis. Especially members of the Dendronotoidea (here Tritonia Cuvier, 1798 and Dendronotus Alder & Hancock, 1845) are characterized by epithelial glandular cells in which the vacuole is filled with homogenously stained contents (Fig. 7F). Aeolidoidea have so called cnidosacs at the tip of their notal cerata that represent the apical parts of the digestive glandular tubes running within the cerata. In these cnidosacs, the cnidocysts from their cnidarian prey are stored and can be used against potential enemies (Fig. 6B).
Other groups in the cladogram
According to the phylogeny presented in Fig. 3, several taxa are united in a monophylum. Systematic relationships of some groups have been discussed for a long time (Acochlidiacea, Rhodopidae), others have been considered to belong to the Cephalaspidea (Philinoglossidae, Runcinidae). Their sister-taxon relationship is not solved yet and the presented cladogram is debatable. Nevertheless they have in common some evolutionary traits, e.g. the complete loss of the shell, their small size compared to other opisthobranchs and their food preference for diatoms and detritus. According to histological results, no particular defensive glands could be detected and defensive strategies probably lay in habits. Many of them burrow in sand and/or are cryptic in colour.
Discussion
In accordance with published phylogenies based on morphology [18-20], or based on genes [14,21,22] all major clades presented here are monophyletic (Acteonoidea – but see the study by Mikkelsen [23], Cephalaspidea with Anaspidea included, Sacoglossa, Tylodinoidea, Pleurobranchoidea, Nudibranchia, Anthobranchia, Cladobranchia). Concerning relationships of major groups, several congruencies with former analyses can be observed: The sister-taxon relationship of the Nudibranchia and Pleurobranchoidea [14,24] is found in nearly all analyses. This group nowadays is called Nudipleura Wägele & Willan, 2000. A further consistent grouping is formed by Cephalaspidea s.str. and Anaspidea. This relationship was already discussed by Mikkelsen [20,23]. All other presented groupings are still under debate. In our morphology-based tree, Elysia represents the most basal taxon within the Sacoglossa. This contradicts other available phylogenetic analyses and has to be considered with care. Jensen presented a thorough phylogenetic analysis on Sacoglossa [25]. According to her results, taxa with a shell are more basal and shell reduction occurred at least twice within the Sacoglossa. In the discussion below, we follow the results of Jensen and Mikkelsen and consider the shell-bearing sacoglossans as more basal [23,25].
Despite these incongruities, a discussion of shell reduction in the different groups and its implications on life history (habitat, feeding and defensive strategies) can be undertaken, and will serve as a guideline for further investigations.
Implications on life style
A shell is generally considered to be a protection against predators, such as fish, crabs and other vagile organisms. "If the shell of a whelk (e.g. Buccinum, a "prosobranchiate" caenogastropod, annotation of the authors) is broken away and the soft animal is then offered to a hungry cod, it is eaten readily." (p: 115) [26]. Reduction, internalisation or loss of the shell within Opisthobranchia implies other defensive strategies. Shell reduction within molluscs is uncommon, and occurs mainly in the highly mobile cephalopods. In gastropods, shell loss is rare in paraphyletic prosobranchs, and known only from few groups of Pulmonata, e.g., the Gymnomorpha and the stylommatophoran groups Arionidae and Limacidae. However, shell reduction occurred many times within the different subgroups of Opisthobranchia. Here, an internalization or complete loss occurs within the Cephalaspidea s.str, Anaspidea, Sacoglossa, Acochlidiacea and Pleurobranchoidea (Fig. 3). Whereas complete loss of the shell is not known from any member of the small taxon Tylodinoidea (about 15 species), this character state occurs in the stemline of the Nudibranchia and Gymnosomata. When estimating species numbers with no shell or a rather tiny internal shell and comparing this to the number of species with a larger external shell, the former outnumber the latter by far.
Loss of the shell therefore can be assumed to have advantages compared to the presence of a protective but heavy shell. Advantages probably lay in the exploration of new habitats, which are more difficult to reach when being protected by a shell. This can be observed e.g. in a subgroup of the Cladobranchia. The Aeolidoidea are able to graze on fragile hydrozoans (Fig. 8E). This kind of prey is used by few other invertebrates, e.g., Solenogastres, members of the Pycnogonida and of the Amphipoda [27-29]. Burrowing forms with a shell, e.g., Scaphander or Acteon Montfort, 1810, have an elaborate cephalic shield that partially covers the shell and renders them streamlined. Loss of the shell probably enables slugs to search for food in sandy or muddy habitats more easily. This is the case for members of the Cephalaspidea s.str. and Acochlidiacea.
Figure 8 Examples of cryptic nudibranch species. A Discodoris atromaculata Bergh, 1905 (Anthobranchia) from the Mediterranean, attached to the roof of a cave between Parazoanthus, B Jorunna tomentosa (Cuvier, 1804) (Anthobranchia) from the Northern Sea, attached to rocks with corralineacean red algae and mimicking a sponge (Halichondria), C Phyllidia flava Aradas, 1847 (Anthobranchia) from the Mediterranean Sea, feeding on Axinella cf. cannabina and incorporating the dyes. D Phyllodesmium briareum (Bergh, 1896) (arrow, Dexiarchia, Aeolidoidea) from the Indo Pacific, mimicking its food, the soft coral Briareum violacea. E Flabellina affinis (Gmelin, 1791) (Dexiarchia, Aeolidoidea) from the Mediterranean, crawling on its food Eudendrium racemosus (Cnidaria, Hydrozoa).
Basal members of the Sacoglossa have retained a shell, but more derived ones have lost it. Shell loss allowed evolution of a phenomenon that is unique in the animal kingdom. Sacoglossa in general feed on algae by piercing the cells with their tooth and sucking out the contents of the cell. The cytoplasm is digested, but in many species (e.g. Elysia timida (Risso, 1818), Placobranchus ocellatus) the chloroplasts are stored in distinct branches of the digestive gland. Here they are stored for a period of several days to months [30]. For this phenomenon, the term "cleptoplasty" is used by several authors [25]. The functioning chloroplasts continue with photosynthesis within the slug and provide nutritional metabolites for the metabolism of the gastropod [3,13,31,32]. Penetration of light into the slug would be hindered by the possession of a shell. A similar system is observed in members of the Nudibranchia, e.g., in Phyllodesmium jakobsenae Burghardt & Wägele, 2004, or Melibe bucephala Bergh, 1902) [33,34]. Here unicellular algae (zooxanthellae) from the coral food or from the free water column are stored in the digestive system and metabolites of these zooxanthellae are used for the slug's own purposes [35-37]. According to published phylogenies and to our own results (unpublished data of both authors) on Sacoglossa and Nudibranchia, it can be assumed that uptake of chloroplasts or zooxanthellae first enhanced crypsis (Fig. 1D) [25]. The short-term storage allows a continuation of the photosynthetic activity of chloroplasts within the slug. Storage over a longer period allowed the reduction of food uptake with the possibilities to search for new and/or less frequent prey organisms [2,38]. The most effective symbiotic relationships are known for the sacoglossan Elysia chlorotica Gould, 1870, which can survive eight months without food [3], the aeolid Pteraeolidia ianthina (Angas, 1864) and the dendronotoidean Melibe bucephala, both of which survived in our aquaria for 10 months without food (Burghardt & Wägele unpublished data).
Implications on defence
Loss of a shell as a protective structure led to an array of different defensive structures. Some of these traits can be observed as a combination in one and the same species.
Crypsis can be observed in many groups and is very often achieved by incorporation of the same dyes from the food (Fig. 8C, Phyllidia flava Aradas, 1847). Cryptic appearance also is achieved by mimicking the same patterns or even outline of the substrate. Corambe pacifica MacFarland & O'Donoghue, 1929 perfectly mimics the colour patterns of its prey, the bryozoan Membranipora de Blainville, 1830. Phyllodesmium jakobsenae mimics the feathered polyps of the soft coral Xenia Lamarck, 1816, on which it lives [33], whereas the cerata of P. briareum (Bergh, 1896) are smooth like the tentacles of its prey, the soft coral Briareum Blainville, 1830 (Fig. 8D). Zebra effects are achieved by patterns with blotches like that in Peltodoris atromaculata Bergh, 1880 (Fig. 8A) or by stripes. Looking like unpalatable sponges (Fig. 8B, Jorunna tomentosa (Cuvier, 1804)) is very common in spicule-bearing dorids. According to Gosliner [39], the cryptic species are rather basal taxa, whereas the taxa with aposematic colour patterns are more derived – a hypothesis that has yet to be proven by thorough phylogenetic analyses that include all species of the subgroup in question.
A unique defensive strategy within animals is the storage of cnidocysts ("cleptocnides"), which is typical for nearly all members of the cladobranch Aeolidoidea [4,9,40]. This group mainly feeds on cnidarians, with priority on Hydrozoa. The mechanisms of the uptake of cnidocysts, so that explosion is not triggered during consumption, are still not understood. It is assumed that the slug exudes a mucus to hinder explosion [9,26]. Investigated aeolids, like Aeolidia papillosa (Linné, 1761), have a highly glandular oral tube (Fig. 6C) that supports this hypothesis. Another theory implies that there occurs a kind of acclimation process, similar to that discussed between sea anemones and anemone fish [41]. According to the investigations of Greenwood and Mariscal [42] only immature cnidocysts are stored in the cnidosac, whereas mature ones are digested. But, histological investigation of many aeolids directly collected from their food have not revealed high numbers of exploded cnidocysts in the stomach (unpublished data of HW). Only Notaeolidia schmekelae Wägele, 1990 from the Antarctic Ocean has been observed to have many exploded cnidocysts in its digestive tract [43].
Presence of spicules in the notum as a defensive strategy was discussed by several authors [10,44]. Spicules are present in many shell-less Anthobranchia and Acochlidiacea, but also in members of the Pleurobranchoidea, which sometimes have an internalised small shell. Spicules never occur in opisthobranchs with a larger shell. Cattaneo-Vietti et al. investigated the mineral composition of dorid spicules and found calcite (CaCO3) and brucite (Mg(OH)2) [45]. Smaller spherules are composed only of calcite. Harris described feeding experiments offering various opisthobranchs to specimens of Navanax Pilsbry, 1895 (Cephalaspidea), who is a ferocious predator on opisthobranchs [10]. This species rejected all spiculose dorids.
Another evolutionary trait for defence, and discussed as a prerequisite for shell reduction at least in sacoglossans [13], is the uptake or de novo synthesis of secondary metabolites that are toxic to possible predators [5,46]. Uptake by feeding on toxic prey (mainly algae, Porifera, Bryozoa, Tunicata and Cnidaria) is the major source of compounds, whereas de novo synthesis is known only from few taxa [5]. When dietary derived, Avila called these cleptochemicals, following the terms cleptoplasts and cleptocnides for incorporation and use of chloroplasts in Sacoglossa and cnidocysts in Aeolidoidea [5]. Literature on chemical compounds in opisthobranchs is numerous. Some reviews summarize our knowledge [5,7,46-49]. Compounds mainly belong to the terpenoids, especially the insoluble sesquiterpenoids and diterpenoids. Little is known about the function of the biological compounds, although their defensive tasks are very often postulated [5-7,46]. Few feeding experiments have been performed in the past, demonstrating a toxic effect on crustaceans and/or fish [26,50]. Also the translocation from prey into the slug, and the transformation by changing the chemical structures either by degradation through digestion, or by an active mechanism into a more effective chemical, is hardly understood [5,7]. Location of the compounds is investigated only for few species, by analysing certain parts of the body [51], or even by isolating larger organs, like the MDFs [52]. Tracing the compounds within the tissue, or even cells, using immunohistochemical methods has never been done. Therefore, it is not possible to correlate chemical bioactivity with certain histological structures, except for the mantle dermal formations in the species Hypselodoris webbi (Chromodorididae) [52].
Inorganic compounds, like sulphuric acid are produced in few groups. Their function and location is better known due to the extensive work of Thompson [53-55]. He analysed the production of sulphuric acid in different members of Gastropoda, including members of the Pleurobranchoidea, Cephalaspidea and Dorididae. The exudated acid contains inorganic chloride and sulphate anions, and traces of organic substances. He was able to localize the acid by histochemistry within the large vacuoles in the median buccal gland (Fig. 7C) and the subepithelial glands of Pleurobranchoidea (Fig. 7D). There, the acid is held in active form [53]. Gillete et al. investigated the role of the central nervous system and peripheral nerves for exudation and showed positive feed back [56].
Broad histological investigations of the Opisthobranchia show that many species are characterized by a large array of glandular structures [8,11,57-59]. These comprise single glandular cells lying in the outer epithelia, or subepithelially. Glandular follicles composed of several cells usually lie subepidermally and open via a duct to the outside. Larger organs are the MDFs, or the glandular tubules of the median buccal gland in the Pleurobranchoidea. Some of these structures have been known for a long time and their defensive tasks were discussed in more detail by Hoffmann [8]. Well known are the ink gland (Blochmann's glands) and the opaline gland (Bohadsch gland) in the Anaspidea. Both glands exude substances that have been shown to be toxic to cnidarians [1]. Probably these substances also caused severe damage of the liver of a 40-year-old man, who ate Aplysia kurodai Baba, 1937 [60]. By experimental studies it was shown that the repellent substance in the ink gland is a monomethyl ester of phycoerythrobilin and is derived from phycoerythrin from the consumed red algae [61]. The role of the opaline gland is less known. According to Carté, the prosteroglandine with the highest known biological activity is Dolastatin 10, a natural product extracted from the anaspidean Dolabella auricularia (Lightfoot, 1786) [62]. This large species of more than 10 cm lives on the intertidal flats in the tropical Indo-Pacific, where it would represent an ideal food for birds and fishes, if not for that highly toxic chemical. This substance is already applied in medical treatments (see ), and seems to be one of the most potent anticancer agents.
Information on other glandular structures are rare, and nearly nothing is known about their contents and their functions. At the moment we are not able to trace the different substances in these glands to find out whether there are any constraints concerning structure (and therefore function) and the stored chemicals.
Only few hypotheses are formulated concerning acquisition of toxicity and loss of the shell. Faulkner & Ghiselin assumed that chemical defence based on metabolites derived from food preceded the reduction of the shell and that chemical defence has been a driving force behind the evolution of Opisthobranchia [13,46,63]. Cimino et al., by analysing the different compounds and their origin, came to the conclusion that evolution within the Sacoglossa started with the uptake and storage of sesquiterpenoids from algae in species still having a shell [7]. Within the shell-less members of the family Elysiidae, diterpenoids from the algae were stored, whereas in highly evolved forms, like Elysia timida, the slugs switched to a de novo synthesis of polyproprionates. Cimino & Ghiselin also mentioned that handling and utilization of a particular kind of defensive metabolite allowed the switch to food with similar compounds quite easily, and therefore has driven adaptive radiation [46]. As an example they named the dorids and in particular the family Chromodorididae, which show a large array of usage of biochemicals from different sponges. Again, the bio-synthesis of compounds, as observed in Dendrodoris Ehrenberg, 1831, is considered to be the most derived form of defence within the Anthobranchia.
Information on defensive strategies, as listed above, is available now for several groups of the Opisthobranchia. More and more reliable phylogenies are becoming available, which allow the identification of well-supported branches and stemlines. Combining this knowledge, it becomes evident that several defensive systems evolved before the loss of the shell (several glandular structures, e. g., the hypobranchial gland, mantle rim glands, Bohadsch gland). Here we would like to extend the hypothesis of Cimino et al. by addressing the problem of excretion [7]. It can not be ruled out that certain glandular structures evolved as a kind of excretory system to get rid of ingested toxic substances. Therefore it is not storage in special organs that preceded the use of toxic substances, but the necessity to expel them. By analysing phylogeny, it is evident that many defensive structures evolved after the internalisation or loss of shells (e.g. acid glands in the notum, cleptocnides, MDFs).
But we still have to identify the location of the compounds for a better understanding of the evolutionary history concerning the acquisition of toxicity, which certainly was a driving force in the evolution of these fascinating opisthobranchs. New techniques, e.g., the oligonucleotide aptameres, could help to solve this question [64]. We also have to keep in mind that chemical substances might not only play a role in defence (allomones), but also in reproduction and development (pheromones).
Conclusions
In this review it is shown that shell loss led to the evolution of a wide array of defensive strategies in Opisthobranchia. Nevertheless, it is not ruled out that some defensive mechanisms have evolved prior to complete loss of shell. This is evident when analysing the Acteonoidea. Members of this taxon have a rather thick and large shell, but also different glandular structures along the mantle fold, as well as in the mantle cavity. One working hypothesis for future research is that defensive glands evolved from simple storage organs while feeding on toxic prey. Evolution of special structures where toxic substances could be stored without further effect on the body and that functioned as a kind of excretory system could have been the prerequisite for employing these structures as defensive devices. To solve this question, thorough phylogenetic analyses are needed. Tracing toxic substances from the food into the different organs or glands by histochemistry or new analytical methods like aptameres will help to understand the tasks of these glands, and their role as excretory or as defensive organs, or as pheromone-producing organs involved in reproduction and development. Additional field and laboratory experiments with potential predators from the natural surroundings are necessary to understand the functioning of chemicals in the slugs.
Methods
Material collection
About 300 different species of Opisthobranchia have been collected and investigated by the authors in the past 20 years. Collecting was performed from the intertidal (e.g. Australia, Helgoland), to the sublitoral zones (e.g. Mediterranean Sea, North Atlantic, tropical waters in the Red Sea, Australia and others) down to depth of 1000 m (Antarctica). Collecting techniques comprised hand collecting in the intertidal and while diving, or using trawls, like the Agassiz trawl in Polar Seas. Specimens were preserved in 4 – 10 % formaldehyde/seawater for histological investigations, or 96% ethanol for molecular investigations.
Investigation techniques
Investigation of morphology and anatomy was performed by macroscopical and histological techniques. For histological investigations, entire animals (when small in size) or parts of the animals were embedded in hydroxyethylmethacrylate for serial sectioning (2 μm). Sections were stained with toluidine blue and investigated by light-microscopy. Toluidine blue stains acid mucopolysaccharides in various shades of red to violet, whereas neutral mucopolysaccharides are staining in blue colours. Observation of living animals aided the understanding of external characters and life strategies. Data used for the phylogeny comprise 79 taxa and 110 characters based on morphology and histology. Polarity of characters was obtained by outgroup comparison with Littorina littorea. Due to some trivial characters, an all-zero outgroup was chosen. The characters are explained in full detail by Wägele & Klussmann-Kolb (in prep). Analyses were performed by PAUP 4.0 beta 3a (Swofford, 1999) [65]. Parameters of maximum parsimony analyses were: ACCTRAN, all characters unordered and unweighted; heuristic search options: stepwise addition = random, branch-swapping option = TBR.
Authors' contributions
HW and AKK together carried out the phylogenetic analysis based on morphological and histological characters published here for the first time. HW drafted the manuscript. AKK helped to draft the manuscript. Both authors read and approved the final manuscript.
Supplementary Material
Additional File 1
Data matrix of characters – 79 taxa and 110 characters are included. N = non applicable. This implies that the character is not present and can therefore not be coded. ? = character state not known. Genus with an asterisk indicate that information on this genus is extracted from literature. Detailed information will be given elsewhere in Wägele & Klussmann-Kolb (in prep.).
Click here for file
Additional File 2
Characters – Characters and coding of character states used for phylogenetic analysis presented in Figure 3.
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Acknowledgements
Many colleagues helped to collect the animals. We just mention our long cooperation with Gilianne Brodie (Townsville/Australia) and Michael Schrödl (Munich/Germany). Thanks go to Conxita Avila (Blanes/Spain), with whom HW had numerous discussions on natural products.
Sincere thanks to the German Science Foundation for continuous support to HW (Wa 618/1 through Wa 618/8). AKK was also supported by the DFG (KL 1303/1). Research on glandular structures was additionally supported by the DAAD (Acciones Integradas Hispano-Alemanas).
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| 15715915 | PMC554092 | CC BY | 2021-01-04 16:38:34 | no | Front Zool. 2005 Feb 16; 2:3 | utf-8 | Front Zool | 2,005 | 10.1186/1742-9994-2-3 | oa_comm |
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Lipids Health DisLipids in Health and Disease1476-511XBioMed Central London 1476-511X-4-51573723910.1186/1476-511X-4-5ResearchL-histidine inhibits production of lysophosphatidic acid by the tumor-associated cytokine, autotaxin Clair Timothy [email protected] Eunjin [email protected] Malgorzata [email protected] Russell W [email protected] Lance A [email protected] Elliott [email protected] Mary L [email protected] Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA2005 28 2 2005 4 5 5 8 2 2005 28 2 2005 Copyright © 2005 Clair et al; licensee BioMed Central Ltd.2005Clair et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Autotaxin (ATX, NPP-2), originally purified as a potent tumor cell motility factor, is now known to be the long-sought plasma lysophospholipase D (LPLD). The integrity of the enzymatic active site, including three crucial histidine moieties, is required for motility stimulation, as well as LPLD and 5'nucleotide phosphodiesterase (PDE) activities. Except for relatively non-specific chelation agents, there are no known inhibitors of the ATX LPLD activity.
Results
We show that millimolar concentrations of L-histidine inhibit ATX-stimulated but not LPA-stimulated motility in two tumor cell lines, as well as inhibiting enzymatic activities. Inhibition is reversed by 20-fold lower concentrations of zinc salt. L-histidine has no significant effect on the Km of LPLD, but reduces the Vmax by greater than 50%, acting as a non-competitive inhibitor. Several histidine analogs also inhibit the LPLD activity of ATX; however, none has greater potency than L-histidine and all decrease cell viability or adhesion.
Conclusion
L-histidine inhibition of LPLD is not a simple stoichiometric chelation of metal ions but is more likely a complex interaction with a variety of moieties, including the metal cation, at or near the active site. The inhibitory effect of L-histidine requires all three major functional groups of histidine: the alpha amino group, the alpha carboxyl group, and the metal-binding imidazole side chain. Because of LPA's involvement in pathological processes, regulation of its formation by ATX may give insight into possible novel therapeutic approaches.
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Background
Lysophosphosphatidic acid (LPA) is both an intracellular and an extracellular signaling molecule that affects biological processes such as cell proliferation, rescue from apoptosis, cell migration, neurite retraction, wound healing, platelet aggregation and vascular remodeling [1]. As a cytokine affecting such varied and important functions, LPA production is normally tightly regulated. Its dysregulation is implicated in a number of pathophysiological states, including certain cancers and atherogenesis. Intracellularly, LPA is produced by calcium-dependent and calcium-independent phospholipase A2, acting on phosphatidic acid. Most of the extracellular LPA appears to be produced by a two-step process: production of lysophosholipid from phospholipids by the action of phospholipase A1 or A2, followed by conversion to LPA by the plasma enzyme lysophospholipase D (LPLD) [2,3]. Recently, this plasma LPLD has been shown to be identical to autotaxin (ATX, NPP2) [4,5].
ATX was originally purified as a potent tumor cell motogen [6], an effect that appears to be mediated by LPA [7] acting through the LPA1 receptor [8]. Recent studies have revealed that ATX/LPLD not only hydrolyzes lyso-phosphoglycerolipids to form LPA, but also hydrolyzes sphingosylphoshorylcholine (SPC) to produce sphingosine-1-phosphate (S1P) [9]. S1P can stimulate or inhibit cellular migration, depending upon the context of receptor expression [10]. Therefore, ATX can produce either agonists or antagonists of cell migration. In addition, ATX has been shown to stimulate tumor aggressiveness and to be over-expressed in certain malignancies [11-13]. The link between ATX and its putative 'mediator' LPA has led us to investigate possible mechanisms of regulating the enzymatic action of ATX in generating LPA.
ATX is a member of the nucleotide pyrophosphatase and phosphodiesterase (NPP) family of enzymes. The NPPs are part of the superfamily of alkaline phosphatases, metalloenzymes in which the active site is characterized by histidine residues coordinated around central divalent cations and by a serine, threonine, or cysteine residue, which is utilized to form an intermediate during the reaction [14,15]. Site-directed mutagenesis of human ATX established that a specific residue, T210, [7,16] and 3 histidine residues [7,9], corresponding to similar loci in other members of the alkaline phosphatase superfamily, were essential for the motility and enzymatic activities of ATX.
Histidine has been implicated as a requirement for many metalloenzymatic reactions, presumably by virtue of its imidazole moiety. Reversible reagents such as diethylpyrocarbonate, which react with imidazole, inhibit these enzymes [17]. Such findings led us to investigate whether histidine itself and some of its derivatives could inhibit the activities of ATX, perhaps by destabilizing the putative metallic cation-imidazole complex at the active site of ATX. Since the migratory effects of ATX depend upon its ability to generate LPA or S1P, focusing upon ATX as a target for regulation of tumor cell motility presents an attractive strategy for therapeutic intervention in metastasis.
Results
Effect of L-Histidine upon Cell Motility
ATX, isolated as a motility-stimulating protein, is a member of the NPP family of metalloenzymes. Both its motogenic and its enzymatic activities appear to require the same active site since both are abolished when T210 [16] or any of three histidines (H316, H360 or H476) are altered by site-directed mutagenesis [7]. Because of the crucial role that these histidines play in the ATX activities, we tested the effect of adding exogenous L-histidine upon ATX-stimulated cell motility.
Fig. 1 shows that L-histidine inhibited ATX-stimulated migration of both A2058 human melanoma cells and SKOV-3 human ovarian carcinoma cells in a concentration-dependent manner. Addition of 10 mM L-histidine to motility assays resulted in a 90 – 95% reduction in stimulated motility, whereas equivalent concentrations of control amino acids, glycine or alanine (data not shown), had no effect. In contrast, adding the same concentration of L-histidine failed to inhibit LPA-stimulated motility. These concentrations of L-histidine did not affect cell viability in either cell line, as measured by Trypan Blue exclusion. Whether the inhibitor was added to both wells of the assay (i.e. to cells and to chemoattractant) or just to the lower well (chemoattractant) did not affect the result.
Figure 1 Effect of histidine on ATX-stimulated motility. The modified Boyden chamber motility assays are described in Materials and Methods. L-Histidine inhibits the motility response of A2058 and SKOV-3 cells to ATX but not to LPA (concentrations shown). All data are shown as Mean ± SEM. Means were analyzed utilizing the ANOVA/Tukey's post-test (GraphPad Prism, San Diego, CA): * (p < 0.001) for histidine-treated vs. untreated controls.
ATX was pre-incubated with 200 nM LPC with or without addition of various concentrations of L-histidine, then heated to abolish its enzymatic activity [9] and added to the bottom chamber of motility assays utilizing A2058 cells as responders. As seen in Fig. 2A, addition of L-histidine to the pre-incubation mixture resulted in a 75% inhibition of motility for A2058 cells at concentrations similar to those utilized in Fig. 1. These data, which were identical in SKOV-3 cells (data not shown), suggest that the L-histidine acts upon the ATX-catalyzed LPLD reaction to produce its inhibitory effect by preventing the formation of LPA from LPC. Previously, we have shown that SPC is an alternate substrate for ATX: pre-incubation of SPC with ATX, followed by heat-inactivation of ATX, resulted in production of the inhibitor of LPA-stimulated A2058 cell motility, S1P [9]. As seen in Fig 2B, inclusion of 20 mM L-histidine in the ATX + SPC pre-incubation step prevented the formation of an inhibitor of A2058 cell motility. L-histidine therefore appears to inhibit the hydrolysis of both glycerophospholipids and phosphosphingolipids.
Figure 2 Histidine appears to act by inhibiting the ATX LPLD activity. The modified Boyden chamber motility assays with A2058 cells are described in Materials and Methods. In these assays, pre-incubations were in DBA media for 3 hr at 37°C. A) L-histidine decreases formation of a chemoattractant by ATX. Shown concentrations of L-histidine were pre-incubated with ATX plus 200 nM LPC, then ATX was heat-inactivated prior to utilizing the resulting mixture as chemoattractant in a motility assay (solid black bars). Data are compared to identical treatments of 100 nM LPA as chemoattractant (white bars). B.) L-histidine decreases formation of a motility inhibitor by ATX. Shown concentrations of L-histidine were pre-incubated with (ATX + 100 nM SPC + 100 nM LPA). ATX was heat inactivated prior to utilizing the mixture as chemoattractant. Controls are shown for comparison: DBA, 100 nM LPA, (100 nM LPA + 100 nM S1P), (100 nM LPA + 100 nM SPC). All data are shown as Mean ± SEM. Means were analyzed utilizing the ANOVA/Tukey's post-test (GraphPad Prism, San Diego, CA): * (p < 0.001) for histidine-treated vs. the appropriate untreated control.
Effects of Histidine and Metal ions upon ATX Activities
The PDE activity of the NPPs has been shown to be dependent upon the presence of metal ions [15]. Tokumura and co-workers [18] demonstrated a similar requirement for a metal cation in the action of LPLD. Both Co++ and Zn++ were markedly effective in restoring enzymatic activity to LPLD in EDTA-treated plasma. In fact, addition of Co++ to EDTA-treated heparinized plasma resulted in a recovery from 37% of the untreated value in the presence of EDTA alone to 154% in the presence of Co++ and EDTA. Since histidine plays an important structural role in the catalytic process, we determined first that addition of free histidine has an inhibitory effect upon ATX-catalyzed hydrolysis of both nucleotide and lyso-phospholipid substrates. After establishing the concentration requirements for histidine inhibition, we next examined the capacity of different metal cations to reverse this inhibition. Finally, we determined how histidine and metal cations affect the kinetics of the LPLD reaction.
As seen in Fig. 3A, histidine inhibited both LPLD (with LPC as substrate) and PDE (with p-Nitrophenyl-TMP or pNp-TMP as substrate) activities of ATX in a dose dependent manner. At a concentration of 20 mM, this inhibition was virtually complete for both reactions. The normalized curves coincided closely with each other and the IC50 values (4.0 ± 1.1 mM and 4.5 ± 1.1 mM for LPLD and PDE activities, respectively) were not significantly different from each other.
Figure 3 Histidine inhibition of ATX enzyme activities and sensitivity to zinc. For these reactions, 1 mM LPC served as substrate for LPLD activity and 1 mM pNp-TMP as substrate for 5'-nucleotide PDE activity; approximately 6 pmoles of ATX was added to each reaction. A.) Product was measured in the presence of variable concentrations of L-histidine. Data are shown as Mean ± SEM. The two reactions were not significantly different from each other. B.) Different metal cations (0.5 mM) were compared for their capacity to abrogate L-histidine (10 mM) inhibition of enzymatic reactions. C.) Effect of Zn++ on LPC- and ATX-stimulated motility. Utilizing A2058 cells as responders and either ATX or 25 nM LPA as chemoattractant, 20 mM histidine or 0.25 mM ZnSO4 or both were added to the bottom wells throughout the motility assay. Data are shown as Mean ± SEM. Means were compared with ANOVA/Tukey's post-test (GraphPad Prism, San Diego, CA). For B), comparisons were between histidine-treated vs. the equivalent untreated reactions with * (p < 0.001). For C), comparisons were as follows: * (p < 0.001) vs. ATX alone, ** (p < 0.001) vs. ATX + His, # (p < 0.01) vs. ATX alone.
We tested the effects of a number of divalent cations in both the presence and absence of histidine upon the PDE and LPLD activities of ATX (Fig. 3B). Neither Mg++ nor Ca++ significantly reversed the inhibitory effect of histidine although Ca++ alone enhanced both reactions by at least 30%. However, Zn++ and Co++ each significantly overcame inhibition caused by free histidine. In the case of Zn++, the presence of the cation alone increased both reactions by up to 50% above control, and virtually abolished the inhibition caused by histidine, with recoveries of 90 – 100% of the activity measured in the presence of Zn++ alone. In the case of Co++, the cation alone increased the PDE activity by 50% and the LPLD activity by 300%; however, histidine still inhibited PDE by ~70% and LPLD by 50% compared to adding Co++ alone. Zn++, therefore, appears to be the most effective cation in reversing the inhibition by histidine. It should also be noted that none of the cations at the levels tested had any inhibitory effects upon the enzymatic reactions of ATX. Histidine therefore inhibits a process that is required for the hydrolysis of both nucleotides and phospholipids. This may be a rate-limiting cation-dependent step in their common reaction mechanism [7].
Since Zn++ reversed the inhibitory effect of histidine upon ATX enzymatic reactions, we tested its effect upon the histidine-induced inhibition of ATX- and LPA-stimulated motility. As shown in Fig. 3C for A2058 cells, 20 mM histidine, 0.25 mM Zn++, or both together had no effect upon LPA-induced motility. In contrast, ATX-induced motility was affected in a more complex manner. Histidine (20 mM) alone inhibited ATX-induced motility by ~65%, while 0.25 mM Zn++ alone increased this same motility by ~30%. When these same concentrations of histidine and zinc were added together to an ATX-stimulated motility assay, the histidine inhibition was largely reversed with greater motility than seen with ATX alone, though not quite up to levels seen with ATX + Zn++. However, statistical analysis of these results revealed no statistically significant difference between ATX + Zn++ + histidine vs. ATX + Zn++. In contrast, there was a statistical significance between ATX + Zn++ + histidine vs. ATX + histidine (p < 0.001).
Fig. 4 shows a concentration curve of the ATX-catalyzed LPLD reaction with LPC as substrate, both with and without addition of 10 mM histidine. The two reactions had Km values (0.49 ± 0.05 mM untreated vs. 0.66 ± 0.18 mM with histidine) that were not significantly different from each other; however, Vmax was reduced more than 50% by the addition of histidine. Also shown are the effects of 0.25 mM Zn++, alone or in addition to histidine. Zn++ alone had no significant effect on the Vmax or Km of the reaction. In the presence of histidine, Zn++ reversed the inhibition, restoring Vmax to untreated levels. It might be noted that the pattern of inhibition seen when histidine is added is typical of a non-competitive inhibitor.
Figure 4 Reaction rate vs. substrate concentration curves in the presence of L-histidine or zinc LPLD determinations were carried out in the presence of 10 mM L-histidine or 0.25 mM Zn++, as indicated. Curves were analyzed with GraphPad Prism (San Diego, CA) to calculate Km and Vmax, as shown.
Effect of Chelating Agents upon the ATX-stimulated Reaction
Because the histidine-induced inhibition of ATX activities could be reversed by addition of appropriate metal ions (i.e., Zn++), the possibility arose that the imidazole ring of histidine acts as a weak chelation agent, competing with the enzymatic active site for binding to the metal ion. EDTA has been shown to inhibit the nucleotide phosphodiesterase and pyrophosphatase activities of PC-1/NPP1 [19]. We, therefore, utilized EDTA, as well as the metal chelating agent 1,10-phenanthroline, in order to examine their effect upon the LPLD and PDE activities of ATX. We also tested the reversibility of their effects with Ca++ and with Zn++.
In a series of preliminary experiments (data not shown), we found that, under the conditions of our enzymatic assays, addition of 10 – 20 mM Na4-EDTA resulted in profound inhibition of both LPLD and PDE activities with an IC50 of approximately 5 mM. We also found that 5 mM Na4-EDTA required very nearly equimolar concentrations of metal cations to reverse its inhibitory effects. As shown in Fig. 5, addition of equimolar concentration of Zn++ resulted in essentially complete recovery of activity (range 90 – 110% recovery) for both LPLD and PDE activities, while an equimolar concentration of Ca++ resulted in ~75% recovery of control activity.
Figure 5 Effect of chelating agents on ATX's LPLD activity The LPLD activity of ATX was compared in the presence of 0.5 mM phenanthroline A (phenA), 5 mM EDTA, or 10 mM L-histidine. The effect of adding Zn++ or Ca++ was compared for each chelating agent. Results are expressed as Mean ± SEM. Statistical analysis of results was performed utilizing GraphPad Prism (San Diego, CA). Data that is significantly different from untreated control is shown by: * (p < 0.001), ** (p < 0.05); recovery with metal cations is shown as statistically different from appropriate inhibitor-treated samples: # (p < 0.001).
Addition of 0.5 mM 1,10-phenanthroline also profoundly inhibited the LPLD activity of ATX (Fig. 5). The IC50 for 1,10-phenanthroline in this reaction was approximately 0.25 mM (data not shown). As has been reported previously, a significant recovery from 1,10-phenanthroline-induced inhibition requires less than equimolar concentrations of Zn++ [20]. Addition of 0.05 mM Zn++ resulted in a 70 – 80% recovery compared to control values, while adding up to 5 mM Ca++ had no significant effect upon phenanthroline-induced inhibition of activity. As can be seen in Fig. 5, the pattern of recovery from 1,10-phenanthroline-induced inhibition is similar to the pattern seen with histidine-induced inhibition of LPLD.
Testing the effect of these chelating agents on motility was problematic because chelation agents have multiple, complex effects upon living cells. For example, Na4-EDTA treatment killed our responder cells (A2058) in about 15 – 20 min, and 1,10-phenanthroline inhibited cellular adhesion at concentrations utilized for inhibition of LPLD and PDE activity. Therefore, we were unable to obtain motility data in the presence of these agents.
Effects of Histidine Analogs upon the LPLD Reactivity of ATX
A number of histidine-derived agents, as well as histamine and imidazole (Fig. 6A), were tested for their effects upon the LPLD activity of ATX. Since 15 – 20 mM L-histidine gave similar levels of inhibition, all reactions were carried out in the presence of 15 mM inhibitor.
Figure 6 Effect of histidine analogs on LPLD activity A.) Chemical structure of histidine analogs compared. B.) Histidine analogs (15 mM) were added to the LPLD assay and analyzed for their effect on LPLD activity with (white bars) or without (black bars) addition of 1.0 mM Zn++. Results are shown as Mean ± SEM. Means, for each histidine analog vs. appropriate untreated control, were analyzed utilizing ANOVA/Tukey's post-test (GraphPad Prism, San Diego, CA): * (p < 0.001), ** (p < 0.01).
As seen in Fig. 6B, D-histidine and L-histidine are the most effective inhibitors of the ATX LPLD activity, resulting in approximately 75% reduction. They were not statistically significant from each other. Nearly as inhibitory was histidine methyl ester, which has an esterified alpha carboxyl group and which reduced LPLD activity by 65%. Other compounds with substitutions on the alpha carboxyl group, histidinamide and histidinol, were markedly less effective as inhibitors. Histidinol, in which the carboxyl group is reduced to a hydroxy-methyl group, was not statistically different from untreated control reactions. Histidinamide, with amidylation of the alpha carboxyl group, was mildly inhibitory, reducing activity ~20%. In contrast, histamine, which lacks the alpha carboxyl group, altogether, was slightly stimulatory to LPLD activity. Histidine analogs with methyl groups added to the alpha nitrogen were also less active than histidine itself. N, N-dimethyl histidine was not statistically different from untreated control, while N-methyl histidine, resulted in just a 30% reduction in LPLD activity. Surprisingly, at the tested concentrations, the metal-binding agent imidazole had no effect on the LPLD activity of autotaxin. Likewise, amino acids that resemble histidine without its imidazole ring (e.g., glycine and L-alanine) also lack inhibitory activity (data not shown).
Among the five histidine analogs found to inhibit ATX LPLD activity, the relative inhibitory activity is: D-histidine = L-histidine > histidine methyl ester >> N-methyl histidine > histidinamide. The effects of all of these agents are reversed by zinc. In contrast, glycine, L-alanine, imidazole, histidinol, and N, N-dimethyl histidine had no significant effect on activity; and histamine appeared to be slightly stimulatory. Most of these histidine analogs, including D-histidine, histidine methyl ester, histidinamide, N, N-dimethyl histidine, and histamine, were toxic to cells. N-methyl histidine resulted in reduced adhesion to our gelatin-coated membranes, precluding an effective motility assay. Only imidazole could easily be tested for its effect on ATX-stimulated motility. At 10–20 mM concentrations, imidazole had no significant effect on ATX-stimulated motility (data not shown).
Discussion
We have shown that L-histidine, at concentrations that are innocuous to cells, can inhibit the motility-stimulating action of ATX upon two tumor cell lines derived from human melanoma (A2058) and human ovarian carcinoma (SKOV-3). This inhibition is largely abrogated by addition of 0.25 mM Zn++ to the motility assays. Pre-incubation of ATX and LPC in the presence of L-histidine, followed by heat-killing the ATX, also resulted in much decreased activity, suggesting that L-histidine inhibits ATX-stimulated motility by ablating its LPLD activity. This hypothesis was in fact confirmed both by direct enzymatic studies in which L-histidine inhibited both PDE and LPLD activities and by the failure of ATX, in the presence of L-histidine, to produce an inhibitor of LPA-stimulated motility. Again, addition of Zn++ abrogated this inhibition of enzyme activity. These data provide further evidence that the LPLD activity of ATX, which results in the production of the mediator LPA, is essential for ATX stimulation of cellular motility.
The role of metal cations in the activation of ATX was further investigated by first comparing the capacity of Ca++, Mg++, Zn++ and Co++ to reverse the histidine-induced inhibition of ATX enzymatic activities. Neither Ca++ nor Mg++ reversed the inhibitory effect of histidine, whereas Co++ alone stimulated a 50% increase in PDE and a 300% increase in LPLD activities of ATX. Histidine partially reversed both of these Co++-stimulated increases: PDE activity was reduced to below uninhibited levels of product while LPLD activity, though reduced by 50% compared to treatment with Co++ alone, remained at levels above untreated controls. The mechanism by which cobalt activates ATX remains unexplained; however, Co++ has long been reported to replace one or more zinc cations in a variety of zinc-bound metalloenzymes [21-23], sometimes with a notable increase in activity [24,25].
In contrast, Zn++ at concentrations as low as 0.25 mM abrogated the inhibitory effects of 10 mM histidine upon both the PDE and LPLD activities of ATX. This 40:1 ratio of ligand to cation appears to be inconsistent with a simple stoichiometric coordination of the cation with the metal-binding imidazole moiety. This would predict that the maximum number of ligand groups to coordinate with a Zn++ would be six. Our result is, therefore, difficult to explain as simple chelation in solution between cations and histidine. Perhaps, Zn++ acts upon ATX in a manner that blocks the entry of histidine into the active site. For example, Zn++ might coordinate with an electronically available locus in proximity with the active site of ATX or it might induce conformational changes in ATX that lowers the affinity of free histidine for the active site. Another possibility is that Zn++ replaces another metal ion, resulting in a binding conformation with a lower affinity for L-histidine. Interestingly, 0.25 mM Zn++ appeared to have a slightly stimulatory effect upon ATX-induced motility, but not LPA-induced motility. This suggests that Zn++ might act in a way that stabilizes ATX in an active configuration.
Many members of the alkaline phosphatase superfamily of metalloenzymes have Zn++ or Zn++ plus Mg++ incorporated into their catalytic site, although a number of other metal requirements have been documented [14,26]. The chelation agents, Na4-EDTA and 1,10-phenanthroline both inactivate ATX, confirming that it is a metalloenzyme. Na4-EDTA complexes with a variety of cations, including transition metals as well as alkali and alkaline earth metals, and is thought to inactivate metalloenzymes by removing the metal cation from its binding site. Under the conditions of our assays, the IC50 of EDTA was ~5 mM. This inhibition was completely reversed by addition of equimolar Zn++ and significantly reversed (about 75% recovery) by addition of equimolar Ca++. In contrast, 1, 10-phenanthroline has more complicated and diverse mechanisms of action. When utilized with metalloenzymes, it can form mixed complexes with the metal ions as well as other cationic sites on the protein, resulting in inactivation of the enzyme but not necessarily removal of the metal cation [27]. Like histidine, 1,10-phenanthroline requires less than equimolar concentrations of Zn++ (approximately 1:10) to reverse its inhibitory effect on ATX. Based on all of our data with metal cations, it appears that Zn++, Co++ and perhaps Ca++ can function as the metal moiety in at least one of the two metal binding sites [15] of the active metalloenzyme ATX, although we do not know the predominant metal cations in the native enzyme.
In order to determine what portion of the histidine molecule was responsible for its inhibitory activity, we utilized a number of commercially available histidine analogs. These analogs were predominantly substituted on the alpha carboxyl and alpha amino groups of histidine; but they also included histamine, which lacks the alpha carboxyl group altogether, and imidazole, the weakly chelating ring that makes up the major portion of the histidine side chain. Except for D-histidine, none of the other imidazole-containing compounds were as inhibitory as L-histidine. Among the 5 agents found to have significant inhibitory activity, the relative potency is as follows: D-histidine = L-histidine > histidine methyl ester >> N-methyl histidine > histidinamide. Methylation of the alpha amino group of histidine resulted in a stepwise loss of potency, with a single methylation (N-methyl histidine) resulting in about a 40% reduction in activity and a double methylation (N, N-dimethyl histidine) not significantly different from untreated control reactions. Since these two compounds should retain the positive charge properties of the native alpha amino group, this could be a steric effect. Similarly, change in the carboxyl group (particularly amidylation or reduction) also decreased the inhibitory potency, though that of esterified histidine methyl ester was only slightly reduced. Interestingly, imidazole itself, a moiety postulated to coordinate with the cation at the active site of ATX, was not effective at the tested concentrations. Likewise, amino acids, which lacked imidazole on their side chains (e.g. glycine and L-alanine), were also ineffective as inhibitors. Clearly, three functional groups of histidine are required for full inhibitory activity: the alpha amino group, the alpha carboxyl group, and the metal-binding imidazole side chain. Recent work with Zn++-binding groups in matrix metalloproteinase inhibitors indicated that slight changes to the Zn++-binding groups can result in major changes in efficacy of inhibition [28], suggesting an approach for developing more potent, and perhaps highly specific, pharmacologic inhibitors.
ATX, the major source of plasma LPA, has been implicated in a number of physiological and disease-related processes [29], including tumor metastasis [30] and angiogenesis [31]. A pharmacologic agent that inhibits ATX activity could have major therapeutic implications. Other than the highly non-specific chelation agents [18], L-histidine is the first reported inhibitor that acts on the LPLD activity of ATX rather than acting on its downstream activation cascade. This inhibition, requiring millimolar concentrations of L-histidine, is reversible by Zn++ metal cations. Our data suggest that the L-histidine-induced inhibition of the ATX enzymatic activities is predominantly non-competitive, i.e. Vmax is decreased significantly, while Km is not significantly affected. Furthermore, the mechanism of action of L-histidine does not appear to be simple stoichiometric chelation of the metal cations within a metalloenzyme but is more likely a complex interaction with a variety of moieties, including the metal cation, at or near the active site. At concentrations required for an inhibitory effect, most available histidine analogs resulted in loss of cell viability. Free L-histidine, therefore, appears to be unique in its ability to inhibit LPLD, and hence regulate a major source of LPA, in living systems. Since LPA has significant pathological effects, the regulation of its formation is of considerable interest. Potential problems with L-histidine as a therapeutic agent include its lack of potency and its conversion in vivo to histamine. While there are known inhibitors of histidine decarboxylase, such as the polyphenols of green tea [32], potent, stable, and non-toxic analogs of histidine would appear to be better therapeutic agents.
Conclusion
L-histidine inhibits the LPLD activity of ATX at millimolar concentrations, reducing its capacity to produce its major mediator, LPA.
Methods
Reagents
LPA (18:1) and S1P were purchased from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA). LPC (18:1), SPC, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (TOOS), 4-aminoantipyrene (4-AAP), horseradish peroxidase, choline oxidase, p-nitrophenyl-TMP, L-histidine, D-histidine, imidazole, histamine, histidinamide, histidine methyl ester, N,N-dimethyl histidine, histidinol, Na4-EDTA, and 1,10-phenanthroline were from Sigma-Aldrich (St Louis, MO). N-methyl histidine was from Bachem Bioscience (King of Prussia, PA).
Cell Lines
A2058 [33] and SKOV-3 cells were maintained in Dulbecco's Modified Essential Medium (DMEM) supplemented by 2 mM glutamine, 1X penicillin/streptomycin and 10% (v/v) heat-inactivated fetal bovine serum. SKOV-3 cells (ATCC # HTB-77) were purchased from American Type Culture Collection (Manassas, VA).
ATX Preparations
Highly purified recombinant human ATX (vATX), cloned from an MDA-MB-435 cell library, was prepared from a Vaccinia viral lysate, as described previously, through the concanavalin A-agarose step [34].
In vitro Motility Assays
Cells were detached from their flasks with a brief exposure to 0.05% trypsin and 0.02% versene and then resuspended at 2 × 106 cells/ml in DMEM supplemented with 1 mg/ml bovine serum album (DBA). Ten min before the start of the assay, appropriate concentrations of potential inhibitors were added to the different treatment groups. Chemotaxis was assayed as described previously in detail [35], utilizing gelatin-coated membranes for A2058 cells and Type IV collagen-coated membranes for SKOV-3 cells. Chemotaxis chambers were incubated 3 hr for A2058 cells and 5 hr for SKOV-3 cells. Migrated cells were fixed and stained, then quantified by cell counting under light microscopy at 200X (SKOV-3 cells) or 400X (A2058 cells).
Enzymatic Activity Assays
Enzyme activities were determined using a modification of the previously described assays [9]. Briefly, a 5 ml aliquot of vATX (adjusted to give ≈ 6 pmoles vATX/reaction) was incubated (50 μl reaction volume) with either 1 mM pNp-TMP or, alternatively, with 1 mM LPC, for 45 min at 37°C in DBA. DBA was utilized in order to mimic the conditions of motility assays. For assays with histidine and its analogues, stock solutions were prepared and adjusted to physiological pH with NaHCO3 or HCl, as appropriate. The inhibitor was added to the reaction mixture; then, reactions were initiated by adding ATX.
For determination of 5'-nucleotide PDE activity, reactions were stopped by the addition of 450 μl 0.1 N NaOH and the nitrophenol product was detected by reading the absorbance at 410 nm (A410 × 64 = nmoles). In order to determine LPLD activity, released choline was detected as follows: a 450 μl cocktail containing 50 mM Tris-HCl (pH 8), 5 mM CaCl2, 0.3 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (TOOS), 0.5 mM 4-aminoantipyrene (4-AAP), 5.3 U/ml horseradish peroxidase, and 2 U/ml choline oxidase was added to the 50 μl reaction mixture and incubated for 20 min at 37°C. Absorbance was read at 555 nm and converted to nmoles of choline by comparison to a choline standard curve (A555 × 17 = nmoles).
List of Abbreviations Used
ATX Autotaxin
DBA DMEM with 1% bovine serum albumin
DMEM Dulbecco's Modified Essential Medium
LPA Lysophosphatidic acid
LPC Lysophosphatidylcholine
LPLD Lysophospholipase D
NPP Nucleotide pyrophosphatase and phosphodiesterase
PDE Phosphodiesterase
pNp-TMP p-Nitrophenyl-TMP
S1P Sphingosine-1-phosphate
SPC Sphingosylphosphorylcholine
Authors' Contributions
TC carried out the enzymatic studies, EK performed cellular studies with A2058 cells, MP performed cellular studies with SKOV-3 cells, and RWB carried out background studies and fine-tuned methodology. ES originated the idea of studying the effect of L-histidine on LPLD activities. LAL and MLS analyzed and interpreted data. MLS drafted and coordinated the manuscript between authors. All authors participated in reading and refining the manuscript drafts.
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| 15737239 | PMC554093 | CC BY | 2021-01-04 16:39:18 | no | Lipids Health Dis. 2005 Feb 28; 4:5 | utf-8 | Lipids Health Dis | 2,005 | 10.1186/1476-511X-4-5 | oa_comm |
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Cardiovasc UltrasoundCardiovascular Ultrasound1476-7120BioMed Central London 1476-7120-3-61574062010.1186/1476-7120-3-6ReviewUltrasound imaging versus morphopathology in cardiovascular diseases. Coronary collateral circulation and atherosclerotic plaque Baroldi Giorgio [email protected] Riccardo [email protected] Lauro [email protected] Institute of Clinical Physiology, National Research Council, Milan and Pisa, Italy2 University School of Medicine and "A. De Gasperis" Foundation, Niguarda Hospital, Milan, Italy3 Cardiovascular Unit, "Campo di Marte" Hospital, Lucca, Italy2005 1 3 2005 3 6 6 14 2 2005 1 3 2005 Copyright © 2005 Baroldi et al; licensee BioMed Central Ltd.2005Baroldi et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This review article is aimed at comparing the results of histopathological and clinical imaging studies to assess coronary collateral circulation in humans. The role of collaterals, as emerging from morphological studies in both normal and atherosclerotic coronary vessels, is described; in addition, present role and future perpectives of echocardiographic techniques in assessing collateral circulation are briefly summarized.
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In the past 25 years, the concept of a compensatory function of the coronary collaterals (or anastomoses) – i.e. vessels that join different coronary arteries or branches – has been practically cancelled from the mind of cardiologists since cineangiography shows that the onset of coronary heart disease (CHD) occurs independently of their presence. The assumption, therefore, was and is that they have no compensatory meaning [1] and coronary obstruction causes ischemia. A crucial and questionable assumption which disregards solid and recognized pathological data and supports invasive therapies, the diagnostic gold standard being the coronary cineangiography. In many cardiological centers, at the first chest discomfort, the latter is the guide for emergency angioplasty + stent or surgical bypass when a coronary ostruction is found; with the belief that a severe coronary stenosis causes angina pectoris, its occlusion an acute myocardial infarct (AMI) or sudden death (SD) and chronic ischemia explains hibernating myocardium.
By injection under controlled pressure of plastic materials through the aorta, casts of coronary arteries, including coronary ostia, in normal and pathological hearts were obtained. They gave an objective tridimensional view of anatomy, different patterns of coronary distribution and overall collaterals in relation to coronary lumen reduction. The method allowed a histologic control of the myocardium [2-4]. The casts of normal coronary arteries showed a smooth surface without identations easily identified when even a minor lumen reduction was present. In hearts of normal people dead by accident without pathological findings at autopsy, homocoronary (between branches of the same coronary artery) and intercoronary (between different coronary arteries) anastomoses were present everywhere joining at any level the intramural branches. Only in two of more than 600 hearts, superficial collaterals between extramural coronary arteries were seen and sampled for histology. The diameter of the innumerable normal collaterals ranged from 20 (maximal penetration of plastic injection) to 350 microns, frequently assuming a corkscrew aspect, possible adaptation to the contraction cycle of the myocardium (Figure 1). The first conclusion was that arterial intramural system, including the terminal bed, is an anastomotic network, at least from the anatomical viewpoint.
Figure 1 Coronary anastomoses or collaterals. A) intercoronary ventricular and (B), atrial. C) homocoronary anastomoses. Note the innumerous collaterals joining different intramural branches at any level of their course. They have frequently a corkscrew aspect (D) visible also histologically (E), as adaptation to cardiac contraction-relaxation cycle.
In hypertrophic hearts with normal coronary arteries and in normal hearts of patients with chronic hypoxia, e.g. anemia, collateral diameter and length were increased in the whole intramural system (500 microns; Figure 2). The more impressive change was seen in presence of coronary stenosis greater than 70% with a diameter and length exceeding 1000 microns and several centimeters respectively (Figure 3). The other peculiarity was that collateral enlargement was strictly related to a stenosis filling distal tract of the obstructed vessel (satellite anastomoses); when more than one severe stenoses exist each one had its own satellite collaterals. However, an identical obstruction located at the same level of an artery might show relatively few highly enlarged collaterals (the only ones visible by cineangiography), or numerous relatively small collaterals (Figure 4). A finding possibly due to a redistribution of blood flow consequent to newly formed severe stenoses or an infarct. In the latter condition, all vessels within the necrotic tissue disappear (avascular area seen in plastic casts; Figure 5) and the surviving collaterals at periphery will further enlarge since the pressure gradient distal to the coronary obstruction persists.
Figure 2 Vessel changes in relation to modification of the cardiac mass. A) atrophic heart with acquired serpentoid form of extramural vessels due to cardiac mass reduction, and minor intramural vascularity. The contrary is seen in cardiac hypertrophy (B) in which the extramural arteries increase in length and diameter (but not in number) to adapt themselves to the greater myocardial mass. Similarly, the same enlargement is seen in the intramural branches. Cor pulmonale, in which condition the right ventricle may become greater than the left one, is an extreme example of adaptation of extramural (C) and intramural, including collaterals (D). No histologic evidence exists of new vessel formation. The cardiac vein show a similar behaviour.
Figure 3 Collateral enlargement in topographical relation (satellite) with severe stenosis or occlusion. A double occlusion of LAD (anterior view) and occlusion of RCA (posterior view) apparently compensated by enlarged collaterals in a non cardiac patient dead from brain hemorrhage. B, similar condition in cases with RCA occlusion (arrow) without corresponding myocardial infarct with numerous homo and intercoronary collaterals of the anterior wall (C), and (D) septum. Occlusion of LAD without evidence of other stenotic changes of the coronary arteries in a 39-year-old woman with rheumatic heart disease and mitral insufficiency. In this case, arteritis was documented histologically by sampling before corrosion. An acute infarct (avascular area at the apex, arrow) was present. F, a single, high enlarged collateral from LCX, supplying the distal tract of an occluded LAD. Note, numerous normal anastomoses. This indicates that ischemia is not the cause (no diffuse enlargement of all collaterals in the whole ischemic area) but rather pressure gradient induces selective compensatory routes.
Figure 4 Different aspects of collateral compensation in presence of the same occlusive pattern of LAD. A, relatively few very enlarged collaterals and (B) numerous relatively small collaterals. This divergency may be due to progressive atherosclerotic obstruction of other main vessels or lost of the intramural vasculature, including collaterals, following an infarct. Chart C shows all the possibilities of flow redistribution. The histology of the enlarged anastomoses corresponds to a capillar-like wall, even in the rare extramural collaterals with rudimentary focal tunica media (C). D), enlarged collaterals in a case of anomalous origin of LAD from the pulmonary artery and (E,G) different aspects of giant capillaries (or plexus) in various stages of an acute/old infarction. The absence of new vessel formation is well documented in recent infarcts associated with endocardial thrombus (G). In the latter numerous new vessels form in the granulation tissue repair of the thrombus in contrast to their absence in infarct (arrow; postmortem coronary injection for vessels identification).
Figure 5 Avascular area of an infarct. By plastic cast (A anterior, B posterior view) or postmortem angiogram (C) the infarcted zone (arrow) lacks of intramural vessel injection ("avascular area"). Stretching of the necrotic myocardium and secondary vascular damage with wall degeneration and thrombosis (D), explain this vascular "sequestration" which occurs in early phase. This may indicate a blockage without possibility of therapeutical intervention via blood flow within the infarcted myocardium. Note that the avascular area in this AMI case documented histologically, depended from LAD without evidence of occlusion or severe stenosis. The occluded vessel (arrow) was (B) the RCA, the distal part of which was filled by numerous anastomoses. No myocardial damage was seen in its territory. By dissection even an expert pathologist, the diagnosis could be of myocardial infarction following occlusion of the RCA. E) obliterative intimal hyperplasia in arterioles around a seven days old infarct with early repair process.
Another satellite collateral system is annexed around and within the atheroclerotic plaque. Plastic casts and histological serial sections showed an extensive vascularization limited only at plaque level and formed by giant adventitial capillary-like vessels filled by intracoronary radiopaque injected material, connecting secondary branches proximal and distal to the stenosis as well as new vessels formed within the atherosclerotic intima i.e. arterioles, with a well developed tunica media, related to angiomatous plexuses which open in the residual lumen (Figure 6). This plaque satellite system may explain why by cineangiography the coronary tract distal to stenosis is immediately filled while in its absence a delay or flow reduction should be expected.
Figure 6 Vascularization of a coronary atherosclerotic plaque showing different aspects of neovascularization. By serial sections of postmortem injected plaques, giant advential capillary-like vessels (A) are connected with secondary branches proximal and distal to the plaque and with new arterioles (B) with a well developed tunica media (indication of functioning blood flow), within the thickened, atherosclerotic intima in turn joined through angiomatous plexuses (C) to the residual lumen (D) E) plastic casts of plaques with different aspects of vascularization.
Both homo-intercoronary and plaque collateral systems are anatomical structures capable to adapt in particular pathological conditions. The question is whether or not they are able to prevent ischemia and compensate an occlusion which by cineangiography appears as a "cut off" of a vessel without imaging of its distal tract. It must be stressed that in postmortem casts with coronary occlusion the latter was always injected through collaterals.
In 87% of AMI patients, within four hours from clinical onset, a cineangiographic occlusion was observed and in 88% of cases undergone emergency bypass surgery, a "layered thrombus" was recovered "proximal to stenosis" [5]; a thrombus due to plaque rupture [6-8] causing the infarct or sudden death.
In discussing this dogma the first need is to review the function of the collaterals.
Collateral function
Capillary function in presence of normal coronary arteries
In normal hearts and in pathologic hearts with normal coronary arteries, the collaterals, due to their capillary structure, participate to the metabolic exchange as terminal capillary bed. This means a much greater extent of the exchange surface which invalidates any "one myocardial / one capillary" model to study the delivery of any substance from capillary to myocardial cell. The myocardial interstitium is crossed by a myriad of "endothelial" vessels in any direction.
Compensatory function in presence of coronary obstruction
The demonstration of tridimensional collateral enlargement by casts indicates, per se, that there was an increased blood flow. Their adequacy to compensate one or more severe coronary obstructions is documented by the following main facts:
1. At the first episode of coronary heart disease (CHD) in apparently healthy people acting their normal life, 89% with a fatal AMI had one or more (47%) severe atherosclerotic stenosis greater than 70% ;65% of sudden and unexpected death (SUD) showed the same finding in one or more (35%) vessels; 66% of non cardiac patients dead from other diseases and 39% of normal subjects dying from accident had the same severe atherosclerotic stenosis in one or more (40% and 16% respectively) coronary arteries (Table 1). At histology, all plaques were old lesions preexisting months or years without any evidence of CHD despite a stressful life and in absence of a myocardial infarct. The only explanation is that the collateral system was able to fully compensate the blood flow reduction consequent to the stenoses.
Table 1 Maximal atherosclerotic lumen diameter reduction and number of main arteries with severe (≥ 70%) stenosis
Source Acute myocardial infarct Sudden death unexpected Non cardiac atherosclerotic Patients Accidental death in normal people
1st chronic 1st chronic
Cases 145 55 133 75 100 97
% Lumen reduction
0 3 - 10 - 7 8
<50 3 1 18 - 10 20
50–69 10 - 18 5 17 31
70–79 30 8 21 8 11 19
80–89 45 11 39 14 24 13
≥ 90 54 35 27 48 31 6
No. arteries ≥ 70%
1 61 16 40 13 26 22
2 49 22 34 26 18 13
> 3 19 16 13 31 22 3
1st episode, in apparently normal people without extensive monofocal myocardial fibrosis Chronic, in subjects with history of coronary heart disease and/or extensive myofibrosis.
2. Myocardial infarct size measured planimetrically was not related to the number of severe coronary stenoses found in each AMI case (Table 2) as should be. More severe coronary stenoses should determine a higher ischemia resulting in larger infarcts.
Table 2 Lack of correlation between number of severe (≥ 70%) coronary stenoses and acute infarct size (% left ventricular mass) in 200 consecutive and selected fatal cases.
Source Acute myocardial infarct
Cases 200
97 103
≤ 20 size > 20
Lumen reduction
< 69 7 10
≥ 70 90 93
in 1 39 38
2 37 34
≥ 3 vessels 14 21
p < 0.05 for trend
3. No relation between the total vascular territory of obstructed coronary artery and infarct size which often extended in territories of non stenosed or occluded vessels. In vivo hypokinetic zones expand in well perfused region [9].
4. The relatively frequent finding of a coronary occlusion without an infarct.
5. In an experiment done in a leading dog lab, a controlled coronary stenosis, maintained for few days and then occluded, did not determine any dysfunction or infarct because a dramatic increase of collateral flow [10-12].
These are the main facts supporting the concept that collaterals shown postmortem succeed in limiting or abolishing ischemia induced by coronary obstruction and question the existence of chronic ischemia due to coronary atherosclerosis since a plaque takes time to develop while collaterals [10,11] adapt itself quickly as soon a pressure gradient between stenosis and distal vessel is established. On the other hand, there is no demonstration of a possible failure, both acute or chronic, of collaterals; including spasm since they have not tunica media.
The inability of cineangio imaging to visualize collateral systems is explained by its very limited power of resolution of all intramural vessels and by the selective injection of radiopaque labelled blood flow in one coronary artery competing with non labelled flow from the other coronary artery. Only very enlarged intercoronary anastomoses can be seen cineangiographically without any value in relation to cardiac dysfunction. Acute ischemia induced by balloon inflation at angioplasty may depend on sudden occlusion by compression of the collateral plaque system.
Active coronary atherosclerotic plaque according to cineangio imaging
Active plaque means an impending infarct expressed by a variety of angiographic signs as irregular lumen, haziness with ill-defined margins, smudge appearance, inhomogeneity, opacification, luciencies, persistence of radiopaque material, etc. Signs difficult to correlate with postmortem findings since terminal changes can not be excluded. They may represent the irregular vascularization of the atherosclerotic plaque opacified by the injected radiopaque material. Worthy of note is that cineangio defects can persist unchanged per years [13].
Cineangio coronary occlusion
The very high frequency of coronary occlusion seen angiographically in AMI patients (see above) does not correspond to that observed in pathological studies in which the mean figure is 50% for AMI and 29% for SUD patients. Nevertheless, different selection of material, divergent definition and an absence of a correct correlation of all pertinent variables give reason of dissimilar conclusions. In 200 selected AMIs and 208 SUD cases the unique cause of occlusion was a thrombus found in 41% and 29% respectively. In AMI group it correlated significantly with a lumen reduction greater than 70% (93%), length of plaque more than 6 millimeters (95%), its concentric shape (100%), prevailing atheroma (84%), medial neuritis (92%) infarct size greater than 50% (86%). SUD cases showed a similar behavior.
In reality, both clinicians and pathologists observe a phenomenon which started before, missing its onset and sequence of events to distinguish whether primary or secondary. In only one case reported in literature [14], this sequence and histological examination of the whole heart was possible in a 45 year old man suffering a two months unstable angina. At coronary cineangiography there were two critical stenoses of the left anterior descending branch (LAD), one proximal and another distal to the origin of diagonal branch and a critical stenosis in the first tract of the right coronary artery (RCA). An antero-septal-lateral hypokinesis was documented. After the fourth left coronary injection, in absence of any symptom or sign and cineangio imaging changes, a first ECG showed downsloped ST segment. The latter persisted for other four LAD injections when the vessel disappeared, again, without any subjective and objective signal. Intracoronary vasodilator and fibrinolytic agents, successful angioplasty in reopening critical stenoses, surgical bypass in rapid sequence were performed without re-establishing flow. Only for few short periods a reflow occurred with an imaging of occlusion which from the distal tract ascended till the origin of LAD (Fig. 7) and not at the site of angioplastically reopened stenoses. An interesting note is that a severe chest pain started after angioplasty, 90 minutes from the first ECG change. The patient survived an extensive myocardial infarction and 12 months later underwent heart transplantation because irreversible congestive heart failure. We had the opportunity to examine the heart removed at surgery confirming a large (40% of the total left ventricular mass) antero-septal-lateral scar, end result of the infarct, scattered foci of fibrosis everywhere, absence of small vessel disease, colliquative myocytolysis expression of congestive failure, severe lumen reduction by sclerosis of LAD (90%) – despite it showed a normal lumen at bypass surgery – and vein graft (80%) without evidence of thrombosis, RCA occlusion by an organized thrombus located in an atherosclerotic plaque with 90% lumen reduction, medial neuritis i.e. lympho-plasmacellular inflammation of nerves closed to the tunica media, in all atherosclerotic plaques, absence of an infarct in RCA territory.
Figure 7 Cineangiographic monitoring in a patient with non occlusive LAD stenosis (A) who developed an extensive infarct without angiographic occlusion. The subsequent imaging of occlusion began distally (B) and ascended to the origin (C) of the vessel (arrow) indicating that the angiographic "pseudocclusion" was due to stasis for increased peripheral resistance and not for primitive thrombosis, not shown morphologically (see text).
One case is only one case but when for the first time shows how the events developed, it becomes a precious mile stone for our knowledge demonstrating that the cineangio occlusion was a pseudocclusion namely a blood flow stasis in LAD secondary to an increased intramyocardial resistance. The first main question is how many of the 87% cineangio occlusion are pseudocclusion and whether the "layered" thrombus recovered at bypass surgery was a true thrombus or a coagulum which frequently show a layering of blood elements not seen in thrombus formation.
"Red" thrombus, namely a coagulum, is frequently and erroneously considered as thrombus. The second question concerns the nature of increased intramyocardial resistance: spasm of intramural arterial vessels or their extravascular compression by an asynergic myocardium? The first sign of CHD is hypokinesis of a myocardial zone which particularly in systole may compress vessels. Any time there is an increase of the intraventricular pressure with bulging of hypokinetic myocardium such a compression may abolish blood flow with subsequent infarction. In the reported patient location and infarct size corresponded to the hypokinetic area observed before the infarct onset.
A last comment deserves the supposition that small atherosclerotic plaques undetectable at cineangio, may rupture causing an infarct. A supposition based on the cineangio finding of a non critical stenosis observed in a vessel tributary of a territory in which an infarct will develop. Since, when the latter occurred, stenoses in other non supplying vessels did not show a further lumen reduction, the conclusion was that even the plaque related to infarction had a non critical lumen reduction [15]. A conclusion that ignores the following two main facts. First that no one pathological study demonstrated the rupture of a small plaque associated with a thrombus occluding a normal or mild stenotic lumen. Second, myocardial asynergy by increasing intramyocardial resistance, promotes plaque progression by an increased dynamic stress on wall of the supplying extramural artery. For instance, in the previous case both LAD and vein graft with a normal lumen at surgery, in 12 months became critically stenotic (90% and 80% respectively). Regional myocardial dysfunction is an important cofactor in accelerating atherosclerosis lesion in related artery.
Target of ultrasound diagnosis: the present and the future
In the past years, clinical methods available to measure collateral flow have been too crude and showed major limitations, thus contributing to debate and confusion about the functional relevance of collateral circulation in the human myocardium. Coronary angiography allows visualization of collateral vessels having a diameter ≥100 μm, that actually prevents the majority of them from being detectable with this technique [16,17]. On the other hand, scintigraphic perfusion imaging techniques have limited spatial resolution [18]. Intracoronary wedge pressure and Doppler flow velocity measurements clearly demonstrated the presence of considerable collateral flow even in patients without angiographic evidence of collaterals [19,20], but they are invasive and not suitable for routine clinical use. With the introduction of new generation echo contrast agents and advanced ultrasound techniques, myocardial contrast echocardiography (MCE), an ultrasound imaging technique that utilizes physiologically inert gas-filled microbubbles as red blood cell tracers, has gained importance for the non-invasive assessment of blood flow at the level of myocardial perfusion [21,22]. Although evaluation of viability is the main clinical application of MCE [23], indirect assessment of collateral derived myocardial perfusion has been described in different clinical and experimental settings. In patients with severe left coronary artery disease, the placement of a graft to the posterior descending coronary artery was found to improve the collateral derived peak contrast effect within the anterior left ventricular wall [24]. In a series of subjects with healed myocardial infarction and total occlusion of the culprit vessel, a correlation was found between angiographic collateral grade and peak contrast effect after contralateral intracoronary contrast injection [25]. Collateral perfusion detected by MCE paralleled changes detected by radiolabeled microspheres during thrombosis and vasodilator administration in a canine model [26]. The usefulness of MCE has been confirmed in subjects without coronary occlusion where it was able to map the myocardial territory perfused by coronary collateral flow and to evidence immediate reduction of perfusion when collateral flow was abolished by angioplasty [27]. In patients with no prior myocardial infarction undergoing coronary angiography, intracoronary MCE effectively quantified coronary collateral flow, as demonstrated by the linear correlation existing between peak echo contrast effect and collateral flow index determined by intracoronary wedge pressure [28]. On the other hand, a strong correlation was reported between collateral receiving area at MCE and regional wall motion score index in patients with coronary occlusion, thus providing evidence that collateral derived perfusion is a good indicator of preserved regional function [29]. Likely, the grade of collateral flow on MCE was inversely correlated to the infarct size and was able to predict functional improvement following coronary revascularization [30]. Using an experimental model of chronic ischemia, it was found that not only the presence of collaterals can be identified by MCE, but also that temporal and spatial development of collateral circulation can be tracked serially [31].
Finally, intravenous MCE has been recently reported to provide qualitative and quantitative evaluation of collateral blood flow in the presence of an occluded infarct-related artery, and to emerge as the only predictor of true collateral blood flow among other markers [32].
All these reports as a whole support the concept that MCE provides important information on collateral flow and represents a promising mean for evaluating the status of coronary collateral circulation in clinical practice. Some important caveat, however, have to be taken into account. First, although the peak contrast pixel intensity has been reported as the most accurate of the variables obtained to measure collateral flow, there is a remarkable scatter in the correlation between peak pixel intensity and true collateral flow [33]. Second, it is known that regional contrast heterogeneity is common, resulting in frequent false positive perfusion defects [34]. Finally, coronary collateral vessels may cause additional dilution of contrast affecting the transit rate calculation. Further technical improvements may contribute in the near future to ensure standardization of the acoustic window and provide a quantitative evaluation of collateral flow. These issues appear to be of crucial importance to turn the echocardiographic assessment of coronary collateral flow into a ready-to-go clinical tool.
Besides the attempt to obtain direct echocardiographic assessment, coronary collateral circulation can indirectly affect the result of diagnostic stress testing with the use of echocardiographic technique. Increased vulnerability to myocardial ischemia induced by pharmacological coronary vasodilation was reported consistently with the hypothesis of a facilitated steal phenomenon in the presence of good collateral circulation [35]. On the other hand, the role of collaterals against echocardiographically-assessed stress-induced myocardial ischemia is controversial, some Authors reporting a protective [36] and others a neutral [37] effect. However, dobutamine-induced wall motion worsening in myocardial territories supplied by occluded epicardial vessels has been reported in case of evident collateral circulation [38], thus emphasizing the importance of a preserved, though reduced, blood flow to distinguish jeopardized myocardium from necrotic tissue. Differently, the ability of low-dose dobutamine stimulation to identify myocardial regions with a high probability of functional improvement after revascularization seems to be independent of both severity of underlying coronary stenosis and degree of collateralization of the involved coronary vessel [39].
The application of low-frequency ultrasound to intravascular microbubble contrast agents has been receiving attention in the last few years due to its potential therapeutic application, primarily as targeted gene delivery systems [40]. Further evidence from experimental studies has shown small capillary ruptures in exteriorized rat skeletal muscle [41], intact mouse muscle [42] and rabbit myocardium [43] to follow the application of ultrasound power. However, capillary rupturing via microbubble destruction with ultrasound is able to enhance arterioles per muscle fiber, arteriole diameters, and maximum nutrient blood flow in skeletal muscle [44]; thus, it may be tailored to stimulate an arteriogenesis response that restores hyperemia blood flow following arterial occlusion [45]. The potential of this method to become a clinical tool for stimulating blood flow to organs affected by occlusive vascular disease and, in particular, to the myocardium represents an interesting track for future research involving the application of ultrasound technology in the ischemic heart disease.
Final consideration on coronary atherosclerotic plaque
Any hypothesis on the pathogenic role of a plaque and its activity and vulnerability should consider all interrelated variables for a correct interpretation of findings. When only one or few variables are investigated erroneous conclusions can be reached. An atherosclerotic plaque is always an active structure since its progression depends on a sequence of events due to a variety of correlated phenomena; while vulnerability is just an hypothesis which believe that some findings indicate a risk of plaque rupture.
The known variables are: degree of lumen reduction, shape, length, satellite collaterals, tunica media changes, inflammatory reaction per se and associated with media nerves (medial neuritis), survival (Table 3) macrophagic repair process, inflammation, vascularization hemorrhage, proteoglicans, atheroma, calcification, smooth muscle cell and elastic fiber hyperplasia, rupture, thrombosis, various factors released from all involved cells, hemodynamic pressure stresses, regional myocardial asynergy, spasm plus still unknown variables to be included.
Table 3 Occlusive coronary thrombus versus significantly main correlated variables. Percentage distribution
Source Acute myocardial infarct Sudden unexpected death
Cases Total 200 208
Cases+occlusive thrombus% 41 15
Lumen reduction%
≤ 69 7 -
70–79 33 16
80–89 35 47
> 90 24 38
Length stenosis mm
≤ 5 6 6
5–20 38 19
> 20 56 75
Concentric 100 94
Atheromatous 84 75
Medial neuritis 92 92
Infarct size %
≤ 10 20 -
11–20 32 -
21–30 48 -
31–40 44 -
41–50 78 -
> 50 86 -
Survival days
≤ 2 29
3–10 51
11–30 45
Survival minutes
< 10 - 12
10–60 - 23
61–180 - 30
Most studies analized few variables mainly observed in animals after hypercholesterol diet or in familial hypercholesterolemia. A pattern [46,47] totally different from that seen in general population and CHD. Furthermore, myocardial infarction is not synonymous of sudden/unexpected death, thrombus is a totally divergent structure from coagulum, collaterals can not be ignored and meaning of the coronary atherosclerotic plaque can be interpreted in another way.
The presence of functioning collaterals induces a particular hemodynamic condition within the residual lumen at the plaque level with proximal flow reduction counterbalanced by distal collateral flow. Any time there is a regional asynergy (Figure 8) with increasing intramural resistance, stasis in related artery will result in blockage of flow within the lumen with the most favourable situation for intimal hemorrhage, rupture, and thrombosis as secondary phenomena and not primary cause of an infarct. It is hard to believe that occlusion of a pinpoint lumen already compensated by collaterals is the cause of an infarct and rupture of a cap causes infarct or sudden death; being clear that any acute coronary syndrome is an etiopathogenetic entity which can not be caged in any unifying theory [48]. In the next review on different types of myocardial damage, this argument will be further reconsidered.
Figure 8 The coronary thrombus is a multivariant phenomenon (A), including medial neuritis. Its location in severe (≥70) stenosis associated with other factors (retrograde collateral flow, reduced fibrinolytic activity, etc, see text) justifies the concept that is a secondary phenomenon. Any time there is an increased peripheral resistance (B) (spasm, intramural extravascular compression following infarction, etc), stasis in related main vessel and in collaterals both outside and within the plaque is expected with hemorrhage, plaque rupture and trombosis (C). On the other hand, it is difficult to accept that acute occlusion of a pin-point lumen bypassed by preexisting functioning collaterals (D) may result in infarct necrosis or sudden death. Even experimentally occlusion of a severe "chronic" (7 days) stenosis does not produce any ischemic dysfunction.
Authors' contributions
Prof. Giorgio Baroldi contributed to the conception and organization of this review and to the final comments. Dr. Riccardo Bigi and Dr. Lauro Cortigiani summarized the use of ultrasound techniques in atherosclerotic plaque imaging.
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| 15740620 | PMC554094 | CC BY | 2021-01-04 16:38:31 | no | Cardiovasc Ultrasound. 2005 Mar 1; 3:6 | utf-8 | Cardiovasc Ultrasound | 2,005 | 10.1186/1476-7120-3-6 | oa_comm |
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Health Res Policy SystHealth Research Policy and Systems1478-4505BioMed Central London 1478-4505-3-31572369410.1186/1478-4505-3-3ReviewThe role of NGOs in global health research for development Delisle Hélène [email protected] Janet Hatcher [email protected] Michelle [email protected] Lori [email protected] Theresa W [email protected] Département de nutrition, Faculté de médecine, Université de Montréal, C.P. 6128 succ. Centre-ville, Montréal, Qc, H3C 3J7, Canada2 Canadian Society for International Health (CSIH), One Nicholas Street, Suite 1105, Ottawa, On K1N 7B7, Canada3 CARE Canada Suite 200 9 Gurdwara Road Ottawa, ON, K2E7X6 Canada4 Canadian Society for International Health (CSIH), One Nicholas Street, Suite 1105, Ottawa, On K1N 7B7, Canada5 Dept. of Epidemiology & Biostatistics, Montreal General Hospital, 1650 Cedar Avenue, Room L-10-420, Montreal, QC H3G 1A4, Canada2005 21 2 2005 3 3 3 22 7 2004 21 2 2005 Copyright © 2005 Delisle et al; licensee BioMed Central Ltd.2005Delisle et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Global health research is essential for development. A major issue is the inequitable distribution of research efforts and funds directed towards populations suffering the world's greatest health problems. This imbalance is fostering major attempts at redirecting research to the health problems of low and middle income countries. Following the creation of the Coalition for Global Health Research – Canada (CGHRC) in 2001, the Canadian Society for International Health (CSIH) decided to review the role of non-governmental organizations (NGOs) in global health research. This paper highlights some of the prevalent thinking and is intended to encourage new thinking on how NGOs can further this role.
Approach
This paper was prepared by members of the Research Committee of the CSIH, with input from other members of the Society. Persons working in various international NGOs participated in individual interviews or group discussions on their involvement in different types of research activities. Case studies illustrate the roles of NGOs in global health research, their perceived strengths and weaknesses, and the constraints and opportunities to build capacity and develop partnerships for research.
Highlights
NGOs are contributing at all stages of the research cycle, fostering the relevance and effectiveness of the research, priority setting, and knowledge translation to action. They have a key role in stewardship (promoting and advocating for relevant global health research), resource mobilization for research, the generation, utilization and management of knowledge, and capacity development. Yet, typically, the involvement of NGOs in research is downstream from knowledge production and it usually takes the form of a partnership with universities or dedicated research agencies.
Conclusion
There is a need to more effectively include NGOs in all aspects of health research in order to maximize the potential benefits of research. NGOs, moreover, can and should play an instrumental role in coalitions for global health research, such as the CGHRC. With a renewed sense of purpose and a common goal, NGOs and their partners intend to make strong and lasting inroads into reducing the disease burden of the world's most affected populations through effective research action.
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"Each country needs to be able to generate knowledge relevant to its own situation, to allow it to determine its particular health problems, appraise the measures available for dealing with them, and choose the actions likely to produce the greatest improvement in health. This should not be seen as the exclusive preserve of universities or research councils, but equally of health/public services, non-governmental organizations, etc." [1].
1 Introduction
Non-governmental organizations (NGOs) have been defined by the World Bank as 'private organizations that pursue activities to relieve suffering, promote the interests of the poor, protect the environment, provide basic social services, or undertake community development'. NGO activities can be local, national or international. NGOs have contributed to the development of communities around the world and are important partners of many governments – while remaining independent from governments. According to the Human Development Report [2], there were in 2002 over 37,000 NGOs in the world, a growth of 19.3% from 1990. Their purposes differ but overall two categories dominate: economic development and infrastructure (26%) and research (23%) . NGOs are generally regarded as valued partners in health research for development, research being viewed as a broad process involving not only the production of knowledge, but also up-stream and down-stream activities needed for its relevance and effectiveness, such as priority setting and knowledge translation. NGOs have made and continue to make substantive contributions through supporting relevant and effective research. In her address at the First Steering Committee Meeting of the International Conference on Health Research for Development in 1999, the (then) Director General of the World Health Organization (WHO), Dr. Gro Harlem Brundtland, voiced her appreciation of NGOs as a partner with WHO in health research [3].
There are several views on what is meant by global health and global health research. In its simplest form, global health is population health on a global scale, and global health research is research which addresses the health of human populations around the globe. Global health also refers to 'inherently global health issues', that is, health-determining phenomena that transcend national borders and political jurisdictions, such as globalization and climate change. In setting global health research priorities, both the burden of disease and inherently global issues should be considered [4,5]. The vision of health research as proposed by the Commission on Health Research for Development [6] is a systems approach driven by equity, focused on country needs and priorities, and within an interactive regional and global framework. This paper will address global health as it was defined in a Canadian consultation paper on global health research held in 2001 , that is, the health of individuals and societies in less developed, less resourced, poorer nations and regions of the world.
A major global health research issue is the inequitable distribution of research efforts and funds directed towards populations suffering the world's greatest health problems. This situation has been referred to as the 10/90 gap because only a meager 10% of all health research funding is being used to address 90% of the world's burden of disease, suffered primarily in developing countries [7]. Because of this imbalance, there have been major attempts at redirecting research efforts and funds to the health problems of low and middle income countries.
One of the roles of health research is to ensure that the measures proposed to break out of the vicious cycle of ill health and poverty are based, as far as possible, on evidence, so that the resources available to finance these measures are used in the most efficient and effective way possible [8]. There are many different types of health research. At the 6th Global Forum on Health Research, held in Arusha, Tanzania in November 2002, Dr. Gerald Keusch, Director of the Fogarty International Center, listed the scope of health research as including: fundamental discovery research, pathogenesis research, epidemiology research, clinical research, product development research, translational and adaptational research, operational research, health services research, policy research and research on health systems [9]. NGOs involved in health research have primarily undertaken operational and action research, but many have also participated in other types of research such as epidemiological research, social science research, product development research, translational research, health services research, and policy research.
The purpose of this paper is to document the role that NGOs have played in global health research and to highlight the need to expand this role. This paper is also intended as a tool to stimulate research activity in NGOs and to advocate for increased NGO involvement in global health research. Following a brief review on the central role of global health research in development, the roles of NGOs at different stages within the research process are discussed and illustrated with a few examples. Key challenges are also identified. The last part of the paper identifies future needs for strengthening the role of NGOs in global health research.
2 Global Health Research and Development
While research means different things to different people, it may best be defined as 'a knowledge loop' from generation of knowledge to its effective use [10]. Indeed, there has been a progressive paradigm shift from narrow 'research' to broader 'knowledge creation and management' [11]. This broad definition is consistent with that of the Organization for Economic Co-operation and Development (OECD) [12] which states that "research and experimental development comprise creative work undertaken on a systematic basis in order to increase the stock of knowledge, including knowledge of man, culture and society, and the use of this stock of knowledge to devise new application". Research is recognized as a fundamental ingredient for action [13,14], and it is essential for development because it informs policies and programs; it also guides the development of human resources in these and related domains (see Figure 1). However, the links among research, policy-making, programming and training, with advocacy constantly in the background, need to be strengthened. It is being increasingly recognized that investments in health research can be economic and social investments [15]. In a WHO discussion paper on knowledge for better health, the emphasis is on research as an investment rather than a cost, on the need to turn research into action, and on the vital part of the civil society ( World report on knowledge for better health 2004).
Figure 1 The relationship between research and development
2.1 Global health research priorities
The call to shift health research priorities from problems of industrialized countries to those affecting populations in developing countries is not new. In 1990, concerns regarding the inequitable distribution of research efforts were first raised in the Report to the Commission on Health Research for Development [6]. Since then, progress has been made to try to correct this gap, and to build capacity in the countries of greatest need. The 2002 WHO World Health Report [16] focuses on risks that contribute to the global burden of disease and death, both in developing and developed countries. Dollar expenditures on health research today, however, remain markedly inequitable in terms of populations served and disease burden addressed. Pneumonia, diarrheal diseases, tuberculosis and malaria, when combined, have been estimated to account for more than 20% of the disease burden in the world (mostly in developing countries), yet they receive less than 1% of the total public and private funds which are devoted to health research. The 10/90 gap is as wide as ever [7].
2.2 Milestones in global health research and development
Several important initiatives have been undertaken to address the global health research agenda. They have been fostered by individuals and groups from local, national and international bodies who shared a common vision in advocating for health research directed towards the low and middle income countries.
2.2.1 Commission on Health Research for Development
The Commission on Health Research for Development declared in 1990 that "For the most vulnerable people, the benefits of research offer a potential for change that has gone largely untapped" [6]. The Commission highlighted several obstacles in undertaking this research, and among others: 1) the insufficient (worldwide) funding of health research directed towards health problems of people in developing countries; 2) the inefficient application of resources; 3) the neglect of major health problems; 4) the lack of individual and institutional health research capacity; 5) the lack of technology transfer; and 6) fragmentation and competition among research initiatives. The challenge to remedy this situation was set down and ultimately led to the establishment of the Council for Health Research in Development (COHRED) in 1993. COHRED works in partnership with WHO, the World Bank and other organizations to strengthen the role of health research at the country level.
Over the years, COHRED has assisted increasing numbers of countries in the exploration and implementation of essential national health research (ENHR) strategies. Networks were created to facilitate national level activities in Africa, Asia, and the Commonwealth Caribbean. For example, AFRO-NETS, the 'African Networks for Health Research and Development', was established in 1997 to facilitate exchange of information among different networks active in this type of research in English-speaking Africa, and to facilitate collaboration in the fields of capacity building, planning and research. Regional and global working groups and projects were established which allowed experiences with ENHR to be shared. Several communication strategies were utilized, including quarterly newsletters, websites and other publications to share experiences and lessons learned. A framework for capacity development, a critical component of ENHR, was established through partnerships and like-minded networks and organizations. The book, 'Forging Links for Health: Perspectives from the Council on Health Research for Development", [14] and the discussion paper 'Health Research for Development: The Continuing Challenge [1] review what has happened in the intervening years since the Commission on Health Research for Development made its first major recommendations in 1990.
Several questions remain unanswered:
• To what extent have the recommendations been implemented?
• Have the recommendations made a real difference in the lives of the countries that carry 90% of the disease burden?
• Has 'Essential National Health Research' worked?
• What is the current situation with regard to health research for development?
• Where and how do we proceed from here?
The 2000 International Conference on Health Research for Development provided COHRED and several partner organizations with an opportunity to review and reflect on their experience with health research, its impact on health and equity and to devise a global strategy for the first years of the coming millennium [14].
2.2.2 Global Forum for Health Research
The Global Forum for Health Research, created in 1998 as a response to the Report of the WHO ad hoc Committee on Health Research Relating to Future Intervention Options [17], has provided a forum for stakeholders to review global health research priorities, promote ongoing analysis of the international health research situation and facilitate coalition building to support its central objective to help correct the 10/90 gap. The Global Forum is managed by a council of 20 members representing government policymakers, multilateral and bilateral agencies, foundations, international NGOs, women's associations, research institutions, and the private sector. It holds funding competitions on targeted global health topics and awards research grants to applicants from low and middle income countries. Its most recent report [18] emphasized the need for action by combined efforts of the public and private sectors. It also recognized the role of NGOs as a partner in contributing to these efforts.
2.2.3 Canadian Coalition for Global Health Research
In November 2001, four Canadian federal agencies, Canadian International Development Agency (CIDA), International Development Research Centre (IDRC), Health Canada, and Canadian Institutes of Health Research (CIHR) signed a Memorandum of Understanding to support national consultation regarding Canada's role in global health research. This marked the first time in Canadian history that Canada's two overseas development agencies, Health Canada and Canada's major federal health research funding agency have collaborated to address global health research.
The Canadian Coalition for Global Health Research (CCGHR) is developing into a network of health researchers, funding agencies, NGOs, and other stakeholders committed to support the pursuit of effective global health research by ensuring that all these groups work together as effectively as possible with researchers in developing countries. This collaborative approach serves as a framework for future research projects in the area of global health, with each organization bringing its own specific area of expertise to the table. It aims to improve the effectiveness of development assistance and to increase the sustainable health gains per dollar of Canadian funds invested in research.
3 Key Roles of NGOs in Global Health Research
Inequities in health are caused by a number of determinants, including the use of or access to health care facilities. Research which addresses these issues requires an intersectoral approach, involving trans-disciplinary teams and methodologies. Building trans-disciplinary teams requires commitment from the research community to seek out colleagues from other disciplines, from the funding agencies to appreciate innovative initiatives, from the community at large as partners and contributors, and from the policy arena to develop strategies for intersectoral policies and programs which may well have the lead outside ministries of health. Indeed, working outside government altogether may well be a solid and sustainable strategy. Understanding and engaging the broader community on these issues comes naturally to communities unrestricted by bureaucratic boundaries. This is where NGOs excel.
NGOs have contributed to all different stages of the research cycle (see Figure 2), namely in advocacy, priority setting, capacity building, resource mobilization, sharing and utilization of research findings, and networking. Traditionally, many NGOs which have undertaken activities that address health issues in resource-poor settings are service-oriented NGOs and concentrate their efforts on implementing "action" programs. This type of NGO finds it difficult to identify resources that would allow them to conduct research. While there are NGOs involved in actually conducting research, for most the focus is usually evaluation. Links with the research community are often weak. Other NGOs undertake innovative field-based experimental research. The effectiveness of these initiatives is often learned by trial and error. Unfortunately, while this enhances effective and efficient implementation in the field, research results are only infrequently analyzed appropriately. There are also barriers to dissemination or sharing of research results to a wider audience (eg. other districts within the same country) and to different audiences (eg. to other researchers, research institutions, etc.). Typically, NGO involvement in research is more downstream of knowledge production and it usually takes the form of a partnership with more traditionally-oriented research organizations such as universities or dedicated research agencies. There is a need to include NGOs in the reconceptualization of global health research to ensure completion of the cycle from generation of knowledge to its effective use.
Figure 2 Research Process
We describe the key roles of NGOs below, using, as a framework, the categories of primary functions of health research systems as recently identified by Butler [1].
3.1 Stewardship
One of the strengths of NGOs has been as advocates for the populations they serve. Health research can make NGOs become more effective advocates. Governments depend on health research for needs assessments, formulation of policy options, implementation of interventions and evaluation of action plans. Empowered citizens and NGOs can demand accountability of the government. They can also encourage international donors to focus on the health priorities of countries and thus facilitate a check and balance mechanism for good governance. Good governance is needed to improve collaboration and cooperation at the international, national and regional levels in order to tackle inequity. High scientific standards are fundamental components of effective health governance, particularly as they relate to health research systems.
The role of research in mobilizing and supporting NGOs, particularly around issues of inequities, is important. NGOs can provide stewardship in terms of the promotion and advocacy for relevant research, shaping research priorities, and the setting and interpretation of ethical frameworks for research. NGOs can often play a more powerful role using the results of research than can the research community itself. Mobilizing communities, utilizing mechanisms for advocacy and acting as an interface between the research community and its wider community will enhance a sense of strong governance and stewardship.
3.1.1 Promotion and advocacy for relevant global health research
There is widespread agreement that health research is not sufficiently valued by many societies as a critical input to human and socioeconomic development. The result is often an environment that is neither conducive to, nor supportive of, research. A culture is necessary that recognizes the value of research and one which builds a supportive environment for research [19].
There is a need not just to allocate funds for research, but also to allocate these funds to areas of research that would have the greatest or maximum social benefit. Advocacy for relevant research, that is, the type of research that will make a difference in terms of equity, health, well-being and development of people, is an important role for NGOs [20]. Not only can NGOs identify researchable topics, but they can also stimulate demand for relevant research.
However, the existing power structure in the research arena often works against NGOs because of a narrow view of research as merely producing new knowledge, with limited consideration of upstream operations (identification of research needs, questions, and priorities), downstream actions (knowledge management, dissemination and translation), and the advocacy efforts required to connect research with policies, programs and training.
Historically, the influence of the biomedical researchers' lobby has been the strongest with regard to agenda-setting and fundraising. Behavioral scientists and social health researchers generally have much weaker potential to influence resource allocation, agenda-setting and policy formulation. Partnerships could be strengthened and supported between NGOs and social science researchers in resource-poor countries to improve influence potential, as the social sector issues that tend to be most relevant to human populations are also of utmost importance to NGOs.
Creating a favorable environment for "relevant" research requires a health system that is supportive and provides financing opportunities. It also requires the existence of a culture of "evidence-generating and evidence-based research". There must be a healthy relationship between communities, researchers and policy makers. Networks to share experiences, lessons learned and policy impact can be enhanced by partnerships with NGOs.
A disproportionately large number of people living in developing countries suffer large disease burdens. Promoting research and development on neglected diseases or issues of global health significance may contribute to bridging the 90/10 research gap, by stimulating research by public or civil society organizations on issues that do not represent marketable research, and are therefore neglected by the private sector. There is a role for NGOs in advocating for more research on these neglected topics (see under 4.1, example of initiative for neglected disease drugs, Médecins sans Frontières [MSF]).
Health research needs to generate knowledge that will facilitate the identification of choices and options to reinforce equity-based policies and programs. In doing so, it also needs to address the difficulties of collecting data that are of primary importance when inequities are discussed. The essential function that data serve will allow tracking and monitoring of resources for research and for improving opportunities for those researchers in more disadvantaged countries. NGOs often have access to information that will highlight inequities and the determinants of inequities.
Similarly, NGOs can advocate for formative and evaluative research on programs that address major health problems, but which are generally a low priority for funding agencies. In doing so, they can contribute to making data available for evidence-based decision-making in policy and program planning. Food system-based approaches to reducing micronutrient deficiencies and malnutrition in general are one of these under-researched areas.
3.1.2 Shaping research priorities
NGOs are well-placed to foster public participation in decisions about health research, as they are close to communities. They can provide the mechanisms by which such public participation is ensured in decision-making processes. Significant progress has been made over the last decade in health research priority-setting for the implementation of ENHR at the country level. Among the lessons learned, it appears that community involvement is in most cases an unresolved issue [21]. What is certain is that, critically at the priority-setting stage of the research cycle, the community must be involved, and NGOs may be instrumental in achieving this.
Defining the research that needs to be done requires the input of civil society and NGOs as much at the beginning as at the end, in terms of dissemination, communication and action.
3.1.3 Setting and interpreting ethical frameworks
NGOs assume a range of roles in research, but a thread that runs through all these is their representation and advocacy for the vulnerable. Broad research roles are described in greater detail in other sections of this paper. This section focuses on the role of NGOs in shaping and interpreting ethical frameworks [22-25], that is, the incorporation of ethical principles in their research partnerships with other organizations. As researchers or research partners, NGOs have a responsibility to ensure that ethical issues are addressed in both the design and conduct of the research. There are distinctive challenges in conducting health research in developing countries, namely to fulfill moral duties of justice and respect in the face of poverty, lack of resources and the potential for exploitation. The Nuffield Council on Bioethics [26] designed an ethical framework for health research in developing countries based on the duty to alleviate suffering, to show respect for persons, to be sensitive to cultural differences, and to not exploit the vulnerable. As NGO research is often conducted among the most vulnerable populations, where power relations are tipped in favor of researchers and those who are literate and eloquent, issues of informed consent and participants' understanding of it and the research, as well as participants having access to the benefits of research, are of special concern. Particularly when research is conducted by first world researchers in resource-limited settings, NGOs who partner in this research at times need to recommend and advocate for reviews from local research and ethics committees, as well as those from industrialized countries. Where relevant, they may also encourage the development of independent national ethics committees and national ethical guidelines, taking account of existing international guidelines [22-25]. This process may involve interpreting cultural ethical frameworks and beliefs, for instance, culturally appropriate means of obtaining informed consent from research participants. In addition, NGOs can make sure that the development of local expertise in health research is an integral component of research proposals.
As watchdogs, NGOs actively seek breaches of ethics and hold researchers to account when the principles of respect for persons, beneficence and justice are not upheld, a role they are well positioned to assume given their understanding of and links to marginalized groups. Watchdogs, as they uncover ethical breaches that may be defined by culture or power relations, have assisted in shaping ethical frameworks to better address ethics when research is conducted among vulnerable groups.
In the communities where NGOs work, they can act as community partner members of and witnesses to research. In this role they can assist with, for example, interpreting research objectives to participants to ensure that consent is informed and the rights of subjects are respected. They may provide researchers with enumerators or local information to expedite the data collection process. NGOs can also monitor the long-term outcomes arising from research, and make sure that the participants benefit from successful intervention.
As knowledge translators, NGOs interpret the knowledge generated by research to their constituents, a key role in working towards the vulnerable having access to the benefits of research that could improve their lives. This may be research conducted in these communities or globally.
3.2 Mobilizing resources for research
While current levels of financial resources are not sufficient to adequately respond to the demonstrated need for health research, there are many sources of "funds" for health research. Some are monetary contributions and some are in-kind contributions. NGOs can provide not only direct funding for projects (albeit in a limited manner) but, and perhaps equally important, they can provide valuable in-kind funding. Thus, personnel or materials developed by NGOs can be used in health research projects at little or no cost.
Some NGOs are directly involved in the administration of research grants. Others may be the fiduciary agent for a grant to a research organization that is exploring an issue related to an NGO program. However, most are organizations that work with communities. A major role is therefore to identify resource gaps using networks to link communities, health providers and managers, and funding agencies in a meaningful way so that financing can appropriately be directed to targeted health issues. NGOs may also contribute by identifying other potential sources of funding, for instance, in the local private sector.
3.3 Knowledge generation
Knowledge can be acquired in various ways, by many methods, and by different types of people; there are different cultures of enquiry. Because of their typical 'grass-roots' experience, several NGOs are able to access indigenous knowledge and specific information, which may be less attainable for other types of organizations. This type of knowledge might be very useful when pooled with knowledge acquired by others; in this way, a more comprehensive analysis can occur. NGOs can be particularly adept in conducting formative research (baseline studies, needs assessment), in operational or action research and in process and impact evaluation. This type of research is particularly relevant for setting priorities, for informing intervention, as well as for identifying further research needs.
Although knowledge generation is generally not a primary NGO activity, there may be specific 'knowledge generation' research niches for NGOs. For instance, as suggested by the Canadian Council for International Cooperation (CCIC) and actually carried out by a few NGOs, "There is a need for NGOs to be more involved in policy research even in Canada" (Interview with B. Tomlinson, CCIC).
Figure 3 illustrates the research cycle in the narrow sense of knowledge generation. This cycle applies whatever the research type, and whether the research is conducted by an NGO or an academic institution.
Figure 3 The research (knowledge generation) cycle (adapted from McKenzie [36])
3.4 Utilization and management of knowledge
While asserting that the production of knowledge is the primary function of research, and that levels of knowledge have increased considerably, a discussion paper for the International Conference on Health Research [1] also recognizes that the ability to draw from research in terms of lessons learned, application to interventions, and programming and policies which support the overarching goal of equity, is often lacking.
Inadequacies include the inability of developing countries to access pertinent international research literature and knowledge bases (either as contributors or users), the inability to access new information technologies, and the inability to ensure closer links among the research community, health service managers and health policy makers.
The effective use of research findings and their dissemination is an increasingly important public health policy concern. In 1995, an international research conference was held in Vancouver, Canada, on dissemination research. This type of research is similar to what is now called 'translational research' , that is, the conversion of research findings from basic, clinical or epidemiological environmental health science research into information, resources, or tools that can be applied by health care providers and community residents to improve public health outcomes in at-risk neighborhoods.
NGOs are frequently at the interface of applied research and policy-making, at least at the administrative level, and their potential input into research utilization for policy-making needs to be valued. Research can make a substantive contribution in at least three phases of the policy-making process: agenda-setting, policy formulation, and implementation [27]. It is widely recognized that health research is underutilized in policy-making. The generation of new knowledge is highly valued, but its translation and use does not appear to be valued as much [28], which may partly explain why application of newer knowledge is often a weak link in the research cycle. Factors potentially enhancing utilization can be identified by exploration of priority-setting, activities of the health system at the interface between research and policy-making, and the role of recipients, or "receptors", of health research [27]. There are several models of research utilization in policy-making, but interactive or exchange models may be more conducive to the effective use of research than unilateral models because they bring researchers and decision-makers closer together [10,27].
NGOs often play a critical role in interpreting the evidence and translating its relevance for local communities. Inevitably the level of involvement by the community depends on relevance and opportunity for action and advocacy. Assessing and evaluating opportunities for advocacy and action occur as NGOs work with communities on these issues. Effective involvement of the community and its participation is a "matter of reciprocity and continuing dialogue in which participation takes different forms and influences change in several directions" [14]. Once the evidence has been analyzed and assimilated, NGOs can serve as intermediaries in delivering feedback to communities and in the planning, implementing and monitoring of new interventions, policies or other actions which might have been proposed. The knowledge and information acquired by NGOs can be unique and offer added insight into new ideas for future health research. This is, in part, because of the extensive interrelationships NGOs have forged with different communities, organizations, the private sector and governments, among others, often over decades of dedicated work. Additionally, NGOs are in a good position to test the ability of research findings to be scaled up in a 'real world' environment.
According to Lavis et al [10], while the "knowledge loop" needs to be completed, that is, from knowledge production to knowledge-based decision-making through knowledge transfer or brokering, not all research organizations should become involved in knowledge transfer; if they do, the knowledge pyramid may be shaky. Innovations stemming from research are at the base of the pyramid, and actionable messages are at the top. Individual studies and synthesis of research knowledge are the intermediate layers. Lavis et al contend that it may not be relevant to transfer knowledge from individual studies, but rather, from bodies of cumulative research knowledge, and that knowledge transfer brokers are needed for this purpose. This model of specialized roles is probably more relevant at the macro level and in industrialized countries. In resource-poor countries, polyvalent organizations such as NGOs have a key role in sharing, translating and implementing research findings at the community and country level. They provide channels for the use of research results at the community level, as they are closest to the communities themselves. For that very reason, they may also feel more compelled to complete the research cycle, including application of the findings. Third World Network , for instance, an independent non-profit international network of organizations and individuals involved in issues relating to development, conducts and disseminates research to help organizations around the world participate in and influence international economic and social policy. NGOs may also be involved in testing pilot models of intervention and in their subsequent scaling-up.
3.5 Capacity development
The preliminary examination of the functions performed by the some 125 organizations involved in a significant way in health research reveals that while knowledge generation is a concern shared by most, research capacity strengthening receives relatively little attention [1]. One weakness or inattention in research capacity strengthening activities, for example, has been the lack of a recognized career path for local health researchers which has resulted in diverting promising researchers to other careers or to other countries.
The development and retention of research capacity remains a challenge in many countries [29]. Quality control and assurance requires skills and structures which support these objectives. Skills such as leadership, advocacy, networking and communication are important and need to be built through capacity development. Research management is also a skill which needs to be strengthened and a skill that will improve the quality, appropriateness and timeliness of research and its dissemination.
NGOs in the North and in resource-poor countries often have the capacity for facilitating training and for sharing the lessons learned in needed skills. Partnership with NGOs in such capacity-building needs to be valued and reinforced. The Canadian Society for International Health (CSIH) and the Canadian Public Health Association (CPHA) have participated in capacity-building activities in many countries and continue to share their experiences and lessons learned. Support for such sharing and building capacity makes sense and should be facilitated by donor agencies. WHO, through its creation of a Department of Research Policy and Cooperation within the cluster of Evidence and Information for Policy, has defined as one of its objectives: "the development of initiatives aimed at strengthening research capacity in the developing world with the ultimate aim of enshrining research as a foundation for policy".
A number of other international initiatives have also attempted to address some of these capacity issues: the International Health Policy Program (IHPP), the Applied Research on Child Health (ARCH) project, the Swiss Commission for Research Partnership with Developing Countries (KAPE) and, in Canada, the IDRC. Since 1970, IDRC has been providing financial and technical assistance to academic institutions, government agencies and NGOs in developing countries, as a means of promoting sustainable and practical development and strengthening indigenous research capacity. IDRC's experience provides important and valuable lessons about implementing applied research in partnership with NGOs [30], as summarized in the table 1.
Table 1 Lessons learned from research in partnership with NGOs: IDRC experience
First, applied research should have a practical application, reinforce knowledge and skills, and introduce and promote innovative, effective strategies and approaches for improving human health and well-being. Not only should research results be for local application, they should also be shared and adapted to other venues and contexts.
Second, efforts need to be made to build knowledge and understanding about the benefits accruing from applied research. NGOs, by their very nature, are action-oriented. Applied research is often perceived as of limited use to their ends, an esoteric, academic exercise of limited value to the immediate needs of the poor and disadvantaged. Time and effort need to be invested in nurturing an understanding within the academic community of the value of applied research within the context of development efforts.
Third, applied research should be used to develop and strengthen local research capabilities. NGOs do not, as a rule, possess the internal capacity and skills to design and conduct applied research studies. Attention should be paid to assisting NGOs in making contact with qualified researchers, and increasing NGO knowledge and skills to negotiate the terms of reference for applied research studies. This cannot be achieved simply through providing information about applied research methodologies or organizing a single workshop. Trust has to be developed between the NGO and academic communities, as a means of reinforcing linkages between them and building upon and using their comparative strengths, characteristics and areas of expertise to design and conduct applied research.
Fourth, local communities should be involved in the design and implementation of applied research activities. The local people need to understand the purpose of the proposed research, provide input and advice about its design and conduct, and be actively involved in the application and dissemination of research results. Without the active participation of the community, the utility and eventual application of the research results will be of little value.
Source: [30]
The CPHA, through the CIDA-funded initiative Canada's International Immunization Program – Phase 2 (CIIP2), dedicated 5% of the program's budget to applied research. Part of this funding was used to strengthen primary health care in developing countries through the NGOs that implemented the immunization and primary health care activities through the auspices of CIIP2.
NGOs who wish to become more involved in research generally recognize the need for extramural training and support. Partnering with universities and research institutions may provide such training opportunities. Additionally, there are international institutions such as INTRAC (International NGO Training and Research Centre) that are specifically geared towards meeting the challenges and needs of NGOs in research. Those NGOs that are part of international networks can draw from the body of research conducted elsewhere.
NGOs may also provide substantive input into research training, be it by grounding research methods in reality so that research is more applicable, or by providing research sites and questions for academia and graduate students. NGOs may also be in a good position to identify young scientists and promising investigators in host countries.
Stimulating the demand for research by user groups, rather than supply-driven research, is one of the three strategies identified by Harrison & Neufeld [31] for capacity-building for essential national health research. NGOs and communities as user groups could be the target of capacity-building efforts.
4 NGO involvement in health research
There is a lack of accessible and centralized information on NGO involvement in health research, although the CPHA CIIP2 applied research publication lists over 20 examples of NGO-related applied research carried out in the 1990s. The examples given below are based on discussions with a limited number of Canadian and international NGOs: CARE, World Vision Canada (WV), CECI (Centre d'étude et de coopération internationale), Inter Pares, HKI (Helen Keller International), and CCIC. In the case of AMREF (Africa Medical Research Foundation), ADI (Alzheimer's Disease International), Médecins sans Frontières (MSF) and RITC (Research for International Tobacco Control), most of the information was obtained from their websites and related publications and documents. The interviews and discussions covered the specifics of the implication of the NGO in health research, lessons learned through the experience, and respondents' perceptions on the role of NGOs in global health research, and on the strengths and weaknesses of their organization in this regard. These selected NGOs provide insight into some of the critical issues facing NGO involvement in global health research. It should be kept in mind that this selection is small and not meant to be representative. Nonetheless, all of these NGOs are involved, directly or indirectly, in global health research, and they are all Canadian or present in Canada.
4.1 NGOs and their involvement in global health research: illustration cases
The interviews covered a broad range of cases, from NGOs little involved in research to those actually conducting independent research. The types of involvement are briefly described below. A salient observation is that what is considered as research by different NGOs is, for the most part, unclear and highly variable. This suggests the need for NGOs to develop common views on what is research, the various types of research, and the components of the research process. The interviews also revealed that while some NGOs are reluctant to be involved in research, others are eager to strengthen their capacity to do so.
CECI has long been involved in health research, although it is reluctant to call this 'research'. A major activity is the undertaking of baseline studies that typically include an assessment of the health and nutritional status of populations. The data are used to orient or reorient programs, and to inform communities. In Cambodia, for instance, it conducted an initial assessment for a project aimed at improving the livelihood of rural poor in two sectors: health/nutrition, and agriculture marketing (CECI and Cambodia Researchers for Development: Improving Livelihood of the Cambodian Rural Poor: Strategies in Health, Nutrition and Agricultural Commodity Marketing, 2001). One interesting aspect of its recent work is the 'policy feedback' that it conducts in its large projects. The intent of the analysis is to clearly identify the lessons learned, and to discuss these with decision-makers and technical officers. This may be considered as part of 'knowledge translation' and it can be a particularly useful approach in advancing policies and programs. While CECI is also involved in health projects that do not include research even in a broad sense, it conducts research in areas that are indirectly related to health. For instance, in the IDRC-funded project intended to alleviate poverty in Burkina Faso, Viet Nam and Nepal, it collaborates with local universities and research institutions for the research and training components, notably on adapting the assessment of poverty to the specific context.
CCIC and its member NGOs are involved in international policy research. For instance, Trade-Related Aspects of Intellectual Property Rights (TRIPS) agreements have implications on access to drugs. In the reorientation of CIDA for improved aid effectiveness, there are obvious health implications, including how to respond to health plans as defined by health ministries, and assist with poverty reduction strategies. CCIC sees research on policies as a critical role of NGOs, and considers that NGOs should be more involved in the policy debate both in Canada and globally.
World Vision (WV) Canada is active in research, particularly (but not only) in the framework of its MICAH projects (Micronutrients and Health in Africa) funded by CIDA. It primarily conducts formative and evaluation research (see table 2 for report of findings in Sénégal, published jointly with CIDA). Although it has PhD or MSc level personnel in each of its technical units, it does not have in-house research expertise per se; it partners with research institutions, in the field and in Canada. It does not have the capacity to analyze all the data that it collects and therefore it collaborates with academic institutions in Canada. Graduate students can use the data for their theses. WV officers may also sit on graduate students' supervisory or examining committees. The primary use of the research findings is to reorient programs and inform the community. As programs may have to change their operations as a result of such research, the exercise may, at times, be regarded as threatening.
Table 2 Final evaluation report, World Vision Canada, Micronutrient-for-Health Project in Sénégal (2002)
The objectives of the project initiated in 4 districts in 1997 were to reduce micronutrient malnutrition among women and children, to reduce the incidence of illnesses affecting micronutrient status, and to strengthen local capacity for controlling micronutrient malnutrition. The baseline study revealed a high rate of (iron deficiency) anemia in pregnancy (49%), of low retinol (vitamin A) levels in breastmilk (57%), and of low serum retinol concentrations among preschool-age children. Iodine deficiency was widespread, with 20% of school-age children showing severely low urinary iodine levels. A similar survey was conducted after 4 years of project activities, and included control zones in each district. The final evaluation showed an almost complete elimination of vitamin A deficiency in the project areas, which was primarily attributable to the high coverage of vitamin A supplementation of under-fives and postpartum women. Household use of iodized salt increased from 6% to 14%. Anemia remained high among pregnant women (44%), however, in spite of the iron-folate supplementation scheme. The rate of intestinal parasites declined, but the project did not have an impact on diarrhea. The MICAH project had a positive impact in strengthening the national vitamin A policy of Sénégal. The evaluation report was published by the project and widely disseminated. The survey findings and recommendations were fed into the design of an up-scaling phase of the project, with more emphasis on the reduction of anemia among women.
CARE is directly involved in research, and its involvement covers the whole process from conceptualization of the research question to data management and dissemination of research results. Some offices have staff whose role is specifically research-related, but this varies. They also work with partners. CARE has even been contracted by some donors to conduct research. The research is primarily qualitative, including participatory approaches, as well as operations and action research. CARE also conducts surveys, situation analyses and policy reviews. It receives funding for research from bilateral and multilateral agencies, and from large organizations such as Family Health International and the Population Council.
Helen Keller International (HKI) is a technical assistance NGO that is also directly involved in research as part of its mandate. It addresses the causes of preventable blindness. It also provides rehabilitation services to blind people, and helps reduce micronutrient malnutrition which can cause blindness and death in children. It is involved in most stages of the research cycle, focusing on operations and action research. HKI's focus on blindness and micronutrients is a strength in that its research is more focused than that of other NGOs involved in health and nutrition. Its funds for research come from different sources. A research component may be built into programs, some operational research is conducted with funds for surveillance, or funds are provided for R&D specifically (eg. for FRAT studies [Fortification Rapid Assessment Technique]) and for the development of tools to assess the quality of nutrition interventions leading to adoption of relevant strategies (in Mozambique, Burkina, Mali and Niger). HKI has in-house expertise in research. There are several full-time research positions. In addition, it works with research partners at the local level, as well as with universities in Canada and USA.
Inter Pares was created in 1975 to support NGOs from the South and to provide international development education in Canada. Inter Pares uses its own funds to conduct social research on political and economic issues, primarily action research. For instance, it carried out collaborative research with NGOs in the Philippines and of Bangladesh on family planning policy, and in Africa it has carried out research on economic issues. With Forum Afrique Canada , for instance, it is studying Canadian government trade and aid policy after G-8. It has in-house research expertise, particularly in sociology, although there is no research position as such. It usually works with partners, as it is a small NGO. Inter Pares uses research findings mainly for education and advocacy.
AMREF has been active since 1957 in the field of applied health research and has an extensive bibliography documenting research results in the form of peer-reviewed publications, theses, manuals, reports, abstracts and conference presentations. The focus of AMREF's research activities has been primarily in the operational and applied domains. Many have addressed the important disease burden caused by communicable diseases such as malaria (see table 3) and schistosomiasis, but others have addressed organizational issues such as health information systems and technological issues like field diagnostics.
Table 3 Example of an AMREF research study listed in its extensive bibliography
In 1995, D'Allessandro et al [32]published a study which compared the efficacy of insecticide-treated and untreated bednets in preventing malaria in children living in the Gambia. The survey included 2300 children between the ages of 1 and 4 years; 1500 from villages who had received insecticide-treated bednets within their primary health care and 800 from villages which had not received treated bednets. It was found that the greatest benefit, in terms of reduced malaria morbidity, was observed in children who slept regularly under treated bednets. Measurable benefits were also accrued in children who slept regularly under untreated bednets, compared to children who did not use bednets at all. The conclusion of this study was that educational campaigns might well promote even the use of untreated nets because of the additional health benefits, while ultimately aiming at coverage with insecticide-treated bednets.
Alzheimer 's Disease International (ADI), an NGO affiliated with WHO, specifically provides support for research among its numerous activities. In particular, it supports the research work of the 10/66 Dementia Research Group (the 10/66 refers to the dementia research gap, in which 'less than one-tenth of all population-based research into dementia is directed towards the two-thirds or more of cases living in developing parts of the world [31]). The vision of ADI is that research not only generates awareness, but is the basis for policy which, subsequently, can provide the impetus for development of appropriate services for affected persons. The 10/66 Dementia Research Group divides its research activities into pilot studies, qualitative studies, intervention studies and population-based studies. This group has published a consensus statement [33] and a methods paper [34], and members are now publishing research results (see table 4). This NGO's 10/66 Dementia Research Group has regional networks in India and South Asia, Latin America and the Caribbean, China and South East Asia, Africa and Russia, Eastern and South Eastern Europe which are coordinated by Dr. Martin Prince of the Institute of Psychiatry in London, England.
Table 4 Example of an Alzheimer's Disease International- supported health research study
The results of a population-based study undertaken in Kerala, India to evaluate a community dementia case-finding program was published by Shaji and collaborators in 2002 [35]. Their aim had been to validate a training program where local community health workers (CHWs) were trained to identify possible cases of dementia. The training program consisted of 2 ½ hours of formal instruction. Workers' diagnoses were then confirmed by an experienced psychiatrist. The 19 CHWs identified 51 possible cases among 1979 persons aged 60 and older in their communities. There was expert confirmation for 33 of these cases (65%). Although the remaining 18 did not have dementia, 13 did in fact suffer from other psychiatric illnesses and only 5 had no psychiatric diagnosis at all. The conclusion was that CHWs can play an important role in identifying cases of dementia in a community setting.
Médecins sans Frontières (MSF) was the first NGO to both provide emergency medical assistance and publicly bear witness to the plight of the populations they served. MSF is at the forefront of emergency health care as well as care for populations suffering from endemic diseases and neglect. MSF has undertaken an initiative on drugs for neglected infectious disease which combines advocacy, research and capacity development, and networking. In contrast with private sector research, it is need-driven rather than profit-driven. Five pilot projects are currently underway focusing on capacity building and technology transfer. This initiative started with a review of pharmaceutical research and development outcomes over the last 25 years and of current private and public initiatives. Highlights of the findings and conclusions, published in Lancet [36], are provided in table 5.
Table 5 Analysis of trends, drug research and development for tropical diseases, MSF (2002)
The extensive review revealed that of 1393 new chemical entities marketed between 1975 and 1999, only 16 were for tropical diseases and tuberculosis. All new drugs for neglected diseases represented a clear therapeutic benefit, and all are included in the WHO Essential Drug List, which indicates the importance of new drugs for neglected diseases. In contrast, over the same period, two out of three new drugs offered little advantage over existing ones. There was no indication that drug development for neglected diseases would significantly improve in the near future, however. Private-public partnerships, or else, incentives to encourage private investment towards the development of new cost-effective drugs may help overcome this limitation. For the most neglected tropical diseases which may not account for a large share of the global burden of disease, a new approach is needed. The feasibility of an international not-for-profit network that would focus on the most neglected diseases is being tested in the on-going pilot projects.
Research for International Tobacco Control (RITC) is an International Secretariat based at IDRC headquarters (Ottawa) that funds multidisciplinary tobacco control research projects in developing countries. Its mission is to create a strong research, funding and knowledge base for the development of effective tobacco control policies and programs, through a combination of research, dissemination, strengthening of capacity and coordination. RITC concentrates on research on psycho-social correlates of tobacco use. It provides support to research projects conducted by NGOs, such as, the Youth and Tobacco Survey conducted in Russia by the Russian Public Health Association, with the technical assistance of the CPHA (table 6).
Table 6 Youth and Tobacco Survey, Russian Public Health Association (RPHA), 1999
This study is part of the Global Youth and Tobacco Survey (GYTS) undertaken in several countries around the world. The survey in Russia was designed to provide prevalence data on tobacco use among adolescents in school (13–16 years), and to better understand and assess students' knowledge, attitudes and practices related to tobacco. Information pertained to, for instance, age of initiation of tobacco use, perceived health risks and social benefits, extent of peer and advertising pressure, perception of the tobacco-related curriculum, and likelihood that tobacco users will quit. The survey raised awareness on the issue of smoking and youth. Several recommendations were made by the Association to Parliament on the basis of survey findings, including the adoption of legislation to limit tobacco advertising, to reduce the tar and nicotine content of cigarettes, and to have an impressive warning labeling on packages. Seminars and conferences on the survey results and their implications were held. The Association has prepared a report: "Tobacco or Health in Russia". Additionally, the President of the RPHA, as a result of the GYTS survey and the ground-breaking leadership role the RPHA played in tobacco control among youth in Russia, was a member of the delegation from the Russian Federation to the WHO Framework Convention on Tobacco Control (FCTC) – hence an example of the translation of applied research results into policy action.
CPHA has also provided technical and financial support through its various international initiatives funded by CIDA to its public health association partners to carry out of the GYTS in Burkina Faso, Niger, Haiti, and Cuba; and in partnership with Institutes of Public Health in Bosnia & Herzegovina, Serbia & Montenegro, and in the UN-administered province of Kosovo. The results from the surveys (carried out in collaboration with the Centers for Disease Control and Prevention [CDC] of USA) are being used to develop tobacco control policy and youth-focused smoking prevention and cessation programs.
The experience of CSIH in global health research is described under 5.2.1.
4.2 NGOs' perceived strengths and weaknesses in research
The following summary of perceived NGO strengths (and weaknesses) in health research is based primarily on the data from individual and group interviews that were specifically conducted as inputs to this discussion paper.
It is a common view that NGOs are in a good position to participate in health research because of their knowledge of, and their presence in, local communities. Furthermore, their involvement increases the relevance of research to communities. "NGOs give a human face to research, and they are in a good position also to build on indigenous capacity" (Interview with S. Baker, HKI Africa). Additionally, they may be more compelled to complete the research cycle and apply the findings. Their involvement in research is perceived as a motivation to use the research to design, develop and respond to circumstances affecting development. Evaluation research, in which they are frequently involved, tells them whether or not they have an impact.
Since they are closely connected with communities, they have the ability to see the application of their research results. For this reason, NGO-initiated research is often more likely to be translated into practice in a timely manner as it is almost always directly related to practice. The NGO structure brings concreteness and a style which is guided by values and beliefs with an action orientation.
Another major strength of some NGOs is their international networks which give them access to technical information and support. Finally, NGO values of ethics, solidarity and dialogue are important for health research to contribute to reducing inequities and for empowerment.
4.3 Constraints to greater NGO involvement in health research
4.3.1 NGO views on research and its congruence with their mandate
Many reasons that account for the reluctance of NGOs to become (more) involved in research activities pertain to NGO perceptions on research (Center for Advanced Studies of International Development. Symposium on NGO/Academic Linkages. East Lansing: Michigan State University; April 16–17, 1993; Edwards M, Griffiths M: Terms of Reference for the DSA [Development Studies Association] Workshop on the Academic Practitioner Interface. London: DSA, 1994). In the past, research was an academia-driven and based, elitist and theoretical exercise, the results of which are of little use to NGOs and the communities with which they work. Traditional research strategies and approaches were seen as top-down, non-participative and controlled by external actors. Research activities were regarded as requiring special technical expertise, much time and effort, access to professional journals and research literature, and substantial human and financial resources, characteristics not typically found in NGOs. Finally, the scientific rigor demanded by researchers was believed to be difficult to achieve in field-based situations, where unpredictability and subjectivity are the norm.
What NGOs perceive as research and as their role in this respect varies widely. For instance, there is some hesitation and even reluctance in including baseline studies or project evaluation under 'research'.
Another obstacle is the fact that some NGOs that raise funds from the public are afraid to go against the expectations of the donors if the money is reallocated for research, and particularly for policy research in Canada: "Donors do not want to hear that NGOs are doing research as they are implementation organizations" (Interview with C. MacDonald, World Vision Canada).
4.3.2 Lack of training opportunities, funding, time and motivation
Among other barriers, interview respondents mentioned lack of training opportunities, lack of funding owing to their (limited) mandate, priorities of funding agencies, and time constraints.
Because of lack of training or of specialized researchers, NGOs may not be in a position to conduct top quality research, and scientific rigor may be lacking in certain instances. Lack of access to scientific literature when in the field can also be a major shortfall.
It is often difficult to secure research funding from certain donors. Many Canadian NGOs rely heavily on negotiated contracts with CIDA, which leaves little time and place for research. However, CIDA does fund some research (in a broad sense), particularly formative and summative evaluations. These are encouraging trends, but the aim should be for bilateral agencies to openly fund some research, like in the UK and some Scandinavian countries.
Lack of interest or of a clear view of the whole research process can also be considered as impediments. As several NGOs do not see research as part of their mandate, they may not be willing to get involved in research: "NGOs do not have a research mandate, and therefore we do not foresee developing research expertise in-house. Linking for instance with universities is feasible for development-driven research" (Interview with R. Hazel, CECI). NGOs may have to change their structures and priorities in order to support autonomous research.
4.3.3 Scale and type of NGO research
Because NGO research is often conducted on a small scale and is usually of a qualitative nature, it often goes unrecognized by governments, and even by research organizations and funding agencies, which tend to favour large scale quantitative research.
NGOs interested in pursuing a research profile require a type of mentorship in terms of standard performance indicators in the research domain. For example, publication has traditionally not been a strength and much NGO research does not reach beyond the gray literature or report level. Because scientific publications are an important means of transfer or dissemination of research results, NGO capacity to publish their findings needs to be strengthened.
4.3.4 Weak links with the international research community
There is not enough networking and collaboration between NGOs and the international research community, including academia. This has traditionally been due to a dichotomy in the interests of NGOs and the academic community, in that NGOs are more oriented towards a development agenda, while academics tend towards special research interests.
5 Future needs
In light of the above issues and concerns, and in order to foster greater interest and participation of NGOs in research, the barriers of lack of interest, lack of funds, lack of training and lack of recognition, among others, need to be addressed. We discuss some strategies below.
5.1 Opportunities to build NGO capacity in research, in Canada and overseas
Substantial global health academic capacity has developed within NGOs both in the U.S. and the U.K. For example, Family Health International (FHI) has integrated research, training, and development capacity on an evidence-based foundation. It also embodies many of the competitive aspects of private sector-led initiatives that can allow creativity and innovation.
As emphasized by Harrison and Neufeld [31], however, capacity building efforts for health research have been of most benefit to industrialized countries. In order to ensure that less developed countries are the principal beneficiaries, they recommend, as part of a three-pronged strategy, the nurturing and support of multi-stakeholder problem-oriented learning, and research networks which include NGOs. The other components of the strategy are research investments that explicitly reduce the high cost of knowledge translation in developing countries, and the stimulation of demand-driven research.
A peer-learning process is among the strategies for NGO capacity building in research. NGOs can draw on expertise already developed in research-based NGOs. The learning process should be shared with NGOs from the North and from the South. Additionally, NGOs should consider taking the initiative in organizing scientific activities (seminars, workshops, symposia) on global health research topics, which could serve as a catalyst in bringing together different stakeholders.
5.2 Building partnerships and alliances
5.2.1 Creating and facilitating networks that support global health research
The creation of networks which have the common goal of supporting global health research is one way to strengthen partnerships and to consolidate valuable resources from each partner. Leadership and governance issues are necessary hurdles which can be overcome by focusing on the ultimate gains in terms of supporting and conducting successful research activities. NGOs can assist in the establishment and functioning of these networks, particularly by providing stable infrastructure support. One of the greatest challenges is in making the network function effectively through different leadership turnovers in the different partner organizations.
CSIH has had global health research as part of its mandate since its formation. CSIH is an active member of the Canadian Coalition for Global Health Research (CCGHR), and a key challenge in this capacity is to develop a strong foundation of understanding and mutual respect amongst all players in global health research, including NGOs. CSIH experience in global health research is described in table 7.
Table 7 The experience of CSIH in global health research
In the early years, CSIH partnered with the Canadian University Consortium for Health In Development, which represented all the major universities in Canada and their partners in research and development. This partnership represented a strong and vital part of CSIH's operations. Following the decrease in funding for such a partnership, there was a decision to disband the Consortium and establish a network of universities and colleges that would promote and support academic and research interests within the Society. This network, which was formally given the status of a Division for a few years, has been and continues to be functional but not as a strong advocacy unit. This was largely due to the fact that funding for the network was cut by CIDA in 2000. Nevertheless the network is an important source of technical support for CSIH in its projects and advice.
Following the Thailand meeting in October 2000, Canadians were challenged to explore the role that they could play in diminishing the 10/90 Gap in Global Health Research funding available to Low and Middle Income Countries (LMIC). To this end an interest group was formed, of which CSIH was part in order to carry on the momentum of Thailand and future explorations.
Key people met with decision makers during the spring and summer of 2000 and September 11, 2001, marked the inaugural workshop in Vancouver to discuss global health research and the 10/90 Gap. CSIH was one of two NGOs who attended. Following that meeting, CSIH was invited to participate in a new Canadian Coalition for Global Health Research (CCGHR). CSIH was active in suggesting that the concept of a coalition was a way to emphasize the role of advocacy and action that is necessary for global health research initiatives to be successful.
As of October 2001, the Coalition included two NGOs (CSIH and CPHA) who were part of a lobby to expand the mandate of CIHR (Canadian Institutes of Health Research) to include global health research in more than one Institute. To this end, the Institutes of Gender and of Aboriginal Health joined the Institute of Population Health in realizing its global health mandate.
The Global Health Research Initiative memorandum of understanding (MOU) was signed at the 2001 Canadian Conference for International Health (hosted by CSIH). The amendment to MOU as a result of negotiations between CSIH and CIHR included NGOs as one of the important players.
The first formal retreat for the coalition was held in August 2002. CSIH was formally named as a member of the Coalition Steering Group. The Working Group on the Role of NGOs in Research was affirmed as separate from the Advocacy Working Group. CSIH agreed to take the lead to collaborate with other key NGOs to develop a paper and case studies.
CSIH as part of CCGHR lobbied in the spring and summer of 2002 to the G-8 for the inclusion of a commitment to global health research within NEPAD (New Partnership for Africa's Development). Support for global health research in Africa was announced and funds were set aside for this new initiative.
The first Annual Meeting of CCGHR was held at the Canadian Conference on International Health (CCIH) in October 2002. The Working Groups reported at that meeting and CSIH announced the formation of a Research Committee and invited its members to participate. The Executive Director drafted an outline of a background paper on the Role of NGOs in Global Health Research and presented to the plenary session of the annual CCGHR meeting for comment and feed-back. An ad hoc Working Group on Research was formed to draft the background paper with a view that it will be a position paper for CSIH and provide a background working paper for the Coalition.
In the autumn of 2002, the first request for proposals for global health research grants was released. Despite the fact that NGOs were named as important partners, they were not invited to be part of the review panel for this round. It was noted as a deficiency in the review of the process by CIHR.
To date, CSIH has been an active and welcomed participant of all key meeting of CCGHR Steering Committee meetings. CSIH remains actively engaged in working groups on Governance to determine options for institutionalization of the CCGHR. In collaboration with CIHR and IDRC, CSIH is actively planning the Second Annual Global Health Research Meetings at CCIH and the integration of significant research and development content in the conference.
5.2.2 Partnerships with universities and other research institutions
Partnerships with universities and other research institutions is one means of strengthening the research capacity of NGOs, and also of academia. NGOs and research organizations each have a unique 'value-added' contribution to make to global health research and therefore, partnering among them amplifies their individual strengths. Such partnering may be a real challenge for NGOs, however, as their institutional culture is so different. NGOs may be invited by universities to partner, but plans are often already laid out, so that the NGOs may only be involved in executing the plans. What NGOs want is to be part of the research process from the start. An interesting initiative to document and promote South-Canada health research partnerships is currently underway [37].
NGOs are open to partnerships with academia, but the goal has to be development-oriented. Experience suggests that it is often difficult to reconcile the academic and development framework, for instance when integrating MSc or PhD students in development projects. Nonetheless, integrating graduate students in NGO projects should be a good strategy for balanced and equal partnerships.
As less than 1% of university-based health research in Canada is directed towards the problems of global health according to a Canadian Consultation on Global Health Research held in 2001 , the prospects of university-NGO research links are constrained by funding. Nevertheless, the recent Global Health Research Initiative of the coalition of Canadian institutions funding global health research is promising as it opens new avenues for research collaboration between the North and the South, and hopefully also between universities and NGOs.
During the 1990s, there were several attempts to bridge the NGO/academic gap with respect to health research in developing countries. Save the Children UK, Oxfam and some US-based institutions supported workshops and symposia that aimed at bringing together representatives from both communities as a means of building links and forging partnerships in support of increasing the scale, scope and relevancy of health research in developing countries (Edwards M, Griffiths M: Terms of Reference for the DSA [Development Studies Association] Workshop on the Academic Practitioner Interface. London: DSA, 1994). CPHA, through the CIDA-funded Canada's International Immunization Program – Phase 2 (CIIP2), supported applied research carried out by Laval University, Université de Montreal and University of British Columbia. At the time, these were quite innovative approaches to applied health research, linking the universities with local NGOs. In 1995 CPHA also organized a Symposium on NGO/University Linkages for Health Research [30].
One mechanism to expand the use of research generated by NGOs is to improve the linkage between NGOs and universities. Each complements the other in the area of health research. NGOs offer proximity to people and situations, reality-based and context-specific research environments, opportunities to develop and assess innovative strategies and research methods, a means of disseminating and popularizing the results of research projects, and credibility outside of academia. Universities and other research organizations offer expertise in research design and application, an environment for reflection, access to and knowledge about most recent literature, a tradition of scientific rigor and interest in new, innovative research methods and approaches, and a high degree of credibility. Academics can also provide guidance and advice on how to prepare research proposals and to carry out research studies, guidance in the preparation of reports and publishing of research results, and training for NGO staff in research methods.
The participants of the CPHA Symposium on NGO/University Linkages for Health Research in Developing Countries [30] identified several mechanisms that could help bridge the gap between NGOs and universities as a means of facilitating future collaborative research initiatives. There must be first and foremost a real willingness on the part of both parties to modify their attitudes about the role and capabilities that each can offer. Mechanisms to achieve this end include conferences and seminars, newsletters, and the use of e-mail and the Internet. Another suggestion called for the use of "field-friendly" research methodologies. It was also recommended that, although the objective is not to transform NGOs into research institutes, they should receive more training in research methods and proposal development. It was suggested that exchanges take place wherein university researchers use sabbatical leave to work with NGOs and NGO personnel be seconded to universities to provide a field perspective. Additionally, research results need to be disseminated quickly and in a format that ensures maximum access by those in the field who are to apply the knowledge generated. Otherwise, research creates expectations within the NGO community and study population that remain unsatisfied.
The development of innovative North-South research partnerships is the focus of a working paper prepared for the CCGHR by Neufeld et al [37]. As emphasized in this document, such partnerships are not an end in themselves, but rather, they are to contribute to sustainable health research systems and to health development. Principles of research partnerships, and a useful model to assess these, are proposed. Although types of partnerships are not specifically detailed, it is implied that NGOs are important research partners.
Finally, as mentioned earlier, stronger partnerships between NGOs and social science researchers in particular should be sought in resource-poor countries. Lessons learned from these partnerships, in the areas of action and indeed policy and legislation (for example, in the tobacco or environment fields) show how evidence can be transformed into action with the right partnerships between researchers and NGOs.
5.2.3 An NGO Network for Global Health Research?
NGOs may benefit in various ways from developing a global health research network. First, many NGOs already operate at national and international levels and understand the challenges of coordination and communication which this entails. There is a need to identify overarching principles of NGO contribution to health research. Second, NGOs must be both proactive and interactive within the framework of the health research agenda. Roles must be clearly understood by each partner. In any case, advocacy for relevant research and use of results would be a critical function of the network (see training modules on advocacy [38]). Such networks may enhance the ability of NGOs to partner with other research stakeholders in multisectoral coalitions, and even to initiate partnerships with research organizations.
In the framework of an NGO network of this sort, the following discrete activities could be envisaged by the lead NGO:
• To invite NGOs to post on a selected website success stories, as well as their experience/opinions/needs/priority research issues, using a template adapted from the one developed for this purpose in the UK
• To organize workshops for NGOs who are, or who wish to become, involved in research, with research organizations where deemed appropriate.
The purpose would be:
• To link these NGOs in order for them to interact on research issues;
• To share lessons learned and success stories of research involvement of NGOs and their partners
• To enhance understanding of, and collaboration with, potential research partners;
• To set-up a core group of NGOs involved in global health research to convey NGO views to global health research fora and organizations.
The following are a few key questions that could be addressed by an NGO network:
1. How can NGOs contribute to the framing of the research questions if we were to support the necessary equity-based research for improving the overall performance of the health system?
2. How do we balance this with the necessity for research which documents and monitors sustained and emerging inequities which may have a greater impact on the health and well being of individuals than the health (care) system will ever have?
3. How can we ensure that NGOs influence research priorities so that they are reflective and evaluative of overseas development assistance (ODA) direction and priorities such as national poverty reduction strategies. For example what is the impact of PRSPs on equity and health? How will researchers monitor this? What could be the potential role of NGOs in partnership with researchers to begin to monitor and evaluate this new direction in overall aid policy?
4. How can NGOs be best represented within the international research community?
The new millennium offers many challenges. In order to maximize the potential benefits of health research, all partners including NGOs must share a common vision and recognize and appreciate the strengths of each. Participation in health research needs to be a coordinated effort. One key challenge will be to establish better communication among all partners in health research. This can only be achieved by a willingness to share in leadership, ownership and in the conduct of health research activities. Another key challenge will be to explore ways in which funding for health research can be strengthened. Leveraging must be seen as a strategic tool of NGOs to maximize dollars allocated to health research.
Conclusion
Several NGOs have had impressive track records in global health research. Other NGOs have expressed an interest in becoming more involved in global health research. Their contribution to more equitable, ethical, relevant and effective research is crucial and needs to be strengthened. Research has to be regarded as a broad loop system rather than restricted narrowly to the production of knowledge. This is particularly critical for global health research whose primary goal should be to improve health and its determinants in low and middle income countries. NGOs principal roles in the process pertain to shaping research priorities, advocacy for more relevant research, translating and using research findings, in addition to generating new knowledge in areas where they may have a comparative advantage, notably qualitative, social, action, evaluative, and policy research. NGO partnerships with research organizations should be seen as means of a mutual enhancement of health research capacity and contribution to development. NGOs should be instrumental in building with other stakeholders coalitions for global health research with the aim of closing the 10/90 health research gap.
List of Abbreviations
ADI Alzheimer's Disease International
AFRO-NETS African Networks for Health Research and Development
AIDS Acquired immunodeficiency syndrome
AMREF African Medical and Research Foundation
ARCH Applied Research on Child Health (ARCH) Project
CCIC Canadian Council for International Cooperation
CCISD Centre de coopération internationale en santé et développement
CCIH Canadian Conference on International Health (hosted by CSIH)
CECI Centre canadien d'étude et de coopération internationale
CCGHR Canadian Coalition for Global Health Research
CDC Centers for Disease Control and Prevention
CHW Community health worker
CIDA Canadian International Development Agency
CIHR Canadian Institutes of Health Research
CIIP2 Canada's International Immunization Program – Phase 2
COHRED Council on Health Research for Development
CPHA Canadian Public Health Association
CSIH Canadian Society for International Health
DFID Department for International Development (UK)
DSA Development Studies Association
ENHR Essential National Health Research
FCTC Framework Convention on Tobacco Control (WHO)
FHI Family Health International
FRAT Fortification Rapid Assessment Technique
GYTS Global Youth and Tobacco Survey (WHO and CDC)
HKI Helen Keller International
IDRC International Development Research Centre
IHPP International Health Policy Program
INTRAC International NGO Training and Research Centre
KFPE Commission for Research Partnerships with Developing Countries
LMIC Low and middle-income countries
MICAH Micronutrients and Health in Africa (WV project)
MSF Médecins sans frontières
NEPAD New Partnership for Africa's Development
NGO Non-governmental organization
OECD Organization for Economic Co-Operation and Development
ODA Overseas Development Assistance
PRSP Poverty Reduction Strategy Papers
RITC Research for International Tobacco Control
RPHA Russian Public Health Association
SHARED Scientists for Health and Research for Development
TRIPS Trade-Related Aspects of Intellectual Property Rights
UNDP United Nations Development Programme
WHO World Health Organization
WV World Vision (Canada)
Authors' contributions
HD designed the outline of the paper, conducted interviews with NGO representatives, wrote the first complete draft, and coordinated the review process within the CSIH. JHR drafted some sections and provided comments on the successive versions of the papers. MM provided international NGO and a field based perspectives to the paper, in addition to conducting group discussions with NGO personnel. LJ designed the figures, and edited and formatted the text. TG contributed to the conceptualisation and content of the manuscript. She wrote several sections and edited others, and she provided case studies.
Acknowledgements
The authors acknowledge the valuable contribution to the paper of the following members of the Canadian Society for International Health, many of whom are also members of its research committee: Adrijana Corluka, Jim Chauvin, Don Juzwishin, Montasser Kamal, Marleny Munoz, Vic Neufeld, Lynn Skillen, Bernadette Stringer, and Peter Tugwell.
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| 15723694 | PMC554095 | CC BY | 2021-01-04 16:37:16 | no | Health Res Policy Syst. 2005 Feb 21; 3:3 | utf-8 | Health Res Policy Syst | 2,005 | 10.1186/1478-4505-3-3 | oa_comm |
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BMC Med GenetBMC Medical Genetics1471-2350BioMed Central London 1471-2350-6-71570748210.1186/1471-2350-6-7DebateTheories of schizophrenia: a genetic-inflammatory-vascular synthesis Hanson Daniel R [email protected] Irving I [email protected] Department of Psychiatry, VA Medical Center (116A), One Veterans Drive, Minneapolis, MN, 55417 and Departments of Psychiatry & Psychology, University of Minnesota, USA2 Departments of Psychiatry & Psychology, University of Minnesota, Minneapolis, MN 55454, USA2005 11 2 2005 6 7 7 26 7 2004 11 2 2005 Copyright © 2005 Hanson and Gottesman; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Schizophrenia, a relatively common psychiatric syndrome, affects virtually all brain functions yet has eluded explanation for more than 100 years. Whether by developmental and/or degenerative processes, abnormalities of neurons and their synaptic connections have been the recent focus of attention. However, our inability to fathom the pathophysiology of schizophrenia forces us to challenge our theoretical models and beliefs. A search for a more satisfying model to explain aspects of schizophrenia uncovers clues pointing to genetically mediated CNS microvascular inflammatory disease.
Discussion
A vascular component to a theory of schizophrenia posits that the physiologic abnormalities leading to illness involve disruption of the exquisitely precise regulation of the delivery of energy and oxygen required for normal brain function. The theory further proposes that abnormalities of CNS metabolism arise because genetically modulated inflammatory reactions damage the microvascular system of the brain in reaction to environmental agents, including infections, hypoxia, and physical trauma. Damage may accumulate with repeated exposure to triggering agents resulting in exacerbation and deterioration, or healing with their removal.
There are clear examples of genetic polymorphisms in inflammatory regulators leading to exaggerated inflammatory responses. There is also ample evidence that inflammatory vascular disease of the brain can lead to psychosis, often waxing and waning, and exhibiting a fluctuating course, as seen in schizophrenia. Disturbances of CNS blood flow have repeatedly been observed in people with schizophrenia using old and new technologies. To account for the myriad of behavioral and other curious findings in schizophrenia such as minor physical anomalies, or reported decreased rates of rheumatoid arthritis and highly visible nail fold capillaries, we would have to evoke a process that is systemic such as the vascular and immune/inflammatory systems.
Summary
A vascular-inflammatory theory of schizophrenia brings together environmental and genetic factors in a way that can explain the diversity of symptoms and outcomes observed. If these ideas are confirmed, they would lead in new directions for treatments or preventions by avoiding inducers of inflammation or by way of inflammatory modulating agents, thus preventing exaggerated inflammation and consequent triggering of a psychotic episode in genetically predisposed persons.
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Background
When the solution to a clinical or scientific puzzle eludes us for more than a century, as with schizophrenia (formerly dementia praecox), we need new ways of thinking about the problem [1,2]. Efforts to understand schizophrenia have focused on neurons and, especially, the role of presumed excess dopamine neurotransmission. We believe that genetic, environmental, and stochastic factors combine with epigenetic factors to create episodes of the illness [3-5]. Thus, the syndrome of schizophrenia is viewed as an endpoint in a dynamic process variously conceptualized as degenerative or developmental or alternating at different points in the process [6-10].
Degenerative models imply that after a period of normal development, the organism, or one of its parts, takes a wrongful turn in its trajectory and begins to malfunction. This describes the eventual outcome for all life forms and is a biological restatement of the second law of thermodynamics. Since degeneration is universal, stating that an illness is degenerative is not particularly helpful. What would be helpful is to determine when in the life course the degeneration begins and how the degeneration is initiated and proceeds. Answers to the "when?" and "how?" questions would then describe the degenerative process in developmental terms.
Developmental models of schizophrenia implicate abnormalities of early brain development predisposing to future schizophrenia. The proponents of the model further argue that the perturbations of development are limited to the early times of development and are discontinuous. Without this qualifier, developmental models are indistinguishable from degenerative models where the degeneration commences early in the life span. The early abnormalities are not necessarily the cause of schizophrenia, but, instead, create a state of risk for a future episode of schizophrenia. That is, a diathesis or predisposition is not a disease. Consequently, there must be factors later in life that convert the vulnerability to an illness. These additional factors are presumed to damage development in such a way that a predisposition becomes actualized. To gain a complete understanding of the syndrome, we must again return to the question of " what happens?"
Following this line of reasoning, the distinction between degenerative and developmental models blurs. In fact, a medical-behavioral condition can be both developmental and degenerative as exemplified by Down syndrome [11-13]. Individuals born with trisomy 21 exhibit a number of developmental anomalies including cardiac malformations, abnormal dermatoglyphics, skeletal changes, and muscular hypotonia, to name a few. As trisomy 21 infants mature, most exhibit degrees of mental retardation. By about age 50, these individuals invariably develop Alzheimer-like CNS degenerative changes that can be seen at autopsy [13].
Schizophrenia involves both developmental and degenerative features. From the time of Bleuler [14] and Kraepelin[15], "It is certain that many a schizophrenia can be traced back into the early years of the patient's lives..." [14] p. 252. The 'follow back' studies of schizophrenia support these views [16]. Likewise, prospective studies of children at high risk for schizophrenia report developmental anomalies in motor skills, cognition, and attention long before the onset of overt illness [17-19]. Overt psychotic symptoms for some individuals usually start in the late teenage years or early twenties, but the illness can start as early as middle childhood [20] and may, more rarely, start in old age [21] p 73].
The evidence suggesting early developmental perturbations in schizophrenia is compelling. At the same time, there certainly are examples of deterioration reminiscent of Kraepelin's suggestion for some people with schizophrenia. However, deterioration in clinical course may not indicate CNS deterioration. Instead, the decline could be a secondary consequence of an illness that disrupts education, economic achievement, and social functioning leading to a downward spiral in all aspects of adult life. Consistent with an early degenerative process, there are reports of declining cognitive function preceding onset of psychosis [22]. Proponents of neurodevelopmental models suggest that the premorbid cognitive abnormalities are developmental risk factors for future schizophrenia (c.f [23]) and argue that such abnormalities show little evidence of decline after onset [6,24]. Whether developmental or degenerative, the premorbid cognitive deficits seen in schizophrenia are also seen in other disorders [25] and lack specificity and sensitivity thus detracting from the concept that the cognitive abnormalities seen in schizophrenia are useful endophenotypes [26]. The strongest evidence for a neurodegenerative phenomenon comes from imaging studies showing progressive loss of brain volumes [27-29]. Neuropathological studies fail to find widespread classic signs of neurodegeneration such as gliosis though there are exceptions to this generalization [30]. Observations of abnormal dendritic arborization [31,32] are consistent with the neuroimaging evidence suggesting abnormal connectivity between brain regions [29]. As a cautionary note, most of the neuroimaging and neuropathology results are subject to confounds from the effects of medications and various other treatments, post-mortem intervals, possible effects of diet, smoking habits, as well as a myriad of other potential confounds associated with glucocorticoid mediated stress following chronic illness and associated life's limitations [33,34].
The symptoms of schizophrenia are highly variable. Within families (and thus presuming relative homogeneity of genetic and environmental factors) symptoms can vary widely over time, as illustrated by identical quadruplets concordant for schizophrenia [35]. Even within affected individuals, symptoms will wax and wane and may even remit [36] suggesting a life long process.
The major behavioral symptoms of schizophrenia include alterations in cognition, memory, perception, thought (inferred from language), motor functions, and affect. People with schizophrenia may show abnormal dermatoglyphics and other minor physical anomalies [37-42]. Other oddities to be incorporated in a comprehensive explanation of schizophrenia include highly visible nail fold capillaries [43,44] and the rarity of rheumatoid arthritis among schizophrenic persons [45]. These physical characteristics suggest the need to look beyond the nervous system per se to have a comprehensive view of the illness.
The fact that the schizophrenia syndrome, as currently defined, is relatively common provides important information about the frequency of causal factors. About 1% of the population will experience schizophrenia during the lifespan. Except for a few rare exceptions, this 1% risk is remarkably constant around the globe regardless of culture, geography, or ethnicity. Men and women are affected equally. These facts mean that the risk factors for schizophrenia must also be common and ubiquitous. Given that the concordance rate for schizophrenia in identical twins [46] is only about 50%, there must be at least two global risk-increasing categories for schizophrenia, i.e., something(s) genetic and something(s) environmental. Assuming these risk factors are independent of each other, the joint probability of acquiring both risk factors is the product of their population frequencies that, for schizophrenia, equals about .01. To make a simplifying assumption to allow easy calculations, let us say that the two risk factors are present with about equal frequency in the population. With this simplification, straightforward mathematics indicates that the individual frequencies of these factors are close to the square root of the population frequency of 1%. That would mean that about 10% of the population would encounter at least one risk factor. The math indicates that the greater the number of independent risk factors, the more common they are. [See [47] for further elaboration].
Our challenge is to develop a theory of schizophrenia that can plausibly explain an illness that affects all domains of behavior (thought, affect, motor performance, etc), that has elements of developmental perturbations early in life leaving clues such as minor physical abnormalities, and also has elements of degenerative changes. At the same time, the defect is so subtle that we can't find the cause(s) with our best modern technology. Furthermore, in spite of brain-wide dysfunctions, many individuals with schizophrenia remain sufficiently intact that, with good treatment and a bit of luck, can maintain jobs and function usefully in society. Thus, we need to find frequent and ubiquitous factors that can affect virtually all brain functions as well as creating somatic signs, but they operate in ways that leave these functions only slightly "off kilter" as compared to the complete disruption seen in strokes, or classical degenerative disorders such as Alzheimer, or as seen in Down syndrome where the behavioral pathology is apparent from earliest stages. As we try to explain schizophrenia, we must account for most all of the developmental and degenerative features of schizophrenia.
To account for the panoply of signs and symptoms seen in schizophrenia, any complete theory of schizophrenia must include organism wide systems. In addition to the nervous system, the immune system and the vascular system are defensible candidates. Both are invoked in the following theory: Some schizophrenia psychoses are the result of damage to the micro-vascular system in the brain initiated by genetically influenced abnormal inflammatory processes acting in response to ubiquitous environmental factors that trigger inflammatory responses, including infection, trauma, or hypoxia. It is the relative infrequency of the vulnerable genotypes in the population [48] that results in only a small proportion developing overt psychosis.
We wish to emphasize that our hypothesis specifically identifies the microvascular system as the critical site of inflammation. We postulate that the inflamed micro-vessels lose their coupling with astrocytes, leading to disrupted regulation of cerebral blood flow and damage to the blood brain barrier. These disruptions in homeostatic mechanisms then lead to abnormal signal processing. Our focus on inflammation of the vessels differentiates our hypothesis from models of widespread parenchymal inflammation such as seen in psychotic syndromes following, for example, encephalitis lethargica, or paraneoplastic syndromes. Many acute inflammatory disorders of the brain involve inflammation of both the parenchyma and the vasculature. By contrast, we are proposing a chronic, smoldering, inflammation of the vessels alone. And, finally, we distinguish our hypothesis from the theories of schizophrenia implicating direct parenchymal infection of the brain (c.f. [49]) and also differentiates our hypothesis from speculations about schizophrenia that invoke infectious agents altering DNA [50].
Many prior debates about inflammation in the brains of people with schizophrenia have focused on the presence of absence of gliosis (see [51] for review). The consensus opinion is that gliosis, though present in some cases, is not a consistent feature of the neuropathology of schizophrenia. However, as Harrison [51] points out, evaluating gliosis is fraught with a multitude of problems and is not a definitive indicator of degenerative/inflammatory changes in the brain. More recent efforts have demonstrated activation of microglia in the brains of some individuals with schizophrenia implying an ongoing immunopathological process in addition to what ever happened early in development [52]. Ongoing neurodegenerative processes are suggested by increased levels of S100B, a small calcium binding astrocytic protein that is involved in inducing apoptosis and modulating proinflammatory cytokines [53-55].
It is likely that the current clinical syndrome of schizophrenia is etiologically heterogeneous. We do not pretend to explain all (DSM or ICD) cases of syndromal schizophrenia. Instead, we put forward our hypothesis as an attempt to define a psychiatric syndrome in terms of a particular pathophysiology. Following this course may then help refine our nosology (see also section on 'specificity' below) and cause us to recalculate basics 'facts' such as prevalence rates.
Discussion
A primer on CNS blood supply
Neurons derive their energy from oxygen and glucose delivered by the vascular system, plus lactate and glycogen derived from astroglia [56]. The combination of neurons, astroglia, and micro-vessels form a metabolic trio [56] whereby the glia extend processes interacting with neurons on the one hand and, on the other, form endplates interdigitated into capillary walls. Rather than being passive conduits, the CNS vascular system is the most precisely managed and the most complex fluid dynamic system known. Regulation of cerebral blood flow (CBF) is managed primarily by a coupling between astrocytic glial cells [56-59] and capillary endothelium [60-65]. Astrocytes sense local neuronal metabolic activity and adjust blood flow as needed. Cerebral vessels change caliber in response to vasoactive substances released by astrocytes activated by glutamate receptors [56,66,67]. Serotonin [68], acetylcholine [69] and dopamine [66,70,71] transmission between astrocytes and micro vessels also play roles. When the neuronal activation of discrete areas is sustained over longer periods, vasoactive substances stimulate angiogenesis resulting in increased capillary density [67] thus enhancing local neuronal circuitry. Conversely, decrease in capillary density is likely to reduce the functional capacity of brain areas so affected [67]. Consequently, capillary beds in the cortex are not distributed in uniform fashion [72]. There are close relationships among local neuronal activity, density of capillary bed, and the distribution of valve-like flow control structures [73].
Developmentally, the CNS vascular system originates from capillary endothelial cells that migrate into developing neuro-ectoderm under the influence of trophic factors such as vascular endothelial growth factor (VEGF) [74] and erythropoietin [75] both produced by astroglia. The developing micro-vasculature, although comprising only 0.1% of the entire brain, and operating under the influence of genetic directives, has a key role in the development, maintenance and repair of the brain [76]. In turn, VEGF has trophic effects on neurons and glial cells, and the activity of VEGF influenced angiogenesis is directly proportional to the high metabolic activity of neocortical development [77]. Thus, angiogenesis and neurogenesis occur simultaneously and synergistically [78-80]. In addition to formation of capillaries themselves, intricate anastomoses between micro-vessels further 'fine tune' the metabolic support of developing glia and neurons [81]
The genetics of infectious & inflammatory diseases
When infectious agents give rise to inflammatory vascular disease, the nature of the infectious agent may be less important that an individual's genetically influenced inflammatory response. The concept that infectious disease may have a genetic component is, of course, not new. Many agricultural geneticists make their livings by breeding disease resistance into both plants and animals [82,83]. One of the founders of behavioral genetics, Franz Kallmann [84], showed genetic factors influenced acquiring tuberculosis (DZ concordance = 26%, MZ concordance = 87%), an observation that was confirmed in modern times [85,86]. Many other infectious diseases appear to have genetic factors influencing susceptibility or resistance to the infection [87-97]. Mechanisms for genetically mediated responses to infection occur through genetic variations in immune mediators such as cytokines[96] and HLA factors [98,99].
Familial Mediterranean Fever (FMF) [100,101] provides a heuristic Mendelian example. The gene for FMF is located on the short arm of chromosome 16 and produces pyrin (marenostrin) that functions in a negative feed back loop to suppress inflammation. Absence of pyrin leads to exaggerated inflammatory responses. Vasculitis is one of the consequences [102]. Additionally, very high rates of rheumatic fever (RF) or rheumatic heart disease (RHD) are found in relatives of patients with FMF[103]. Having even one mutant gene appears to lead to immune hyperactivity to streptococcal antigens. We also know that antibody [104] production and cytokine activity [105] in RF patients is more marked than non-rheumatics. It is clear that genes influence the host's response to infection. A similar line of reasoning applies to other inducers of inflammation such as traumatic injury [106] or hypoxia [107,108].
Just as the CNS blood supply is highly regulated, the inflammatory systems in the brain require 'fine tuning.' Given the limited ability for adult brain to regenerate, and assuming there is little tissue to spare, it would make sense that the brain should be protected from overabundant inflammatory reactions [109]. Astrocytes play a key role in the expression of inflammatory cytokines, chemokines, and growth factors involving the modulation of gene expression for these factors [109-111].
Let us suppose that schizophrenia develops following an infection (or trauma or anoxia – the environmental contributors) but the host's response is determined by genetic factors regulating the nature and degree of inflammation. That infectious agents may be operative in schizophrenia is supported by several of lines of evidence. Summaries can be found in numerous sources [49,50,112-116]. The same concept applies to trauma [106] or anoxia [79,107] that may also stimulate inflammatory processes.
Vascular disease and psychopathology
The syndrome of schizophrenia is likely to be etiologically heterogeneous and a multitude of CNS disorders can give rise to schizophrenic-like psychoses [117]. The idea that CNS micro-vascular diseases, in particular, are factors in psychotic disorders is also an old idea [118,119] that deserves a second look in light of new perspectives offered by developments in the genetics of inflammatory diseases. There are many examples of psychoses resulting from micro-vascular CNS disease including lupus and Sjögren syndrome [120]. Neuroimaging and neurocognitive deficits in these disorders are similar to those seen in schizophrenia [121]. Psychoses associated with substance abuse are also associated with CNS vasculitis [122]. Furthermore, infectious agents such as syphilis [123] and rheumatic fever (RF – see below), lead to micro-vascular disorders of the CNS that are associated with psychiatric symptoms including psychoses. Thomas, et. al. [124] also demonstrated small vessel abnormalities in the depressed elderly. At the same time, there is growing interest in cytokines and other inflammatory agents in psychoses[125] as well as growing awareness that inflammatory reactions are modulated by neuropeptides [126].
Inflammatory processes often damage the precise regulation of cerebral blood flow. The wide spectrum of clinical conditions thought to be created, in part, by inflammatory CNS micro-vessel disease include Alzheimer disease where it is thought that inflammatory processed damage the micro-vascular endothelium causing insufficient blood flow leading to oxidative stress, a build up of amyloid, and eventual cell death [127-135]. Cerebral palsy is also conceptualized as an infectious-inflammatory-vascular disorder where the vascular lesion is complete thrombosis [136]. Neurotoxic effects of methamphetamine and cocaine appear to be due to induction of inflammatory genes in small vessel endothelial cells [122,137], thus explaining the vascular damage seen in amphetamine and cocaine abuse that was previously attributed to contaminants of injected drugs [122,138-140].
Returning to the early stages of life, we have seen that the development of the neurons and glia are intimately associated with, and dependent on, the parallel development of the CNS vasculature. If the stated theory is correct, and given the developmental perspective of schizophrenia ---early developmental perturbations of the CNS set the stage for later schizophrenia--- we would expect to find support for the idea that inflammatory events early in life affect CNS vascular function. Such is the case. Whether the early insults are traumatic, infectious, or hypoxic; inflammatory process are involved in the attempts to protect and repair by modulating angiogenesis [141-148]. Thus, the reports implicating pregnancy and birth complications (anoxia, trauma or maternal infections) in the development of some cases of schizophrenia [149,150] could all be mediated by the common pathway of inflammatory-vascular mechanisms. Individuals who's genes created perturbations in inflammatory-vascular regulation would continue to experience abnormalities of protection and repair in response to subsequent CNS insults. Over time, the accumulation of 'hits' could lead to brain dysfunction to the extent seen in psychoses. The greater the number and duration of 'hits,' the greater the risk for a deteriorating /degenerative course. That neuroleptics may alter the permeability of the blood brain barrier and modify immunoregulation in the CNS [151] strengthens the argument for early treatment as a strategy to prevent deterioration.
Alterations of cerebral blood flow in schizophrenia
Since the time of Seymour Kety's pioneering efforts [152,153], there has been interest in altered cerebral blood flow in people with schizophrenia. An in-depth review of this large literature is beyond the scope of this paper. The interested reader is referred to discussions of reduced anterior cerebral perfusion leading to the concept of 'hypofrontality' in schizophrenia [154,155] and to more recent reviews [156-158]. Bachneff's [159] review and theory about defects in regulation of CNS microvascular systems is particularly relevant. These reviews summarize a consistent body of evidence showing reduced cerebral blood flow in brains of people with schizophrenia especially to anterior regions. Flow deficits are seen in medication-naive new onset cases [160,161] and more established cases free of neuroleptics [162] suggesting that flow perturbations are neither the consequence of duration of illness nor treatment. Neuroleptics can alter cerebral blood flow [163,164] although the effects may be regionally and drug specific [165,166]. Decreased frontal flow is often associated with negative symptoms [167,168]. In addition to the frontal cortex, flow abnormalities in people with schizophrenia have been noted in the cingulate cortex [169,170], thalamus [171], basal ganglia [172], parietal cortex [167,170] and cerebellum [171]. Furthermore, in some instances, flow rates are increased [160,170]. Rather than a simple hypothesis of hypofrontality in schizophrenia, theorizing is evolving toward a concept of "dysfunctional circuits"[160] or "inefficient dynamic modulation" [173] of cerebral metabolism which is supported by other examples of abnormal modulation of cerebral blood flow in response to activation tasks [171,174]. Disturbances of blood flow in schizophrenia are well documented but are not limited to schizophrenia. Disturbed cerebral blood flow is also reported in obsessive compulsive disorder [175] and depression [176,177] as well as in Alzheimer disease (cited earlier). The usual interpretation is that alterations of blood flow arise as a consequence of abnormal neuronal metabolism. The theory proposed by this paper turns the causal arrow around to suggest that abnormalities of blood flow lead to altered neuronal-glial function that, in turn, leads to psychopathology. There has been scant direct visualization of the vascular system in schizophrenia, but at least one laboratory has found evidence of atypically simplified angioarchitecture and failure of normal arborization of small vessels [32].
Post- streptococcal behavioral syndromes as a model
Post-streptococcal neuropsychiatric syndromes include Syndenham chorea, the PANDAS/obsessive compulsive syndrome, tics including Tourette syndrome, and possibly, ADHD [178-184]. Psychotic disorders are also implicated [183,185] and see citations below.
Sydenham chorea is the best-known neuropsychiatric complication following streptococcal pharyngitis. The association of psychoses and Sydenham chorea as well as with RF even in the absence of chorea, was discussed in the 17th and 18th centuries starting with Sydenham himself (see [186]). The interest in psychoses associated with RF continued throughout the 1900's [187-197]. People with a history of Sydenham chorea and/or rheumatic fever are at high risk for developing psychopathology later in life [198,199] with a relative risk for schizophrenia as high as 8.9 in a 10 year follow-up of 29 Sydenham patients [200]. There is a suggestion that the family members of Sydenham patients are also at higher risk for psychosis [201].
During the 1940's-1960's when RF was still quite prevalent, people with psychoses appeared to have higher than expected rates of histories of RHD or RF)[195,202,203] or rheumatic chorea [204]. Psychotic patients with RHD more often had early (<age 19) onset, movement disorders, progressively insidious courses and poor long-term outcomes [203]. Preliminary data from a Minnesota study also finds increased rates of RHD in psychotic patients, a pattern of increased psychiatric hospitalization following an epidemic of RF, and a clinical course for "rheumatic psychoses" that disproportionately led to a severe and continuous decline in function [205]. Although schizophrenia-like psychoses were the most common psychopathology related to rheumatic syndromes, manic-depressive, involutional, and senile psychoses were also observed [183,197].
An inflammatory reaction of the CNS vascular endothelium (vasculitis) is a common denominator in the both acute and chronic cerebral consequences of rheumatic fever. [186,187,190,195,197,206-209]. The microvascular lesions suggest both an obliterating process likely due to micro-emboli from rheumatic cardiac valves and an inflammatory process involving irregular proliferative changes in the vascular endothelium, dilatation of the lymphatic spaces surrounding the capillaries suggesting increased permeability of the capillary endothelium, and inflammatory cell infiltrates. Disruption of the blood brain barrier suggested by the evidence of increased permeability of the small vessels could compromise the immunological protection of the brain leading to the formation of the anti-neuronal antibodies seen in post-streptococcal CNS syndromes. In parallel fashion, people with schizophrenia show evidence of altered blood brain barrier and consequent alterations in immunological markers [210]
The post-strep psychopathologies provide a precedent for the hypothesis of this paper by demonstrating that an infectious process can trigger a series of inflammatory reactions that lead to a variety of somatic and psychiatric syndromes, including psychoses where vascular pathology is implicated. The pathogenicity of a strep infection is a function of the strain (genotype) of the bacterium and the genetically mediated inflammatory mechanisms of the host [211] and illustrates how a ubiquitous and often relatively benign environmental factor can create more serious sequelae in a limited number of genetically predisposed individuals-true genotype by environment interaction.
Summary
The ideas here are not completely new. Eugen Bleuler [14] remarked: "The fragility of the blood vessels which appears in many schizophrenics, both acute and chronic, seems to indicate a real vascular pathology (p.167)." We bring old ideas forward into the light of new understandings offered by molecular genetics and inflammatory diseases. Since the late 1800's there has been evidence of inflammatory neuro-vascular abnormalities in psychiatric illness that were initiated by infectious agents. CNS lues (syphilis) is the best-known example. This paper expands the concept to suggest that a variety of environmental insults (infection, trauma, anoxia) may follow a common final pathway to psychopathology by stimulating inflammatory processes that damage the capillary-glial-neuron triad as illustrated in Figure 1.
Abnormal behaviors develop as a result of disruptions in astroglial mediated coupling of cerebral blood flow to neuronal metabolic needs. These subtle disruptions are hard to find, as the microvasculature comprises only about 0.1% of the brain and are of a scale more appropriate for electron microscopy. None-the-less, the hemodynamic perturbations have sufficient impact to cause subtle but widespread disruption of the normally harmonious coordination of CNS function leading to a condition variously conceived as a "neurointegrative defect"[212], "synaptic slippage" [213], "abnormal signal transduction" [4], "inefficient dynamic modulation" [173] or "synaptic destabilization" [214]. The ultimate impact would lead to psychopathology including psychoses as the vascular-glial-neuron triad is progressively damaged over time after repeated inflammatory episodes. The resultant failure to regulate the delivery of oxygen and energy adequately would lead to oxidative stress [215-217]. Oxidative stress, in turn, can further damage the microvasculature and the blood brain barrier [218-220]. The astroglial-capillary partnership that protects the integrity of the blood brain barrier would be compromised, thus exposing neural tissue to damage from immunological attack [221]. Known precedents of such processes are found in the behavioral changes seen in CNS vascular inflammatory diseases such as lupus and the post-strep syndromes described above.
This theory could explain how developmental events such as prenatal infections [150,222], and other birth and pregnancy complications [149] including anoxia [223] are linked to later schizophrenia – infection, trauma, or anoxia all stimulate inflammatory processes [224]. The data suggesting an (statistical) influence of season of birth [116] is also consistent with the hypothesis as infectious epidemics often follow seasonal patterns. Some of the minor physical anomalies such as unusual scalp hair patterns and dermatoglyphic changes are explained because the development of these phenomena are linked to each other [225], to the development of the central nervous system [226], and are developmentally modulated by the pleiotropic effects of the same substances that modulate brain vascular development (e.g., vascular endothelial growth factor/vascular permeability factor [227] and epidermal growth factor [228]). The waxing and waning of symptoms would correspond to waxing and waning of inflammations as individuals are exposed, recover, and then re-exposed in conjunction with other physiological and hormonal influences, as seen in lupus [229]. The nature and severity of symptoms would depend on where in the brain the inflammation takes place and this may be stochastic. As the micro- vascular system is everywhere in the brain, lesions could produce the variety of symptoms seen in schizophrenia including dysfunctions of thought, emotion, memory, motor skills and autonomic regulation. The developmental age of the individual will also make a difference. Inflammatory processes that alter angiogenesis during prenatal development will likely have more dire consequences than inflammatory reactions that start after CNS maturation although even the adult brain remains susceptible [230]. We have attempted to schematically illustrate this dynamic process in Figure 2.
This theory also captures many of the little oddities observed in schizophrenia. For example, the reported abnormalities of the nail fold capillary beds seen in some people with schizophrenia [44] are also seen in people with inflammatory disorders such as FMF [231] and rheumatoid arthritis [232]. Another oddity is the negative association between schizophrenia and rheumatoid arthritis [45]. There are parallels in the post-streptococcal syndromes where RF and acute post-streptococcal glomerulonephritis very rarely occur in the same patient [233]. Some strains of group-A-streptococci identified by their M-protein serotypes are rheumatogenic while others are nephritogenic [233,234]. Phage or phage-like elements inserted into the streptococcal DNA are a major source of variation between streptococcal strains and these elements determine pathogenicity [235]. Additionally, host variation in humoral and cellular immune response shape the outcome of infection[211] By analogy, individuals with vascular/CNS involvement following, for example, streptococcal infections may be systematically spared from joint involvement as a function of both the invading strain and the individuals susceptibilities. Alternatively, as postulated for Alzheimer disease (cited earlier) that is also less common in people treated for arthritis, the anti-inflammatory treatments for arthritis might reduce the risk of inflammatory brain disease.
Another line of evidence compatible with this theory is the observation that genetic linkages for schizophrenia coincide with sites for glial growth factor cell regulators [214] and, as we have seen, the glia are key intermediaries of CNS inflammation and vascular regulation. More specifically, emerging data demonstrate associations between schizophrenia and genetic polymorphisms in regulators of inflammation such as tumor necrosis factor alpha genes [236,237] and interleukin-1 genes [238]. Another piece that fits into the puzzle is the fact that neuroleptics have inflammatory modulating properties [239-244] and neuroleptic treatment may be synergized by addition of anti-inflammatory drugs [245].
It may well be that the environmental components of psychiatric illness such as schizophrenia are relatively minor, ubiquitous, or chance events [246,247] that have the potential to stimulate the inflammatory systems. However, the nature of the insults may be less important than individuals' genetically influenced and idiosyncratic responses to the insults, similar to individuals with FMF who have an exaggerated inflammatory response. Thus, the genetic components of the inherited predisposition to mental illness may lie "upstream" in the immune system rather than in the CNS per se. The possibility that the environmental agents may be nearly universal (e.g. who has not had a strep throat or viral syndrome?), will mean that the prevalence of the etiological factor will be similar in control and experimental groups thus making it too easy to dismiss key environmental factors in null hypothesis designs [47,248]. Rather than focus on the environmental contributors that could be non-specific and ubiquitous, it will be more productive to look for genotypes that respond abnormally to triggers of inflammation and microvascular dysfunction (cf[48]). These individuals would be the ones who are at high risk for psychiatric illness. However, the inflammatory processes involve a cascade of steps involving many genes. But this, too, fits with the polygenic features of schizophrenia [249]. Identification of high-risk individuals, combined with such tools as immunizations or anti-inflammatory agents may promote prevention of much psychiatric morbidity. Already, the cytokine regulator and vascular growth factor erythropoietin is suggested as a possible neuroprotective factor in schizophrenia [250]
Future directions
The speculations about psychoses developing from vascular/inflammatory processes provide direction for future research across many domains. In addition to pursuing direct evidence of altered activities in inflammatory/immune systems in people with psychoses, the inflammatory/vascular theory has implications for epidemiology, genetics, neuroimaging and neuropathology. For the epidemiologist, the challenge will be to detect relatively small signals against a very noisy background. We hypothesize that the triggers for inflammation can be many and varied and are common factors in the environment. Imagine starting with the clinical syndrome of Sydenham chorea and comparing the rates of strep throat in those affected vs. comparison sample of people without Sydenham chorea. Null hypothesis testing with small sample sizes and nearly ubiquitous etiological agents are clearly not adequate. A second epidemiological challenge is to cast a broad enough net to capture the wide variety of possible contributing factors. Rather than taking a one by one approach to exploring the etiological contributions of, say, virus titers, anoxia, physical trauma, the epidemiologist should look for any and all. It would be predicted that individuals with multiple "hits" (e.g. in utero exposure to virus and low Apgar scores and childhood head trauma) would be at greater risk than those exposed to just one event. If in utero inflammatory processes are active in the genesis of schizophrenia we would also predict an increased rate of fetal deaths in families of schizophrenic probands. A third epidemiological opportunity lies in the search for non-psychiatric inflammatory-related disease or traits in people with psychosis. If something is askew in the inflammatory process in schizophrenia, the effects will show up in other parts of the body. Though requiring replication, the association of psychosis with hemolytic anemia in lupus [251] provides an illustrative example. In addition to rheumatoid arthritis, the associations of diabetes and cancer have been explored in schizophrenia; one of is exploring rheumatic heart disease [205]. Population-based health registries should be used in a search for co-morbid physical illness.
For geneticists, the proposed theory obviously points to linkage/association studies using inflammation genes; a few examples were cited previously [236-238]. A simple step with extant data might start with a meta analysis defining chromosomal "hot spots" for linkage with schizophrenia and search the gnome maps for immune regulators at these sites as Moises, et al [214] have done for glial growth regulators. Family, twin, and adoption methodologies can all be applied to the issue of co-morbid or co-segregating physical conditions.
The inflammatory/vascular theory has much to suggest to neuroimaging research especially in the realm of reinterpreting regional perturbations in metabolic activity as primary disturbances of flow regulation rather than intrinsic neuronal metabolic abnormalities. It would be interesting to assess the impact of vasoactive compounds and inflammatory modulators on neuroimaging studies of regional blood flow. Likewise, further pursuit of neuroimaging evidence of disrupted blood brain barrier, as initiated by Dysken, et al [252], and with manipulation of inflammatory systems as suggested by Mueller and Ackenheil [253] would test our hypothesis.
The neuropathology of schizophrenia, focused mostly on the neurons, is notable for inconsistencies in findings (see [51,254] for reviews). Such inconsistency is exactly what would be predicted by an inflammatory/vascular theory where the lesions are truly functional in the sense that the function of the brain alters in relation to perturbations in blood flow regulation. Only the more prolonged and serious inflammation will leave visible traces of neuronal damage and such damage may be patchy and inconsistent from one patient to another. However, over the early years of CNS development, alterations in cellular organization or migration may result from disrupted angiogensis that must go hand in hand with neuronal and glial development. The location and extent of CNS change will be a function of severity of inflammation and timing during development. Such consequences will be hard to demonstrate in human post-mortem tissues and animal or in vitro models may be more fruitful areas for study the effects of inflammation on neurogenesis and blood flow regulation. To our knowledge, human post mortem studies have not utilized vascular cast methodology and this should be considered, perhaps casting one half of a specimen brain while subjecting the other half to cellular analysis.
Specificity
Because of our interests and expertise, we have focused our attention on schizophrenia as the behavioral phenotype resulting from inflammatory-vascular pathology but the theory presented here is likely to be more general. Indeed, our use of examples of psychoses associated with known inflammatory- vascular pathologies (e.g. autoimmune CNS vascular disease or infectious CNS vascular disease as seen in syphilis) makes it clear that a vascular-inflammatory theory may apply to a wide range of psychotic conditions that may also include psychoses associated with mood disorders. Whereas, the classical genetic studies support the separateness of schizophrenia and mood disorders [255], there are modern molecular signs that schizophrenia and mood disorders share genetic elements in common [256,257]. Furthermore, mood disorders, like schizophrenia, show evidence of frontal lobe pathology, enlarged ventricles, abnormal cerebral blood flow [33,258] and vascular abnormalities [124]. To what extent all of these changes are epiphenomena of being psychotic (treatment effects or stress, etc) remain debatable [259].
However, finding similar brain changes in a variety of psychotic conditions does not necessarily mean these changes are epiphenomena. Examples from neuropsychiatry teach us that the underlying pathology does not necessarily define the behavioral symptoms. Thus, psychoses with Huntington disease may be affective-like or schizophreniform. Similar pathophysiological mechanisms may underlie a variety of psychotic phenotypes. The evolution of behavioral symptoms for any given pathophysiology may depend on a variety of moderating variables such as an individual's developmental age when the disease process begins, gender, hormones, genetic 'landscape' upon which the disease process unfolds, along with the nature, frequency, and intensity of successive triggers of inflammatory response.
Reprise
A broad spectrum of observations leads to a working hypothesis that schizophrenia and, possibly, other psychiatric syndromes are the result of genetically mediated inflammatory reactions that damage the neuron-glial-capillary triad with resultant loss of ability to fine tune regional brain metabolism. This hypothesis incorporates genetic, epigenetic [260], and environmental factors. Furthermore, an inflammatory/vascular theory can explain the variety of behavioral symptoms seen in schizophrenia, the variable course of the illness, and the numerous other puzzling observations such as an excess of minor physical anomalies. Should this theory prove heuristic, it would point to the use of inflammatory modulators in treating the illness. Perhaps more importantly, identifying individuals who were at high risk for the disorder in high genetic risk families as well as the general population, because of abnormalities of their inflammatory systems, holds hope for prevention through early intervention using inflammatory modulators.
List of abbreviations
ADHD attention deficit hyperactivity disorder
BDNF brain derived neurotropic factor
CBF cerebral blood flow
CNS central nervous system
DZ dizygotic
FMF familial Mediterranean fever
MZ monozygotic
NGF nerve growth factor
NO nitric oxide
PANDAS pediatric autoimmune neurological disorder associated with strep.
RF rheumatic fever
RHD rheumatic heart disease
VEGF vascular endothelial growth factor
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
This article was the joint effort of both authors with input as noted below.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This work was supported by a grant to DRH from The Stanley Medical Research Institute. We gratefully express our appreciation for the important suggestions by N. Mueller and H. K. Manji in their reviews of an earlier version of this manuscript. All remaining deficiencies remain the responsibility of the authors.
Figures and Tables
Figure 1 Simplified schematic illustrating the interconnected vascular-glial-neuron triad and how inflammatory processes may disrupt normal function.
Figure 2 Schematic illustration of how inflammatory processes, from conception onward, may lead to CNS damage or dysfunction that dynamically alters the epigenetic landscape (reaction surface) thus affecting the liability for developing schizophrenia. Blue planes intersecting the reaction surface indicate levels of liability above which symptoms become manifest. Measurable factors in the middle of the figure are good candidates for endophenotypes. Adapted from [261,262].
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| 15707482 | PMC554096 | CC BY | 2021-01-04 16:03:33 | no | BMC Med Genet. 2005 Feb 11; 6:7 | utf-8 | BMC Med Genet | 2,005 | 10.1186/1471-2350-6-7 | oa_comm |
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BMC Cell BiolBMC Cell Biology1471-2121BioMed Central London 1471-2121-6-61571323510.1186/1471-2121-6-6Research ArticleMammalian cells lack checkpoints for tetraploidy, aberrant centrosome number, and cytokinesis failure Wong Connie [email protected] Tim [email protected] Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA2 Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA2005 15 2 2005 6 6 6 26 9 2004 15 2 2005 Copyright © 2005 Wong and Stearns; licensee BioMed Central Ltd.2005Wong and Stearns; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Mammalian cells have been reported to have a p53-dependent tetraploidy checkpoint that blocks cell cycle progression in G1 in response to failure of cell division. In most cases where the tetraploidy checkpoint has been observed cell division was perturbed by anti-cytoskeleton drug treatments. However, other evidence argues against the existence of a tetraploidy checkpoint. Cells that have failed to divide differ from normal cells in having two nuclei, two centrosomes, a decreased surface to volume ratio, and having undergone an abortive cytokinesis. We tested each of these to determine which, if any, cause a G1 cell cycle arrest.
Results
Primary human diploid fibroblasts with intact cell cycle checkpoints were used in all experiments. Synchronized cells exhibited G1 arrest in response to division failure caused by treatment with either cytochalasin or the myosin II inhibitor blebbistatin. The role of tetraploidy, aberrant centrosome number, and increased cell size were tested by cell/cell and cell/cytoplast fusion experiments; none of these conditions resulted in G1 arrest. Instead we found that various drug treatments of the cells resulted in cellular damage, which was the likely cause of the arrest. When cytokinesis was blocked in the absence of damage-inducing drug treatments no G1 arrest was observed.
Conclusions
We show that neither tetraploidy, aberrant centrosome number, cell size, nor failure of cytokinesis lead to G1 arrest, suggesting that there is no tetraploidy checkpoint. Rather, certain standard synchronization treatments cause damage that is the likely cause of G1 arrest. Since tetraploid cells can cycle when created with minimal manipulation, previous reports of a tetraploidy checkpoint can probably be explained by side effects of the drug treatments used to observe them.
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Background
Cell cycle checkpoints preserve genome integrity by monitoring the fidelity of DNA replication and segregation. In mammalian somatic cells, the best-characterized checkpoints are the DNA damage/replication checkpoints and the mitotic spindle checkpoint. The DNA damage/replication checkpoints result in cell cycle arrest if DNA is not fully replicated, or is damaged [1]. The mitotic spindle checkpoint results in cell cycle arrest prior to anaphase if the spindle is not properly assembled [2].
There is also evidence that defects in events relating to cell division itself can result in cell cycle arrest. Lanni and Jacks [3] and Casenghi et al.[4] found that mammalian cells that had adapted to microtubule depolymerization and exited mitosis without undergoing cytokinesis arrested in G1 of the subsequent cell cycle. Kurimura and Hirano [5] and Andreassen et al. [6] reported that inhibition of cytokinesis with the actin-depolymerizing drug cytochalasin resulted in a similar arrest in G1 of the subsequent cell cycle. These treatments resulted in cells that were tetraploid, and Andreassen et al. [6] proposed that the cell cycle arrest was triggered by ploidy, terming this effect a "tetraploidy checkpoint".
Other evidence suggests that mammalian cells are not sensitive to tetraploidy. Rao and Johnson used cell fusion to study the regulation of DNA synthesis and mitosis by fusing cells at different cell cycle stages [7,8]. Binucleate tetraploid cells resulting from fusion between cells in different cell cycle stages were able to progress through the cell cycle. Uetake and Sluder ([9], reviewed in [10]) reported that inhibition of cytokinesis with a low dose of cytochalasin also allowed cell cycle progression. Most strikingly, there are rare cases of human infants born with fully tetraploid karyotypes [11]. Although these individuals have severe defects, their existence argues against tetraploidy as a trigger for cell cycle arrest.
Here we investigate whether tetraploidy or other cellular defects in binucleate cells lead to cell cycle arrest. We show that neither tetraploidy, aberrant centrosome number, cell size, nor failure of cytokinesis lead to G1 arrest, suggesting that there is no tetraploidy checkpoint. Rather, certain standard synchronization treatments cause DNA damage that is the likely cause of G1 arrest.
Results and discussion
Immortalized cell lines often have altered checkpoints, therefore we used early passage primary cells to investigate the tetraploidy checkpoint. All experiments were performed with human diploid fibroblasts (HDF) from infant foreskin and used prior to passage 10. We had previously developed methods for synchronizing these cells [12], and tested them here for the presence of normal checkpoint mechanisms. First, the levels of p53 were determined by western blotting and found to be similar to other p53+/+ cell lines (not shown). Second, we tested for a functional DNA damage response. G1 phase HDF cells were released from serum starvation and irradiated with ultraviolet (UV) light. The cells were then assayed for entry into S phase by 5-bromodeoxyuridine (BrdU) incorporation. The HDF cells exhibited a normal DNA damage response; at a low dose of UV, cells were delayed by about 12 h for entry into S phase, and at a higher dose most cells did not enter S phase even 36 h after irradiation (Figure 1A). Third, we tested for a functional spindle checkpoint. Exponentially-growing HDF cells were treated with nocodazole for 12 h to depolymerize microtubules, and assayed by light microscopy. Nocodazole treatment caused a 6-fold increase in the mitotic index, indicating that the cells had a functional spindle checkpoint.
Figure 1 Cell cycle responses of human diploid fibroblast (HDF) cells. (A) Response to DNA damage. HDF cells were exposed to 0, 10 or 20 J/m2 ultraviolet light and entry into S phase was assayed by BrdU incorporation. For each bar n ≥ 300 cells. (B) Recovery from nocodazole arrest. HDF cells were arrested in mitosis by double thymidine block followed by nocodazole (left) and released for 30 min. (center and right). DNA, blue; α-tubulin, green; γ-tubulin, red. (C) Example of binucleate cells created by cytochalasin-induced cytokinesis failure. DNA, blue. (D) Cell cycle progression of HDF cells in response to cytokinesis failure induced with 2 μM cytochalasin. Cells were assayed for BrdU incorporation at the indicated times after removal of cytochalasin. "control" cells were not treated with cytochalasin; "cytochalasin (mononucleate)" cells were treated, but completed cytokinesis, and "cytochalasin (binucleate)" cells were treated and failed to divide in cytokinesis. For each bar n ≥ 300 cells. (E) Cell cycle progression of HDF cells in response to cytokinesis failure induced with 12.5 μM blebbistatin. Cells were assayed for BrdU incorporation at the indicated times after removal of blebbistatin. "control" cells were not treated with blebbistatin; "blebbistatin (mononucleate)" cells were treated, but completed cytokinesis, and "blebbistatin (binucleate)" cells were treated and failed to divide in cytokinesis. For each bar n ≥ 300 cells. (F) Cell cycle progression in response to the presence of extra centrosomes. Image shows the product of fusion between a G1 cell and a G1 cytoplast. 24 h after fusion this cell has four centrosomes, indicating that it has undergone centrosome duplication, and has incorporated BrdU, indicating that it has entered S phase. DNA, blue; BrdU, green; pericentrin, red. Punctate blue staining is due to cell surface marker used to identify fusion products [12]. Scale bars represent 10 μm.
We first tested HDF cells for the previously described G1 arrest following cytochalasin-induced failure of cytokinesis [6]. Cells were synchronized in mitosis by double thymidine arrest followed by nocodazole treatment (Figure 1B), then released for 30 min, after which most cells had a bipolar spindle. Cells were then allowed to proceed into interphase in medium containing BrdU, +/- 2 μM cytochalasin. By 10 h after the addition of cytochalasin, both control and cytochalasin-treated cells had exited mitosis; approximately 30% of the cytochalasin-treated cells had two nuclei (binucleate) (Figure 1C) and the remainder had a single nucleus (mononucleate), presumably having completed cytokinesis successfully. Thus there were two types of control cells in these experiments: cells that had not experienced the drug, and cells that had experienced the drug, but remained mononucleate. The cultures were washed at this point to remove drug and allowed to proceed in the cell cycle.
At 6 h after the removal of cytochalasin, 50% of the untreated control cells had entered S phase, whereas only about 10% of either the mononucleate or binucleate cytochalasin-treated cells had entered S phase (Figure 1D); these numbers changed only slightly by 12 h. However, at 24 h after cytochalasin removal, 75% of the control cells and 44% of the mononucleate cytochalasin-treated cells had entered S phase, whereas only 11% of the cytochalasin-treated binucleate cells had entered S phase. Similar results were obtained with 5 μM and 10 μM cytochalasin (not shown). Thus, binucleate HDF cells resulting from cytochalasin-induced failure of cytokinesis did arrest in G1, as previously described for other cells [6].
A potential problem with cytochalasin treatment is that depolymerization of the actin cytoskeleton is likely to have effects other than blocking cytokinesis. Indeed, we found that even at 2 μM, cytochalasin had a strong cytotoxic effect, delaying cell cycle progression significantly, with slow recovery after release (data not shown and [13]). To determine whether the effect was specific to cytochalasin, the above experiment was repeated using two other drugs that inhibit cytokinesis: blebbistatin and aurora kinase inhibitor-1 (AKI-1). Blebbistatin is an inhibitor of non-muscle myosin II, the motor protein that provides the force for furrow ingression during cytokinesis [14]. AKI-1 inhibits the aurora family of kinases, which play important roles in mitosis and cytokinesis [15].
HDF cells were synchronized in mitosis by double thymidine block followed by nocodazole treatment, then released into medium containing BrdU, +/- 12.5 μM blebbistatin. By 10 h after the addition of blebbistatin, most cells had exited mitosis; in the presence of blebbistatin approximately 30% of the cells were binucleate and the remaining cells were mononucleate, presumably completing cytokinesis successfully. Blebbistatin was removed, and cells were assayed for S phase entry over time.
At 6 h after the removal of blebbistatin, 44% of the untreated control cells and 53% of the mononucleate blebbistatin-treated cells had entered S phase, whereas only 18% of the binucleate blebbistatin-treated cells had entered S phase (Figure 1E). By 24 h the fraction of both untreated and blebbistatin-treated mononucleate cells that had entered S phase rose to about 70%, whereas the fraction of binucleate cells that had entered S phase remained at about 20% (Figure 1E). Similar results were obtained with 25 μM and 50 μM blebbistatin, as well as with 5 μM AKI-1 (not shown). This indicates that synchronized mitotic cells that failed cytokinesis became arrested in G1 regardless of the specific inhibitor used.
Cells that have failed to divide after mitosis differ from normal cells in that they have two nuclei, two centrosomes, and a decreased surface area to volume ratio. We tested each of these defects individually for an effect on G1 arrest. To test the role of centrosome number, serum-starved G0 cells were fused with enucleated G0 cytoplasts to create cells with two centrosomes, but only one diploid nucleus (Figure 1F). The cell-cytoplast fusions were released from G0 into BrdU-containing medium and allowed to proceed through the cell cycle. The fused cells were compared to cells in the population that had experienced the fusion treatment but had not fused. At 24 h after fusion, 66+/-15% of cytoplast-cell fusions with an extra centrosome had entered S phase, and 63+/-11% of unfused control cells had entered S phase. Therefore the presence of an extra centrosome at G1 does not delay S phase entry and is not responsible for the G1 arrest in binucleate cells resulting from cytochalasin-induced failure of cytokinesis.
To test the role of tetraploidy, serum-starved HDF cells were fused to create binucleate cells. Creating binucleate cells by fusion avoided disruption of the actin cytoskeleton, allowing us to examine the effect of ploidy alone. The binucleate cells resulting from fusion were both tetraploid and had two centrosomes; we showed above that centrosome number was not a factor in the G1 arrest. As above, unfused cells in the population served as an internal control. At 24 h after fusion, 72+/-2% of the unfused cells and 75+/-1% of the fused, binucleate, cells had entered S phase. Therefore, tetraploidy does not cause the observed G1 arrest resulting from cytochalasin-induced failure of cytokinesis.
Cells that fail to divide at cytokinesis are larger than normal cells. Larger cells have a decreased surface area to volume ratio, which might affect the response to perturbation of the cytoskeleton. Thus, the apparent sensitivity to cytokinesis failure might derive directly from a difference in size. To test this, we created large binucleate cells by fusing serum-starved G0 cells to each other. The fusion products were released into growth medium for 3 h to allow for reattachment to the culture substrate. We then added 25 μM blebbistatin, 5 μM cytochalasin, or 5 μM AKI-1 to cells for 10 h, followed by release into BrdU-containing medium. Figure 2 shows that mononucleate and binucleate cells in the control and drug-treated populations entered S phase with similar kinetics. Note that in the cells treated with cytochalasin there was a significant delay in S phase entry, consistent with the cytotoxicity of cytochalasin that we and others have described [13]. These results demonstrate that binucleate cells are not more sensitive to cytokinesis inhibitors due to their increased size.
Figure 2 Cytokinesis inhibitors do not block the G1 to S phase progression of binucleate HDF cells. Serum-starved G0 cells were fused and released into medium containing BrdU and (A) no drug, (B) 25 μM blebbistatin, (C) 5 μM AKI-1, or (D) 5 μM cytochalasin. Some cells remain unfused after the fusion treatment, and were used as mononucleate controls. Time points were taken to assay for S phase entry.
If cells were sensitive to failure of cytokinesis, one might expect that the sensitivity would be expressed as a delay in exit from mitosis, a time when a cytokinetic defect could be corrected. This would be similar to the known DNA damage, DNA replication and spindle assembly checkpoints [16]. We tested in two ways whether mammalian cells delay the exit from mitosis in response to cytokinesis failure. First, HDF cells were imaged by time-lapse microscopy as they progressed through mitosis in the presence or absence of blebbistatin. Cells were synchronized in mitosis by nocodazole treatment, then released for 30 min, when 25 μM blebbistatin was added. Control cells (n = 5) exhibited cytokinetic constrictions beginning about 60 min after release from nocodazole. These cells flattened and began to spread, signaling the end of mitosis, about 85 min after release (Figure 3A). Blebbistatin-treated cells (n = 9) did not undergo observable cytokinesis, as expected, but did flatten and spread about 110 min after release from nocodazole (Figure 3A). We also tested for a delay in mitotic exit by staining with MPM-2, and antibody specific for mitotic phosphoepitopes [17]. Cells that were MPM-2 positive and had condensed DNA were considered to be in mitosis (Figure 3B). After release from nocodazole arrest, the fraction of mitotic cells declined in both the control and blebbistatin-treated populations with only a slight delay apparent in the blebbistatin-treated cells (Figure 3C). Both assays showed that blebbistatin treatment resulted in only a brief delay in the exit from mitosis, suggesting that failure of cytokinesis does not trigger a checkpoint-like arrest.
Figure 3 Cytokinesis failure does not significantly delay the exit frommitosis. (A) Images from time-lapse series of HDF cells at the indicated times after release from nocodazole-mediated mitotic arrest. "Control" cells were not treated with blebbistatin; "Blebbistatin" cells were treated with blebbistatin beginning at 30 min after release from nocodazole. (B) MPM-2 immunofluorescence as a marker for mitotic exit. Fluorescence image of a mitotic cell with condensed DNA and intense MPM-2 staining (top) and a cytokinetic cell with decondensed DNA and diminished MPM-2 staining (bottom). DNA, blue; MPM-2, green. Scale bar represents 10 μm. (C) Mitotic index of control and blebbistatin-treated cells after mitotic release. "Control" cells were not treated with blebbistatin; "Blebbistatin" cells were treated with blebbistatin beginning at 30 min after release from nocodazole. Mitotic index was determined by MPM-2 staining and DNA morphology, as in (B). For each point n = 100 cells.
Since we had ruled out most of the cellular defects associated with division failure as being the cause of the G1 arrest, we attempted to further characterize the arrest. Andreassen et al. [6] reported that p53 is important in the G1 arrest caused by cytokinesis failure. To test the role of the p53 pathway, we repeated the blebbistatin experiment above with wt, p53 -/-, and p21 -/- mouse embryonic fibroblast (MEF) cells. Wt MEF cells behaved similarly to the HDF cells, arresting in G1 in response to cytokinesis failure (Figure 4A). However, in p53 -/- and p21 -/- MEF cells both binucleate and mononucleate cells entered S phase with the same kinetics (Figure 4A). Thus, the p53-p21 pathway is required for the G1 arrest of binucleate cells.
Figure 4 Cells arrested in G1 by cytokinesis failure enter p53- and p21-dependent premature senescence. (A) p53 pathway dependence of the G1 arrest following cytokinesis failure. Wt, p53-/- and p21-/- mouse embryo fibroblasts were synchronized and treated with blebbistatin and assayed as in Figure 1E. For each bar n ≥ 200 cells. (B) G1 arrested binucleate cells entered premature senescence as assayed by senescence-associated β-galactosidase activity (SA-β-gal). The dark stain in the binucleate cell shown is the reaction product of Xgal cleavage. Scale bar represents 10 μm. (C) Time course of appearance of senescent cells. Mononucleate and binucleate cells were assayed for SA-β-gal at the indicated days after blebbistatin removal. For each point n = 60–100 cells.
p53-p21-dependent G1 arrest often results in either apoptosis or senescence [18,19]. To determine the fate of the G1-arrested products of a failed cytokinesis, binucleate HDF cells were prepared using blebbistatin as described above. Blebbistatin was then removed and the cells were assayed by microscopy. The binucleate cells persisted in the population over the course of more than two weeks, consistent with these cells being permanently arrested in the cell cycle. The binucleate cells did not undergo apoptosis, as assayed by morphology and staining with annexin V, an early marker of apoptosis (not shown). However, the binucleate cells did develop several hallmarks of senescence, including becoming flattened and enlarged, and accumulating senescence-associated β-galactosidase activity (Figure 4B). As the criteria for defining cellular senescence are not firmly established [20], we will refer to this phenotype as "senescent-like". At 4 days after blebbistatin removal approximately 35% of binucleate cells and 10% of mononucleate cells were senescent-like; by 12 days virtually all of the binucleate cells, but only 10% of mononucleate cells, were senescent-like (Figure 4C).
We have shown that failure of division of synchronized cells results in a p53-dependent arrest, but that the arrest is not due to ploidy, centrosome number, or cell size, and that the arrest is not preceded by a delay in mitotic exit, suggesting that it is not a classical checkpoint. The characteristics of the arrest are similar to those of the G1 arrest caused by the DNA damage checkpoint in HDF cells, which respond to irreparable DNA damage by entering senescence, instead of apoptosis [21]. These similarities led us to test whether the binucleate G1 arrest might actually be due to DNA damage suffered during the treatment. Cells were synchronized in mitosis by the double thymidine – nocodazole regimen described above and treated with 25 μM blebbistatin. The binucleate cells were released from blebbistatin for 1 h, 3 days, and 8 days respectively, then stained for γ-H2AX, a marker of DNA damage [22]. As a positive control for DNA damage, asynchronous cells were treated with 1 mM hydrogen peroxide for 30 min, allowed to recover in medium for 1 h, and stained for γ-H2AX.
In the untreated control cells, only 3.4% of cells contained γ-H2AX foci, whereas in the hydrogen peroxide treated cells, 33% of cells contained γ-H2AX foci (Figure 5A). Remarkably, at 1 h after release of synchronized cells from blebbistatin, 52% of the binucleate cells contained γ-H2AX foci in one or both nuclei (Figure 5A), suggesting the presence of DNA damage. However, we found that 32% of the mononucleate cells that successfully completed cytokinesis after blebbistatin treatment also contained γ-H2AX foci. This suggested that the observed DNA damage might not be the result of division failure per se, and therefore might have occurred prior to the addition of blebbistatin, possibly during cell synchronization. At 3 days after release from blebbistatin, 30% of the binucleate cells and 11% of the mononucleate cells contained visible γ-H2AX foci. At 8 days after release from blebbistatin, 29% of the binucleate cells and only 6% of the mononucleate cells contained γ-H2AX foci. Most of the binucleate cells also displayed senescent-like phenotypes at 8 days after the removal of blebbistatin (Figure 5B).
Figure 5 Binucleate cells contain nuclear γ-H2AX foci. (A) Binucleate cells were prepared by synchronization and treatment with 25 μM blebbistatin as described above, then stained for the DNA damage marker γ-H2AX. Untreated control cells (top) did not contain any visible γ-H2AX foci, whereas binucleate cells, after released from blebbistatin for 1 h, (bottom) contained γ-H2AX foci that were similar to those of cells treated with H2O2 (middle). Scale bar represents 2.5 μm. (B) Culture was continued for 8 days after release from blebbistatin. Most mononucleate cells lacked γ-H2AX foci (top), whereas approximately 30% of binucleate cells still contained nuclear γ-H2AX foci (bottom). The binucleate cells were also flattened and enlarged, consistent with a senescent-like arrest. Scale bar represents 10 μm.
The binucleate cells that persisted in culture were arrested in G1, as they did not incorporate BrdU after the previous round of mitosis, and they did not proceed to the next round of mitosis, as evidenced by the preservation of the binucleate phenotype. The presence and persistence of nuclear γ-H2AX foci in the G1-arrested binucleate cells suggested that DNA damage might be the cause of the arrest. However, not all the arrested binuclear cells contained visible γ-H2AX foci, indicating that γ-H2AX-associated DNA damage might not be the only cause of the arrest. The percentage of binucleate cells with nuclear γ-H2AX decreased from 52% to 29% over 8 days of culturing, possibly indicating that some cells were able to correct the DNA damage after being arrested in G1 for several days. In contrast, the percentage of mononucleate cells that displayed γ-H2AX foci decreased dramatically over 8 days of culturing, however this was likely due to proliferation of normal mononucleate cells in the culture rather than a difference in response of the mononucleate and binucleate cells to the treatment.
The presence of γ-H2AX foci in the mononucleate cells that successfully completed cytokinesis after blebbistatin treatment suggested that the DNA damage might have been the result of the synchronization treatments, prior to the addition of blebbistatin. Therefore, we tested whether any of the cell synchronization treatments alone had an effect on cell cycle progression. Asynchronous cells were treated with double thymidine block, nocodazole, or blebbistatin individually, following the same protocols used above in multiple treatments. Cells were released from drug and S phase entry was assayed at time points. Figure 6A shows that none of the drug treatments resulted in a substantial failure of cell cycle progression after release. Most importantly, we found that most of the binucleate cells that resulted from cytokinesis failure with blebbistatin treatment alone were able to enter S phase normally after release. This result indicates that there is no cytokinesis checkpoint, in accord with the results of Uetake and Sluder [9].
Figure 6 The combination of double thymidine block and nocodazole treatment causes DNA damage in HDF cells (A) Asynchronous HDF cells were treated with either double thymidine, nocodazole, or blebbistatin individually, then released into BrdU-containing growth media and assayed for S phase entry. For each bar n ≥ 200 cells. (B) Cells were subjected to the treatments in the order double thymidine, nocodazole, blebbistatin. Samples of cells were taken after release from each drug, and S phase entry was assayed. For each bar n ≥ 200 cells. (C) Cells were treated with double thymidine block followed by nocodazole, then stained for γ-H2AX. Scale bar represents 2.5 μm.
Since none of the single drug treatments resulted in a cell cycle arrest, we reasoned that some combination of the treatments must be responsible. To determine which combination of treatments caused a G1 arrest, cells were subjected to the treatments in the order double thymidine, nocodazole, blebbistatin. Samples of cells were taken after release from each drug, and S phase entry was assayed (Figure 6B). Although neither double thymidine nor nocodazole arrest and release alone resulted in a G1 arrest, the combination of them did; only about 45% of such cells progressed into S phase. The addition of blebbistatin to the treatment did not cause a further reduction in the fraction of cells entering S phase; about 64% of the mononucleate cells progressed into S phase. However, only about 22% of the binucleate cells that failed cytokinesis after blebbistatin treatment progressed into S phase. This indicated that the G1 arrest in our experiments is due to the double thymidine block, followed by nocodazole treatment, and that binucleate cells are more susceptible to this effect.
To determine whether the thymidine – nocodazole combination caused the DNA damage we observed, cells were treated with both drugs as above and stained for γ-H2AX. At 3 h after release from the drug treatments, about 33% of cells had γ-H2AX foci (Figure 6C). Thus the thymidine – nocodazole synchronization treatment caused the DNA damage that resulted in cells becoming arrested in G1. Given these results we suggest a simple model for the increased susceptibility of binucleate cells: those cells that failed cytokinesis are more likely to become arrested in G1 because they contain two nuclei, and thus have twice the chance of inheriting DNA damage compared with cells that successfully divided.
Conclusions
We have shown that tetraploidy, aberrant centrosome number, increased cell size, and failure of cytokinesis do not lead to G1 arrest in primary human diploid fibroblasts. Rather, we found that the observed G1 arrest in cells that have failed to divide is likely due to cellular damage caused by standard synchronization treatments. We note that all published observations of a G1 arrest in response to division failure involved extensive manipulation of mammalian cells in culture. It seems likely that these manipulations resulted in DNA damage, or in other damage, that resulted in a G1 arrest, but was not directly associated with division failure. For example, Uetake and Sluder [9] found that supplementing the culture substrate with fibronectin allowed binucleate cells formed by cytochalasin treatment to progress through the cell cycle, suggesting that cell adhesion was defective in the drug-treated cells. Given that binucleate cells clearly can cycle when formed with minimal manipulation, it is likely that all previous reports of a tetraploidy checkpoint can be explained by side effects of the drug treatments used to observe them.
Methods
Cell methods
Human diploid fibroblasts (HDFs) were from infant foreskin. Wt, p53 -/- and p21 -/- mouse embryo fibroblasts (MEFs) were the kind gift of Laura Attardi (Stanford, CA). HDFs and MEFs were cultured in DMEM (Gibco) with 10% fetal bovine serum. HDFs were used prior to passage 10 and MEFs were used prior to passage 5. HDFs were synchronized in G0 by serum starvation [12] and S phase by double thymidine block [23], as described. In the cell fusion experiments, serum-starved G0 cells, or cytoplasts derived from those cells by centrifugation, were fused with serum-starved G0 cells, as described [12]. Immunocytochemistry was as described [12]. Live cell imaging was with a Nikon Diaphot microscope equipped with an environmental chamber allowing incubation at 37°. Images were collected with a CCD camera (Photometrics) and processed with Openlab (Improvision) and Photoshop (Adobe) software. Senescence-associated β-galactosidase activity (SA-β-gal) was assayed as described [24].
Assay for S phase entry by BrdU incorporation
Cells were incubated with 20 μM BrdU (Sigma) for indicated times and fixed in -20°C methanol for at least 10 min. Fixed cells were treated with DNase I (Boehringer Mannheim) and exonuclease III (New England Biolabs) to expose the BrdU epitope prior to incubation with anti-BrdU antibodies, as described [12]. Nuclei were visualized by staining 4',6-diamidino-2-phenylindole (DAPI). Cells were observed with a Zeiss Axioskop microscope with a Zeiss Plan-Neofluar 100/1.3 objective, and images were collected with a cooled-CCD camera (Hamamatsu) controlled by Openlab software.
Assay for DNA damage by γ-H2AX staining
Cells were fixed with 2% paraformaldehyde at room temperature for 10 min, washed 3× with PBS, then permeabilized with-20°C methanol for 5 min and stained with 27 ng/ml γ-H2AX antibody (Trevigen, MD). Only cells with multiple, clearly labeled foci were counted as being γ-H2AX positive.
Drug-induced cytokinesis failure
Cells synchronized in S phase by double thymidine block were released from the block for 6 h to allow completion of S phase. Nocodazole (100 ng/ml) (US Biological) was then added for 6 h to arrest the cells in mitosis. Cells were released from the mitotic arrest for 30 min, during which time most cells formed a bipolar mitotic spindle (Fig 1B). At 30 min after release from mitotic arrest, 20 μM BrdU was added to the medium, together with the indicated concentration of cytochalasin B (Sigma) or (s)-(-)-blebbistatin (Toronto Research Chemicals). Cells were incubated in this medium for 10 h to inhibit cytokinesis, then changed to growth medium containing 20 μM BrdU but no cytokinesis inhibitor, and assayed for S phase entry at the indicated times.
Authors' contributions
CW participated in the design of the study and carried out the experiments. TS conceived of the study and participated in its design.
Acknowledgements
We thank Laura Attardi for insightful discussion about cellular senescence, Aaron Straight for an initial gift of blebbistatin, and Tarun Kapoor for aurora kinase inhibitor. This work was supported by NIGMS grant GM52022 to T.S.
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| 15713235 | PMC554097 | CC BY | 2021-01-04 16:39:11 | no | BMC Cell Biol. 2005 Feb 15; 6:6 | utf-8 | BMC Cell Biol | 2,005 | 10.1186/1471-2121-6-6 | oa_comm |
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BMC GenetBMC Genetics1471-2156BioMed Central London 1471-2156-6-101572071810.1186/1471-2156-6-10Research ArticleA novel replication-independent histone H2a gene in mouse Nishida Hiromi [email protected] Takahiro [email protected] Yasuhiro [email protected] Yoshihide [email protected] Laboratory for Genome Exploration Research Group, RIKEN Genomic Sciences Center (GSC), RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan2005 19 2 2005 6 10 10 7 10 2004 19 2 2005 Copyright © 2005 Nishida et al; licensee BioMed Central Ltd.2005Nishida et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
An uncharacterized histone H2a-coding transcript (E130307C13) has been cloned from a mouse full-length cDNA library. This transcript is encoded on chromosome 6, approximately 4 kb upstream of a histone H4 gene, Hist4h4. The proteins encoded by this transcript and the human H2afj mRNA isoform-2 have the highest amino acid similarity. In this paper, we characterize it from the expression pattern given by quantitative RT-PCR.
Results
Quantitative RT-PCR indicated that the gene that encodes E130307C13 (E130307C13) is regulated in a replication-independent manner, and therefore it is H2afj. Certainly, H2afj transcript lacks a stem-loop structure at the 3'-UTR but contains a poly (A) signal. In addition, its promoter region has a different structure from those of the replication-dependent histone H2a genes.
Conclusion
The bioinformatics imply that E130307C13 is a replication-independent H2a gene. In addition, quantitative RT-PCR analysis shows that it is replication-independent. Thus, it is H2afj, a novel replication-independent H2a gene in mouse.
==== Body
Background
Replication of the eukaryotic chromosomes requires the synthesis of histones to package the newly replicated DNA into chromatin. Control of the level of histone mRNA accounts for much of the control of histone protein synthesis [1]. Mouse has 18 replication-dependent histone H2a genes [2]. Among these 18 genes, 13 genes are located in the Hist1 cluster on chromosome 13, 4 in the Hist2 cluster on chromosome 3 and 1 in the Hist3 cluster on chromosome 11 [2]. The histone mRNAs that are cell-cycle-regulated increase 35-fold as cells progress from mitosis through G1-phase and into S-phase [3]. The promoters of histone genes contain CCAAT and TATA boxes [4]. The replication-dependent histone H2a genes lack introns and a poly (A) signal. They have a conserved stem-loop structure (5'-GGCTCTTTTCAGAGCC-3') at the 3'-UTR, which plays an important role in mRNA processing and stability [5-7].
Mouse also has two replication-independent histone H2a genes, H2afx on chromosome 9 and H2afz chromosome 3. These two genes encode polyadenylated mRNAs. H2afx mRNA has both a polyadenylated tail and a stem-loop structure [8]. Mouse replication-dependent histone H2a mRNAs and H2afx mRNA have a nuclear export element (5'-ACAACAAGAAGACGCGCATCAT-3') in the protein-coding region that functions to export the mRNA from the nucleus to the cytoplasm [9].
An uncharacterized histone H2a-coding transcript (E130307C13, FANTOM clone ID; NM_177688, Genebank accession number) has been cloned from a mouse full-length cDNA library. E130307C13 lies on chromosome 6, approximately 4 kb upstream of Hist4h4. In this paper, we characterize it from the expression pattern given by quantitative RT-PCR. In addition to this, we compared the structure with the sequences deposited in the international DNA/protein database.
Results and discussion
Comparison of the putative amino acid sequence encoded by E130307C13 with the amino acid sequences deposited in the international DNA/protein database showed that it has the highest similarity to that encoded by human H2afj mRNA isoform-2 (NM_177925, Genebank accession number; Figs. 1, 2). Human has two isoforms of H2afj [10,11]. Isoform-1 (NM_018267, Genebank accession number) is produced after splicing of two introns; isoform-2 does not need intron splicing for maturation. Interestingly, H2afj also lies near a histone H4 gene, on human chromosome 12. According to the nomenclature of histone genes [2], if E130307C13 is regulated in a replication-dependent manner, it is recognized as Hist4h2a. But if it is regulated in a replication-independent manner, it is recognized as H2afj.
Figure 1 Phylogenetic relationships among 44 amino acids sequences from human and mouse histone H2a related proteins. The bar indicates 5% difference of sequence. The numbers at the branches indicate percentage of 1000 bootstrap analyses. (H) and (M) indicate the human sequence and mouse sequence, respectively.
Figure 2 Alignment of the human and mouse histone H2a proteins in Fig. 1.
Each product of the quantitative RT-PCR gave a single band on the agarose gel of the expected size (Fig. 3). Observation using the quantitative RT-PCR showed that the expression pattern of E130307C13 is typical of replication-independent histone gene (Table 1, Fig. 3). The expression pattern of E130307C13 is more similar to that of the replication-independent H2afz than to that of the replication-dependent Hist2h2aa2. The expression of Hist2h2aa2 increased along with cell cycle progression from the beginning of S-phase (0 h), peaked at 2 h, and then decreased (Fig. 3). On the other hand, E130307C13 and H2afz were expressed constantly (Fig. 3). These results suggest that E130307C13 is a replication-independent histone H2a gene in mouse. According to the nomenclature of histone genes [2], E130307C13 is recognized not as Hist4h2a but as H2afj.
Table 1 CT values, ΔCT, and ΔΔCT
Time (h) GAPDH E130307C13_1 ΔCT ΔΔCT Expression E130307C13_2 ΔCT ΔΔCT Expression Hist2h2aa_1 ΔCt ΔΔCT Expression Hist2h2aa_2 ΔCT ΔΔCT Expression H2afz ΔCT ΔΔCT Expression
0 14.4 19.1 4.65 0 1 20 5.61 0 1 18.1 3.63 0 1 19.3 4.9 0 1 16.8 2.35 0 1
1 14.4 19 4.64 -0.01 1.01 20.1 5.71 0.1 0.93 17.5 3.1 -0.53 1.44 19.2 4.79 -0.11 1.08 16.6 2.26 -0.09 1.06
2 14.4 18.9 4.54 -0.11 1.08 19.7 5.32 -0.29 1.22 16.8 2.42 -1.21 2.31 18.3 3.92 -0.98 1.97 16.7 2.33 -0.02 1.01
3 14.3 18.8 4.5 -0.15 1.11 19.7 5.4 -0.21 1.16 16.8 2.48 -1.15 2.22 18.3 3.92 -0.98 1.97 16.8 2.46 0.11 0.93
4 14.4 19.2 4.78 0.13 0.91 19.9 5.49 -0.12 1.09 17.2 2.79 -0.84 1.79 18.3 3.93 -0.97 1.96 16.8 2.43 0.08 0.95
5 14.4 19.2 4.82 0.17 0.89 20 5.61 0 1 17.1 2.66 -0.97 1.96 18.6 4.17 -0.73 1.66 16.8 2.42 0.07 0.95
6 14.1 19.8 5.69 1.04 0.49 20.4 6.33 0.72 0.61 18.4 4.29 0.66 0.63 19.9 5.8 0.9 0.54 16.8 2.71 0.36 0.78
7 14.2 19.7 5.45 0.8 0.57 20.3 6.11 0.5 0.71 17.6 3.35 -0.28 1.21 19 4.83 -0.07 1.05 16.9 2.7 0.35 0.78
8 14.3 19.2 4.87 0.22 0.86 20.1 5.71 0.1 0.93 17.6 3.3 -0.33 1.26 19.2 4.81 -0.09 1.06 16.9 2.55 0.2 0.87
9 14.3 19.3 5.03 0.38 0.77 20 5.76 0.15 0.9 17.8 3.54 -0.09 1.06 19.3 5.01 0.11 0.93 16.6 2.34 -0.01 1.01
10 14.2 19.1 4.83 0.18 0.88 19.8 5.58 -0.03 1.02 17.8 3.6 -0.03 1.02 18.7 4.5 -0.4 1.32 16.6 2.4 0.05 0.97
11 14.3 19.8 5.5 0.85 0.55 20.2 5.87 0.26 0.84 18.3 4 0.37 0.77 19.2 4.91 0.01 0.99 16.2 1.94 -0.41 1.33
Figure 3 RT-PCR products on agarose gel and expression patterns. Lanes 1, 100 bp ladder; 2, RT-PCR product amplified with E130307C13 primer set 2; 3, that with E130307C13 primer set 1; 4, that with Hist2h2aa2 primer set 1; 5, that with Hist2h2aa2 primer set 2; 6, that with H2afz primer set.
Upstream of the 5'-end of E130307C13, no TATA box was found. In addition, the first CCAAT box lies 230 bases upstream of the translation start codon (Fig. 4). The other replication-dependent H2a genes have the first CCAAT and TATA boxes within 100 bases upstream of the translation start codon (Fig. 4). The replication-independent genes H2afx and H2afz also have a TATA box (Fig. 4). Thus, histone H2a genes have a TATA box in the promoters, except for E130307C13. Interestingly, the promoter of H2afz lacks CCAAT box but includes TATA box, on the other hand, that of E130307C13 lacks TATA box but includes CCAAT box (Fig. 4).
Figure 4 Sequences between CCAAT and TATA boxes upstream of the histone H2a and E130307C13 coding regions. Underlines indicate CCAAT and TATA boxes. The ATGs located at the 3'-end indicate translation start codon. Numbers in parentheses represent numbers of bases not shown. The Hist1h2aj is a pseudogene, lacking a start codon.
In addition, the 3'-UTR of the E130307C13 mRNA does not include the conserved stem-loop structure (Fig. 5). But the E130307C13 mRNA has two poly (A) signals at the middle and near the 3'-end. It indicates that the E130307C13 gives rise to two differentially polyadenylated mRNA transcripts. Considering the position at the 3'-UTR, it has a possibility that the poly (A) signal near the 3'-end is functional. Except for the pseudogene Hist1h2aj, the replication-dependent H2a mRNAs have the conserved stem-loop structure at the 3'-UTR. H2afx gives rise to a cell-cycle-regulated mRNA ending in the stem-loop when it is transcribed during S-phase, and a polyadenylated mRNA that is present in G1-phase cells [2,8]. H2afz mRNA lacks the stem-loop structure and has poly (A) signals. H2afz contains four introns in the protein-coding region and needs a splicing mechanism to produce the mature mRNA. Interestingly, E130307C13 lacks introns. Comparing the transcript structure of E130307C13 with those of the other histone H2a genes suggests that E130307C13 has replication-independent characteristics.
Figure 5 Alignment of nuclear export elements and stem-loop structures. ATG indicates translation start codon. TAA or TGA indicates translation stop codon.
Conclusion
The bioinformatics imply that E130307C13 is a replication-independent H2a gene. In addition, quantitative RT-PCR analysis shows that it is replication-independent. Thus, it is H2afj, a novel replication-independent H2a gene in mouse.
Methods
Phylogenetic tree construction
Multiple alignment of 44 amino acids sequences of histone H2a related proteins from human and mouse was created using the CLUSTAL W [12] on the DNA Databank of Japan. The phylogenetic tree by the neighbor-joining method with 1000 bootstrap analyses was constructed based on the multiple alignment using MEGA version 2.1 [13].
Cell cycle synchronization
The cell cycle of Hepa 1–6 cells was synchronized at the end of G1-phase by the addition of thymidine-hydroxyurea. The cell cycle arrest was released by washing out the thymidine-hydroxyurea, then the cells were harvested at intervals of 1 h from 0 h to 11 h.
RNA extraction
Total RNA was extracted by using the RNeasy mini kit (Qiagen) according to the manual for the cell line. After that, each sample was treated with DNase I.
cDNA synthesis
RNA (approximately 0.5 μg) and random hexamer primers were heated to 70°C for 10 min, followed by cooling on ice for 5 min. The cDNA was synthesized using Superscript III First Strand buffer (Invitrogen) according to the manual. The reverse transcriptase was inactivated by a 15-min incubation at 70°C.
Quantitative PCR
The following primers were used: 5'-AACTGTAGCCCGGCCCG-3' and 5'-TTCGTCTGTTTGCGCTTT-3' (primer set 1, product size 100 bp) and 5'-CAACAAGCTGCTGGGCAAA-3' and 5'-TCGCCTTATGGTGGCTCTCC-3' (primer set 2, product size 101 bp) for transcripts of Hist2h2aa2; 5'-ACTCCGGAAAGGCCAAGACA-3' and 5'-GTTGTCCTAGATTTCAGGTG-3' for H2afz, product size 100 bp; 5'-CGTCCTGCCCAATATCCAG-3' and 5'-TCTGCACCCGTCTGTCG-3' (primer set 1, product size 90 bp) and 5'-AAGCAGGGCGGTAAGGTG-3' and 5'-TCCGCGTAGTTGCCCTTC-3' (primer set 2, product size 110 bp) for E130307C13; and 5'-TGTGTCCGTCGTGGATCTGA-3' and 5'-CCTGCTTCACCACCTTCTTGA-3' for GAPDH (glyceraldehyde-3-phosphate dehydrogenase), product size 76 bp. Quantification of GAPDH mRNA was used as a control for data normalization. PCR amplification was performed on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems). The PCR conditions were an initial step of 30 s at 95°C, followed by 40 cycles of 5 s at 95°C and 30 s at 60°C. The SYBR premix Ex Taq (Takara) was used according to the manual. Expression was assessed by evaluating threshold cycle (CT) values. The relative amount of expressed RNA was calculated using Livak and Schmittgen's method [14].
Authors' contributions
HN designed this study, carried out the molecular biological studies, and the molecular evolutionary studies. TS and YT carried out synchronization of cells and quantitative RT-PCR. YH participated in the design of this study.
Acknowledgements
We thank the two anonymous reviewers for their helpful comments. This work was supported in part by a Research Grant for the RIKEN Genome Exploration Research Project, a Research Grant for Advanced and Innovational Research Program in Life Science, and a Research Grant for the Genome Network Project from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of the Japanese Government, and in part by a Research Grant for CREST of Japan Science and Technology Corporation to Y.H., and supported in part by grant 15770055 from the MEXT to H.N.
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| 15720718 | PMC554098 | CC BY | 2021-01-04 16:38:18 | no | BMC Genet. 2005 Feb 19; 6:10 | utf-8 | BMC Genet | 2,005 | 10.1186/1471-2156-6-10 | oa_comm |
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BMC GenetBMC Genetics1471-2156BioMed Central London 1471-2156-6-81571590810.1186/1471-2156-6-8Research ArticleAn interactional network of genes involved in chitin synthesis in Saccharomyces cerevisiae Lesage Guillaume [email protected] Jesse [email protected] Charles A [email protected] Anne-Marie [email protected]énard Patrice [email protected] Shamiza [email protected] Amy Hin Yan [email protected] Charles [email protected] Howard [email protected] Department of Biology, McGill University, Montréal (PQ) H3A 1B1, Canada2 Department of Medicine, Boston University, Boston MA 02118, USA3 Banting and Best Department of Medical Research, University of Toronto, Toronto (ON) M5G 1L6, Canada2005 16 2 2005 6 8 8 20 10 2004 16 2 2005 Copyright © 2005 Lesage et al; licensee BioMed Central Ltd.2005Lesage et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
In S. cerevisiae the β-1,4-linked N-acetylglucosamine polymer, chitin, is synthesized by a family of 3 specialized but interacting chitin synthases encoded by CHS1, CHS2 and CHS3. Chs2p makes chitin in the primary septum, while Chs3p makes chitin in the lateral cell wall and in the bud neck, and can partially compensate for the lack of Chs2p. Chs3p requires a pathway of Bni4p, Chs4p, Chs5p, Chs6p and Chs7p for its localization and activity. Chs1p is thought to have a septum repair function after cell separation. To further explore interactions in the chitin synthase family and to find processes buffering chitin synthesis, we compiled a genetic interaction network of genes showing synthetic interactions with CHS1, CHS3 and genes involved in Chs3p localization and function and made a phenotypic analysis of their mutants.
Results
Using deletion mutants in CHS1, CHS3, CHS4, CHS5, CHS6, CHS7 and BNI4 in a synthetic genetic array analysis we assembled a network of 316 interactions among 163 genes. The interaction network with CHS3, CHS4, CHS5, CHS6, CHS7 or BNI4 forms a dense neighborhood, with many genes functioning in cell wall assembly or polarized secretion. Chitin levels were altered in 54 of the mutants in individually deleted genes, indicating a functional relationship between them and chitin synthesis. 32 of these mutants triggered the chitin stress response, with elevated chitin levels and a dependence on CHS3. A large fraction of the CHS1-interaction set was distinct from that of the CHS3 network, indicating broad roles for Chs1p in buffering both Chs2p function and more global cell wall robustness.
Conclusion
Based on their interaction patterns and chitin levels we group interacting mutants into functional categories. Genes interacting with CHS3 are involved in the amelioration of cell wall defects and in septum or bud neck chitin synthesis, and we newly assign a number of genes to these functions. Our genetic analysis of genes not interacting with CHS3 indicate expanded roles for Chs4p, Chs5p and Chs6p in secretory protein trafficking and of Bni4p in bud neck organization.
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Background
In vegetatively growing cells of Saccharomyces cerevisiae, chitin, a linear polymer of β-1,4-linked N-acetylglucosamine (GlcNAc) residues, is selectively concentrated at the bud neck and is also found as a minor component of the mature lateral cell wall. Chitin is also the main constituent of the primary septum, a structure that separates mother and daughter cells (for reviews, see [1-3]).
Polymerization of UDP-GlcNAc to chitin is catalyzed by a family of three membrane-associated chitin synthases (CS) with specialized activities. CSIII, encoded by CHS3, is responsible for synthesis of the chitin ring at the bud neck and for chitin in the lateral wall. CSII synthesizes the chitin of the primary septum, and is encoded by CHS2, a gene that is essential in many strain backgrounds [4]. CSI, encoded by CHS1, is localized to the plasma membrane and to chitosome vesicles [5] and mutants are hypersensitive to the chitin synthase inhibitor, polyoxyin D, and under acid conditions can form small aberrant buds that are prone to lysis [6]. Disruption of the chitinase gene CTS1 required for cell separation suppresses the chs1 lysis phenotype, leading to the suggestion that Chs1p is involved in chitin repair at cytokinesis [7].
The precise deposition of chitin is achieved through spatial and temporal controls on each chitin synthase which determine their localization and activity. CSII is expressed in a cell cycle-dependent manner, and is transported to the bud neck through the secretory pathway, and subsequently degraded in the vacuole [8,9]. CSI and III are transported to a specialized endosome-derived compartment, the chitosome, from which they are mobilized by regulated secretion to the plasma membrane [5,8,10]. The localization and trafficking of Chs3p require BNI4, CHS4/SKT5, CHS5, CHS6 and CHS7. Chs7p is required for exit of Chs3p from the endoplasmic reticulum [11], while Chs5p and Chs6p are involved in transport of Chs3p from the chitosome to the plasma membrane [12,13]. Chs3p forms a complex with Chs4p/Skt5p, a protein required for Chs3p activity during vegetative growth, and Bni4p localizes this complex to the septin ring at the bud neck [14].
Although accounting for only 1–2% of the wild type cell wall under vegetative growth, chitin can contribute up to 20% of the cell wall under the conditions of cell wall stress found in cell wall mutants or on drug exposure [3]. Indeed, in response to cell wall stress Chs3p activity is up-regulated leading to an increased synthesis of chitin, which can be essential for survival. For instance, CHS3 is essential for maintaining the cell integrity of several cell wall mutants, such as fks1 or gas1 [15-17]. Similarly, defective primary septum synthesis can be compensated for by Chs3p-dependent formation of a remedial septum, resulting in a synthetic lethal interaction between CHS2 and CHS3 [4].
To further explore the relationship between chitin synthesis and other pathways, we assemble a network of 316 synthetic interactions of 163 genes with genes involved in the regulation of chitin synthesis. The relationship of these genes with chitin synthesis was analyzed by measuring the chitin content of the 156 viable deletion mutants and by testing for Calcofluor white sensitivity phenotypes of the 116 deletion mutants in non-essential genes of the CSIII network.
Results
A network of genetic interactions with genes involved in chitin synthase function
To identify genes buffering defects in chitin synthesis, we searched for genes engaged in synthetic interactions with BNI4, CHS1, CHS3, CHS4, CHS5, CHS6 or CHS7 using the SGA methodology [18,19]. Our results identified 163 genes involved in 316 synthetic interactions that form a network in which BNI4, CHS1, CHS3, CHS4, CHS5, CHS6 and CHS7 are connected to 22, 57, 63, 47, 71, 25 and 31 genes, respectively (Table 2). Genes interacting with BNI4, CHS3, CHS4, CHS5, CHS6 or CHS7 tend to be multiply connected, while those interacting with CHS1 form a more distinct subnetwork (Figure 1A). Indeed, just 17 of the 57 CHS1 interacting genes show an additional interaction with at least another query gene (Figure 1B). In contrast, 67/123 genes interacting with BNI4 or CHS3-7 are multiply connected (Figure 1B, green oval), and 55 of those show an interaction with either BNI4 or CHS3-7 (Figure 1B, red oval) resulting in a densely connected CSIII network.
Table 2 Synthetic interactions with BNI4, CHS1, CHS3, CHS4, CHS5, CHS6 and CHS7.
Functional category Gene Interacting partners
Cell wall maintenance BCK1 BNI4, CHS1, CHS3, CHS4, CHS5, CHS7
FKS1, SLT2, SMI1 BNI4, CHS3, CHS4, CHS5, CHS6, CHS7
YPL261C CHS1
ECM21 CHS1, CHS5
CHS2 CHS3
SWI4 CHS3, CHS4, CHS5
CCW12, GAS1, YLR111W CHS3, CHS4, CHS5, CHS7
TUS1 CHS4, CHS5, CHS7
DAN3, PAT1 CHS5
Cell polarity & vesicular transport NBP2, RGD1, SHS1, SPA2 BNI4
EDE1, MYO2, RVS167, VRP1 BNI4, CHS3, CHS4, CHS5, CHS7
ARC40, ARP2 BNI4, CHS3, CHS4, CHS5, CHS6, CHS7
BNI1 BNI4, CHS3, CHS4, CHS7
RVS161 BNI4, CHS5
CYK3 BNI4, CHS7
BUD20, VPS5, VPS17, VPS29, VPS35 CHS1
HBT1 CHS1, CHS3
ARC18 CHS1, CHS3, CHS4, CHS5, CHS6
EMP24 CHS1, CHS3
BEM4, PEA2 CHS1, CHS5
CDC3, CDC11, IES6, SRV2, VAM7 CHS3
CDC12 CHS3, CHS4
FAB1 CHS3, CHS4, CHS5
CLA4 CHS3, CHS4, CHS5, CHS6, CHS7
SAC6, SLA1, TPM1 CHS3, CHS4, CHS5, CHS7
YLR338W CHS3, CHS4, CHS6
SHE4, SMY1 CHS3, CHS4, CHS7
VPS24, VPS67 CHS3, CHS5
AST1, LST4, YPK1 CHS4
SEC22 CHS4, CHS5
AOR1, HSE1, VPS21 CHS5
Suspected role in cell polarity & vesicular transport YPL066W BNI4
ILM1 BNI4, CHS3, CHS4, CHS6
SPF1 CHS1, CHS4
YGL081W CHS1, CHS5
YBR077C CHS3
GUP1 CHS3, CHS4, CHS5, CHS6
OPI3 CHS3, CHS4, CHS7
LSM6 CHS5
IST3 CHS6
Protein modification VAN1 BNI4, CHS3, CHS4, CHS5, CHS6, CHS7
YGL110C CHS1
ANP1, BTS1, MNN2, MNN9 CHS3
MNN10 CHS3, CHS4, CHS5, CHS6, CHS7
UBI4 CHS3, CHS5, CHS6
UBP13 CHS4
BRE1 CHS5
UFD4 CHS5
MNN11 CHS5, CHS6
LAS21 CHS6
Ribosomal function/cell size LGE1 CHS1, CHS3, CHS5
RPL20B CHS3
RPS8A CHS3, CHS4, CHS5
ASC1 CHS3, CHS5
RSA1 CHS4
RPL14A CHS5
Cell cycle CLN2 CHS1
CDC26, DOC1 CHS3
YNL171C CHS3, CHS5
CLB3, CTK2 CHS5
Mitochondrial function MDM38, NUC1, UTH1, YME1 CHS1
YFR045W CHS1, CHS5
MST1, TOM37 CHS3
YTA12 CHS3, CHS4
ATP17 CHS4
RPO41 CHS4, CHS6, CHS7
COQ2, COX11, LAT1, MDM12, PET8, SHE9 CHS5
Carbohydrate and lipid metabolism DEP1, ELO1, HXT8, IPK1, PDA1, PDC1, PFK2, PHO5, PKR1, RPE1, TYR1, YDR248C CHS1
Other functions BRE5 BNI4, CHS3, CHS4
IXR1 BNI4, CHS5
CNB1, HAP2, HIT1, PEX22, PMP3, PRM3, SKI2, WHI2 CHS1
RPA34 CHS1, CHS3, CHS4, CHS5, CHS7
FPS1 CHS1, CHS4, CHS5
GRS1, LEA1, MRE11 CHS1, CHS5
CSF1 CHS3, CHS4, CHS5
PRE9 CHS3, CHS5, CHS7
MUM2 CHS3, CHS6
UME6 CHS4, CHS6
DOT1, PDE2, PEX14, SWI3 CHS5
IRA2 CHS5, CHS6
NUP133 CHS5, CHS6, CHS7
MSN5 CHS6
PEX6 CHS7
Unknown function YBR209W, YDR314C, YEL033W, YIL110W, YMR003W, YNL179C, YOR322C, YPR053C CHS1
YDL206W CHS1, CHS5
YDL032W CHS3
YDL033C CHS3, CHS5
YGL152C CHS5
YNL235C CHS6
YIL121W CHS7
Figure 1 A network of genetic interactions with BNI4, CHS1, CHS3, CHS4, CHS5, CHS6 and CHS7. (A) Global view of the network. Synthetic interactions with any query gene (diamonds) are depicted as edges joining these to nodes (circles). Nodes whose deletion mutant have a decreased, wild type and increased chitin content are colored in green, gray and red, respectively. For the decreased (green) and increased (red) chitin contents, color intensity is proportional to the magnitude of the change. (B) Venn diagram of the CHS1 interaction set with the CSIII network. The number of genes interacting with CHS1 or with any of the CSIII query genes is indicated. The numbers in parentheses indicate the number of interactions for multiply connected genes. Genes showing 2 or more interactions are grouped in green or red ovals, respectively.
The CSIII network
The 123 genes engaged in the 259 interactions of the CSIII network were grouped by function (Figure 2A, outer pie). Some genes show multiple connections, with 55 of these accounting for almost 75% of the interactions. Among this group, 44 genes (Figure 2B) interact with CHS3 and at least one other query gene, reflecting the central role of CHS3 in the network. These 44 genes, involved in 166 interactions, are significantly more connected to the query genes than the remaining 11 multiply connected genes, which have 25 interactions (p < 0.01). Thus, this set of 44 genes defines a core group of multiply interacting genes. In addition, the "core" genes account for 57 of 74 synthetic lethal interactions of the CSIII network [19], highlighting their importance for survival when the CSIII pathway is defective. Grouping "core" genes by functional categories (Figure 2A, inner pie) revealed enrichment for certain functions relative to the overall CSIII network. For example, cell wall assembly and secretory pathway polarization/vesicular transport contain 18% and 43% of "core" genes, respectively, whereas these functional categories represent 10% and 33% of genes in the CSIII network, respectively. In contrast, the category "mitochondrial function" is under-represented in the "core" group when compared to the CSIII network (Figure 2A). Thus, analysis of the "core" of highly connected genes indicates that cell wall assembly and polarization of the secretory apparatus are central processes buffering defects in the CSIII pathway.
Figure 2 Analysis of the CSIII network. (A) Grouping genes of the CSIII network in functional categories. Genes belonging to the "core" group (CHS3 plus at least one other) are underlined. The proportions of the functional categories in the CSIII network and in the "core" group are represented in the outer and the inner pies, respectively. (B) Interactions among the "core" group. Color coding for nodes is as in (A).
CHS1 interaction set
Some 57 genes show synthetic interactions with CHS1 (Figure 1A and Table 2), and while a number of these are embedded in the CSIII network, most interact only with CHS1, indicative of a distinct functional role for Chs1p that is analyzed further in the Discussion.
Chitin content in mutants of interacting genes
To investigate the relationship between the interacting genes and chitin synthesis, the chitin content of the 156 deletion mutants in non-essential genes of the CSIII network and the CHS1 interacting genes was measured (see Additional file 2). To focus on the biologically meaningful changes in chitin level, a set of mutants with marginally altered chitin content were excluded from our analysis despite their having statistical significance. Thus, 51 and 3 mutants with levels above 20 and below 12 nmole GlcNAc/mg dry weight, respectively are discussed below as having altered chitin content. To integrate synthetic interaction and chitin determination data, each node of the interaction network was colored according to the chitin level of its deletion mutant (Figure 1A). Four groups of genes emerged from this analysis.
Group 1 has 33 mutants with an altered chitin level and a requirement for Chs3p function for optimal growth (Figure 3A). All but one of the group 1 mutants have elevated chitin levels, indicating that they trigger the chitin stress response. Nine of these genes are involved in the synthesis of cell wall components such as β-glucan and mannoprotein. Half of the group 1 genes (17/33) are required for polarization of the actin cytoskeleton or have a function in vesicular transport through retrograde transport in the endosomal pathway. The majority of the group 1 genes belong to the CSIII network "core", with just 7 genes interacting uniquely with CHS3 (ANP1, BTS1, DOC1, MNN2, MNN9, RPL20B and YBR077C). Thus, the deletion mutants of group 1 genes are highly sensitive to CSIII pathway perturbation.
Figure 3 Grouping deletion mutants with an altered chitin content according to their interaction pattern. (A) Chitin levels, expressed in nmole GlcNAc/mg dry weight, in wild type, group 1 and query mutants. (B) Chitin levels in wild type and group 2 mutants. Note the different scales in (A) and (B). Hypersensitivity, resistance, wild type and not determined sensitivity to Calcofluor are indicated by black, open, gray and hatched bars, respectively.
Group 2 is composed of 19 and 2 mutants with an increased and a decreased chitin level, respectively, but whose optimal growth does not require CHS3 (Figure 3B). A large fraction of group 2 genes (16/21, 76%) interact with CHS5 and/or CHS6 (Table 2). Group 2 mutants are affected in secretion or mitochondrial functions and in the regulation of transcription and translation. The elevated synthesis of chitin in 19 of the group 2 mutants is probably triggered as a non-specific stress response to the mutation, but unlike group 1, it does not serve to buffer against the deleterious effects of the mutation. For example, a set of 14 group 2 mutants interacting with CHS5 or CHS6 have elevated chitin levels. In these cases, the stress activated chitin response reflects a broader Chs5p- and/or Chs6p-dependent activation that is required for cell wall buffering in these mutants (see Discussion).
The third group of 16 mutants have a wild type chitin content and a synthetic interaction with CHS3 (Figure 4A). These mutants are defective in ubiquitin processing/cell cycle progression, membrane biogenesis and polarized secretion.
Figure 4 Grouping deletion mutants with wild type chitin content according to their interaction pattern. (A) and (B) group 3 and 4 mutants, respectively. Hypersensitivity, resistance, wild type and not determined sensitivity to Calcofluor are indicated by bold, open, gray and underlined characters, respectively.
Finally, group 4 contains deletion mutants in 57 genes with a wild type chitin level and a synthetic interaction with any CHS gene other than CHS3 (Figure 4B). Sixteen of these genes are connected to CHS5, suggesting a broad, and Chs3p-independent, role for Chs5p in their buffering.
Calcofluor white phenotypes of the CSIII network mutants
Calcofluor white is a toxic compound which binds primarily to chitin in yeast, and mutants with cell surface defects frequently show altered sensitivity to it [20-23]. For example, a chs3 null mutant and mutants with a defective CSIII pathway show Calcofluor resistance because they make low levels of cell wall chitin [23]. We thus searched for synergistic interactions between Calcofluor white and the deletion of each gene found in the CSIII network. Mutant strains were spotted on solid medium containing 10 μg/ml or 50 μg/ml Calcofluor white, and scored for sensitivity relative to the wild type. In all, 59% of mutants exhibited an altered Calcofluor sensitivity, with 65 and 4 mutants showing hypersensitivity and resistance, respectively (Figure 3 and 4, and see Additional file 2). As seen in Figure 3, a high fraction of mutants with an altered chitin content also showed an altered sensitivity to Calcofluor. Indeed, 80% (39/49) and 67% (2/3) of mutants with increased and decreased chitin levels, respectively, were hypersensitive and resistant to Calcofluor, respectively. More specifically, 97% of group 1 mutants had a Calcofluor phenotype, revealing the critical role of Chs3p-synthesized chitin in Calcofluor sensitivity. However, Calcofluor toxicity does not correlate strictly with the chitin level or the requirement for Chs3p function. Indeed, 10 mutants with wild type Calcofluor sensitivity have an altered chitin content (Figure 3, gray bars). This set is almost entirely composed of group 2 mutants (Figure 3B), with the optimal growth of 9 of these mutants not requiring CHS3. In addition, 17 mutants with an altered Calcofluor sensitivity have a wild type chitin level (Figure 4, bold and open characters): 8 and 9 of those mutants fall in groups 3 and 4, respectively. The 8 group 3 mutants require Chs3p function but do not trigger the chitin stress response, indicative of a requirement for an additional Chs3p function distinct from lateral wall chitin synthesis, such as remedial septum or bud neck chitin synthesis (see Discussion). The 9 group 4 mutants require integrity of the CSIII pathway but not an increase of chitin level through Chs3p. This subgroup indicates that components of the CSIII pathway function in other cellular processes. Finally, a set of 26 mutants are wild type for both Calcofluor sensitivity and chitin level. Nineteen of them are not connected to CHS3, reflecting chitin-independent functional requirements for CHS4, CHS5, CHS6, CHS7 and BNI4.
Synthetic interactions with SHC1
The SHC1 gene product is 43% identical to Chs4p. While Chs4p functions in Chs3p activation during vegetative growth, the known role of Shc1p is restricted to sporulation [24]. However, overexpression of Shc1p during vegetative growth can compensate for the lack of Chs4p, and reciprocally, overexpression of Chs4p during sporulation partially complements the shc1Δ mutant phenotype [24]. Although Chs4p and Shc1p show structural and functional relatedness they are not an essential redundant pair since the chs4 shc1 double mutant has no synthetic growth defect. We searched for genes required for the optimal vegetative growth of the shc1Δ mutant and found 6 synthetic interactions. In addition, we added the previously reported synthetic interaction between PHO85 and SHC1 [25] to this list. FAB1 and DEP1 are part of the CSIII network and CHS1-interacting set, respectively. The remaining 5 genes (BUD16, DBF2, HOP2, PHO85 and SPT8) interact uniquely with SHC1. The pho85 null mutant was not further analyzed due to its very poor growth. The amount of chitin produced in the 4 remaining mutants was measured and found to be similar to the wild type (see Additional file 2). Genes compensating for a SHC1 deletion form a distinct group from those buffering a CHS4 deletion (Figure 5), and their genetic interactions with SHC1 appear to be independent of a chitin defect. Thus, our synthetic interaction data indicate that SHC1 has evolved new functions that are not shared with CHS4 and which extend the role of Shc1p beyond sporulation to mitotic growth.
Figure 5 Comparative synthetic interaction patterns of CHS4 and SHC1. Synthetic interactions with CHS4 or SHC1 are depicted as connections between these nodes and their respective partners (black and gray nodes, respectively).
Discussion
We globally analyzed a network of 259 interactions among 123 genes required for optimal growth of BNI4, CHS3, CHS4, CHS5, CHS6 or CHS7 deletion mutants. The query genes are highly interconnected, reflecting common requirements in the bni4 and chs3-7 null mutants. This network centers on CHS3 function, with CHS3 sharing most of its interactions with the other query genes.
Grouping CHS3-interacting genes by functional requirement for Chs3p
The genetic interactions observed with CHS3 can be sorted by Chs3p function, which includes synthesis of chitin in the lateral wall, in the remedial septum and at the bud neck.
Lateral wall chitin, the chitin stress response
The Chs3p-dependent synthesis of wall chitin is dramatically stimulated upon cell wall stress, through a stress response pathway involving activation of the chitosome and stimulation of the cell integrity pathway [10,15-17]. Mutants with cell wall defects activate this stress pathway and our synthetic analysis indicates that many of them require Chs3p function (Figure 6). Our work indicates that the extent of this stress response is far greater than previously realized: just 6 of the 26 mutants in this group were previously known to have an altered chitin content. Among such new mutants involved in triggering the chitin stress response are the cell wall protein encoding gene CCW12, and the actin-based polarity genes BNI1, CLA4, SAC6, SHE4, SLA1 and VRP1. Actin patches are crucial for the proper targeting of cell wall synthesis components [26], and their perturbation activates a chitin stress response. Other mutants include CSF1, GUP1 and ILM1 that have growth defects on non-fermentable carbon sources and the putative vacuolar protein encoding YBR077C.
Figure 6 Functional integration of CHS1- and CHS3-interaction sets. CHS1- and CHS3-interacting genes were grouped according to their effects on chitin synthesis. The Venn diagram shows the distinct and overlapping sets for each functional category.
Glucosamine-driven chitin synthesis
Chs3p has an additional role in the synthesis of chitin upon glucosamine addition [27]. The basis for this process is uncertain, but probably relies on metabolic flux changes and appears to be independent of the classic chitin stress response [27]. Deletion of genes compensating for defects in this glucosamine-response pathway may interact synthetically with CHS3 and genes of the CSIII pathway (Figure 6). Candidate genes are MST1, TOM37 and YTA12, involved in mitochondrial function, a process known to be down-regulated by glucosamine exposure: their deletion may lead to metabolic imbalance compensated for by an increased chitin synthesis.
Insight into Chs2p function
Chs2p is responsible for synthesis of the primary septum but a detailed understanding of how this is achieved remains incomplete. Analysis of CHS3 synthetic interactions can give insight into Chs2p function as, in its absence, Chs3p can partially compensate by forming a "remedial septum" [28]. We reasoned that a set of synthetic interactions with CHS3 could occur through perturbation of CHS2 function, leading to the need for CHS3. A group of genes affecting cell cycle progression likely have an impact on septation in this way. For example, mutants in CDC26, DOC1 or YNL171C (which is an apc1 allele) show a delay in exit from mitosis and mutants in ASC1, IES6, LGE1, RPL20B, RPS8A or VAM7 exhibit altered cell size, a phenotype frequently reflecting defects in cell-cycle checkpoints [29,30]. Deletion of any of these genes can uncouple cell-cycle progression and septation, resulting in defective synthesis of the primary septum by Chs2p. The synthetic interactions between these genes and CHS3 likely result from a failure to fully synthesize both the primary septum (as a consequence of a defect in cell-cycle progression) and a remedial septum (Figure 6). Pertinently and consistent with our data, Ufano et al. [31] show that deletion of SWM1, encoding a subunit of the anaphase promoting complex, also leads to an increase of Chs3p-catalyzed chitin deposition.
Chs2p has a cryptic in vitro activity that can be detected only after treatment of a cell extract with trypsin. This suggests that Chs2p may also be produced as a zymogen in vivo and be activated by posttranslational modification [32]. Although proteomic analysis reveals the existence of ubiquitinated and phosphorylated forms of Chs2p [33,34], the effect of these modifications on Chs2p activity is unknown. Mutants with defects in Chs2p activation or turnover may exhibit a low Chs2p activity and depend on a compensatory Chs3p activity. Of the CHS3 interacting genes, the serine/threonine protein kinases Bck1p and Slt2p are candidates for Chs2p activation, while the polyubiquitin gene UBI4, the ubiquitin protease Bre5p and the proteasome subunit Pre9p may be required for Chs2p turnover (Figure 6).
Chitin at the bud neck
Chs3p synthesizes a chitin ring that marks the incipient bud site. Defects in secretion or polarization of the secretory apparatus may lead to abnormal bud neck assembly and/or septation. For example, the genes EDE1, EMP24, FAB1, HBT1, OPI3, RVS167, SMY1, TPM1, VPS24, VPS67 or YBR077C are required for polarization of the secretory pathway, indicating that transport and proper localization of protein(s) to the bud neck are essential for growth of mutants with low CSIII activity. Thus, we identify these genes as candidates for involvement in Chs2p localization and in bud neck integrity (Figure 6).
Functions of Bni4p, Chs4p and Chs5p beyond chitin synthesis
The existence of synthetic interactions with BNI4, CHS4, CHS5 or CHS6 not shared with CHS3 uncovers functions of these genes that are unrelated to Chs3p transport or activity.
Bni4p
Five genes interact uniquely with BNI4, indicating that Bni4p has functions distinct from anchoring Chs3p to the septin ring. Among these, NBP2, RGD1, SHS1 and SPA2 are required for regulation of cytoskeleton organization at the bud neck by the cell integrity pathway. Further, BNI4 shows a unique interaction with YPL066W, which together with the bud neck localization of Ypl066p [35] implicates this gene in bud development. Our data and the finding that localization of Crh2p at the bud neck requires Bni4p [36] indicate that Bni4p has a broad role in bud neck organization.
Chs4p
Of the 7 unique CHS4 interacting genes, 4 are required for trafficking of membrane proteins. Ast1p and Lst4p are required for Golgi to plasma membrane transport of the H+-ATPase Pma1p and the amino-acid permease Gap1p, respectively [37,38]. Spf1p, a putative calcium pump of the endoplasmic reticulum, may also play a role in the translocation of transmembrane proteins [39]. Ypk1p, a serine/threonine protein kinase required for full induction of the PKC1-SLT2 cell integrity pathway under stress condition, is also required for endocytosis [40]. Absence of these genes combined with a CHS4 deletion likely leads to defects in targeting membrane proteins to the septin ring, with resultant synthetic growth phenotypes.
Chs5p and Chs6p
Chs5p and Chs6p are late-Golgi localized proteins involved in targeting Chs3p to sites of polarized growth [12] and to the plasma membrane [13]. Our results for CHS5 and CHS6, showing a strong web of synthetic interactions with CSIII network "core" genes, reflect these roles. Whereas little is known about a Chs6p function besides Chs3p trafficking, Chs5p is also involved in the selective polarization of other surface proteins, such as Fus1p [41] and, at least partially, Crh2p [36]. CHS5 and CHS6 show a large number of CHS3-independent interactions (39/71 and 9/25, respectively), suggesting multiple additional roles for Chs5p and Chs6p in protein targeting. Interestingly, a number of these interacting mutants have elevated chitin levels and fall into group 2. For example, the mutants ira2 and pde2 are synthetic with CHS5 and CHS6 and CHS5, respectively and make 76% and 35% more chitin than the wild type, respectively (Figure 3B). These mutants constitutively elevate the Ras/cAMP pathway and pde2 mutants are known to affect cell wall integrity and to cause slight changes in glucan and chitin levels [42,43]. Our work suggests that the chitin elevation involves increased activity of the Chs5p and Chs6p chitosome pathway. However, the key buffering component in this cAMP response is not chitin, but must be some other component of the activated chitosome pathway, since neither ira2 nor pde2 show a synthetic interaction with CHS3. Thus here, the stress activated chitin response is a gratuitous consequence of a broader Chs5p- and/or Chs6p-dependent activation that is required for cell wall buffering in these mutants.
Regarding the known role of Chs5p in specialized late-Golgi trafficking; several of the CHS5-interacting genes have products that likely work in conjunction or in parallel with Chs5p. These include AOR1, BEM4, HSE1, LSM6, PEA2, RVS161, SEC22 and VPS21. A new candidate is YGL081W that interacts with CHS5, and whose product has been found in a complex containing Cop1p, required for Golgi retrograde transport [44].
CHS5 interacts uniquely with 6 genes involved in mitochondrial function (COQ2, COX11, LAT1, MDM12, PET8 and SHE9) some of which show elevated chitin levels. These mitochondrial proteins may play indirect roles in late-Golgi trafficking; for example, the secretory apparatus and mitochondria exchange lipids [45], and a defect in mitochondrial function may impact on secretory function. Alternatively, in the absence of CHS5, mitochondria may be poorly transferred to daughter cell with their efficient functioning being essential for optimal growth.
Finally, some 9 genes show an interaction with both CHS5 and CHS1 (Figure 1A), indicating some common requirement for these genes. One provocative possibility for this interactional signature is that Chs5p is involved in the targeting of Chs1p.
Analysis of synthetic interactions with CHS1
Although it was the first fungal chitin gene identified, the role of Chs1p has remained unclear. Cell lysis phenotypes of chs1 mutants have led to the view that Chs1p is an "auxiliary" enzyme implicated in the repair of chitinase-mediated cell wall damage associated with cell separation [7]. How such damage is sensed or how the repair process is activated remains unclear. The line between repair and redundant synthesis with Chs2p may be an arbitrary one, and a direct role for Chs1p involvement in septal chitin synthesis on growth in acidic minimal media where cell lysis is more pronounced, also explains the phenotype (see [46] for a discussion). The lysis phenotypes associated with CHS1 deletion also show strain variability. For example, a strain with a recessive suppressor in an uncharacterized gene SCS1 shows no lysis phenotype, indicating the involvement of other genes [7].
Our synthetic approach allows a broad survey of possible CHS1 function. However, CHS1 is part of a family and a synthetic analysis of a gene family can be complicated [47]. Specialized roles for CHS1, CHS2 and CHS3 are likely ancient, predating the genome duplication of S. cerevisiae [48,49], since all three genes are present in Ashbya gossypii, a related fungus that did not undergo the S. cerevisiae duplication event. Our finding that the majority of the CHS1 interactions are both distinct from the CSIII network and do not trigger the chitin stress response (Figure 1A) indicates distinct function. CHS1 and CHS3 mutants do not synthetically interact under our test conditions, so the synthetic effects of CHS1 mutants are not caused by a buffering of CHS3 function. Consistent with this, the CHS1 deletion does not activate the chitin stress response, as chitin levels in the chs1Δ mutant are close to wild type [6], see Figure 3A). One possible cause of synthetic effects of CHS1 mutants is through genes that buffer Chs2p function. A number of unique interactors with CHS1 are involved in bud morphogenesis (BEM4, BUD20, PEA2), and in protein recycling through the endocytic pathway (VPS5, VPS17, VPS29 and VPS35), all could be required for Chs2p function (Figure 6). This hypothesis, supported by the genetic evidence presented here, will require further testing.
In our data we also find interactions with mutants in a number of genes that are singly prone to lysis or show phenotypes consistent with osmotic imbalance (ARC18, BCK1, CNB1, FPS1, and WHI2). CHS1 also shows synthetic interactions with YEL033W and YNL179C, that overlap with and are alleles of YEL034W/HYP2 and YNL180C/RHO5, genes that play a role in balancing cell integrity [50,51], and with YOR322C which has a role in signaling through the cell integrity pathway [52]. In addition the absence of Chs1p is buffered by the presence of 16 genes (BCK1, BEM4, CNB1, ECM21, FPS1, GRS1, HBT1, HIT1, NUC1, PDA1, PFK2, SPF1, TYR1, YGL081C, YGL110C and YPL261C) that show synthetic interactions with the FKS1, GAS1 or SMI1 genes involved in β-1, 3-glucan synthesis [19]. These results provide strong independent support for a function of Chs1p in buffering cell wall robustness through regulated chitin synthesis, and identify many candidates that may participate in the modulation of Chs2p function.
As mentioned above, yeast cells are more dependent on Chs1p to prevent lysis and allow growth on synthetic minimal media [6,53]. The basis for this increased dependence is unknown, though there are data indicating that the partitioning of Chs1p activity between the plasma membrane and the chitosome is somewhat more pronounced toward the plasma membrane in minimal medium [54]. Interestingly, a number of unique CHS1 interactors are involved in metabolism and nutrient utilization (Figure 6), providing functional clues to this aspect of Chs1p function.
Conclusions
Our synthetic network analysis reveals a deep interactional complexity underlying chitin biology. The CHS3-core network is informative in identifying components involved in all aspects of regulated chitin deposition. The chitin stress response that adds chitin to lateral cell walls is now shown to be triggered very broadly by cell wall and actin-based polarity defects and to play a key role in cell wall buffering. The CHS3 core-network also offers insight into Chs2p function by identifying proteins implicated in bud neck localization, and in the cell cycle coordination of septum formation with mitotic exit. Genes involved in secretory trafficking of Chs3p (CHS4, CHS5, CHS6 and BNI4) show many CHS3-independent interactions and these greatly expand the range of trafficking functions for these genes, especially for the heavily interacting CHS5. In contrast to its currently assigned minor auxiliary role, CHS1 shows an extensive web of genetic interactions, most of which are distinct from the CSIII network and which do not trigger the chitin stress response. One set of these identifies components of endocytosis, budding and cell morphology, which may be required for Chs2p function. A second set of 25 interacting genes show that Chs1p is intimately involved in buffering yeast cell wall robustness during vegetative growth.
Methods
Strains, media and drugs
Haploid deletion mutants (Table 1) are available from the deletion project consortium. These strains were arrayed on sixteen 768-format plates using a colony picker [18]. Starting strains for the SGA analysis (Table 1) were constructed as described in Tong et al. [19]. Arrays were propagated at 30°C on standard YEPD (10 g/l yeast extract, 20 g/l bacto-peptone, 20 g/l glucose) or YEPD supplemented with 200 μg/ml G-418 (Invitrogen, Carlsbad, CA). When required, strains were grown on standard SD medium (6.7 g/l yeast nitrogen base, 20 g/l glucose) supplemented with appropriate amino acids [55]. Nourseothricin (ClonNat) was purchased from Werner Bioagent (Jena, Germany).
Table 1 Strains used in this study.
Strain Genotype Reference
BY4741 MATa his3Δ leu2Δ met15Δ ura3Δ [60]
BY4742 MATα his3Δ leu2Δ lys2Δ ura3Δ [60]
BY4743 MATa/α his3Δ /his3Δ leu2Δ /leu2Δ met15Δ /MET15 lys2Δ /LYS2 ura3Δ /ura3Δ [60]
ΔarrayORF MATa orfΔ::KanMX4 his3Δ leu2Δ met15Δ ura3Δ [61]
HAB1122 As Y3656 chs3Δ::NatMX4 [19]
SBY4 As Y3084 chs1Δ:: NatMX4 [19]
SBY5 As Y3084 chs5Δ:: NatMX4 [19]
SBY6 As Y3084 chs7Δ:: NatMX4 [19]
SBY30 As Y3084 chs4Δ:: NatMX4 [19]
SBY70 As Y3084 shc1Δ::NatMX4 This work
SBY105 As Y3656 chs6Δ:: NatMX4 [19]
Y3084 MATα mfα1Δ ::MFα 1pr-LEU2 can1Δ ::MFA1pr-HIS3 his3Δ leu2Δ lys2Δ ura3Δ [18]
Y3638 As Y3084 bni4Δ:: NatMX4 [19]
Y3656 MATα can1Δ ::MFA1pr-HIS3-MFα 1pr-LEU2 his3Δ leu2Δ lys2Δ ura3Δ [19]
Screening for synthetic lethal/sick interactions and data refinement
Synthetic genetic array analysis (SGA) was used to identify genes required for the optimal growth of strains deleted for BNI4, CHS1, CHS3-7 or SHC1, as described [18,19]. From three SGA screens for each "query" gene, ~1,800 potential interactions were identified and 333 synthetic interactions confirmed by random spore or tetrad analysis as described previously [19]. Briefly, spores were germinated into liquid haploid selection medium [SD-His/Arg + canavanine] in a 96-well format. The germinated MATa spore progeny were serially diluted in sterile water and 2 μl for each dilution was spotted onto medium selecting for the query-gene mutation [SD-His/Arg + canavanine/Nourseothricin], the interacting gene mutation [SD-His/Arg + canavanine/G-418], and both the query-gene and interacting gene mutations [SD-His/Arg + canavanine/Nourseothricin/G-418] then incubated at 30°C for ~2 days. Cell growth under the three conditions was compared and double mutants were scored as synthetic sick (SS), synthetic lethal (SL) or no interaction (No) [19]. For tetrad analysis, dissections were performed on solid complete SD medium and growth of individual spores was scored after 4 days incubation at 30°C. Plates were then replicated on YEPD + G-418 or Nourseothricin to identify tetrad type. Growth of double mutants was compared to that of single mutants from tetratype tetrads and then scored as "SS", "SL "or "No". Ten of the 22 previously reported synthetic lethal interactions with CHS3-7 or SHC1 [4,15,25,56,57] were found by the SGA procedure. Of the remaining, 9 engaged genes whose mutant is absent from our deletion collection (CDC3,CDC11, CDC12 and CHS2) or genes whose deletion leads to systematic growth defects in our conditions (ANP1, MNN9, PHO85 and SRV2) and these genes were used in our network analysis. No synthetic interaction between GAS1 and CHS4 or CHS7 was found by the SGA. These discrepancies with other's data [56] reflect differences in strain background. These two synthetic interactions were included in our analysis. An additional set of 57 interactions were analyzed further by random spore or tetrad analysis [19]. Of these, 30 synthetic interactions were confirmed, with the 27 remainder discarded (see Additional file 1). It is important to note that this additional set of tested interactions was not random and was strongly biased toward dubious interactions: for example, a group of 11 interactions with genes closely linked to CHS1 or a set of 16 non-reciprocal interactions (that is gene A found in screen for genes interacting with gene B and gene B not found in the set of genes interacting with gene A).
Chitin assay
Stationary phase cultures were diluted 1:100 into 3 ml of YEPD and grown again for 22-24 h at 30°C. Cells from 1.5 ml culture were colleted by centrifugation (20,000 × g, 2 min). Pellets were then frozen at -20°C until used for alkali-extraction. Dry weights were determined after a 2 day incubation at 37°C. Cell pellets were resuspended in 1 ml 6% KOH and heated at 80°C for 90 min with occasional mixing. Alkaline insoluble material was pelletted (20,000 × g, 20 min), neutralized with phosphate-buffered saline for 10–20 min with occasional mixing. After centrifugation (20,000 × g, 20 min), 200 μl of McIlvaine's Buffer (0.2 M Na2HPO4/0.1 M citric acid, pH 6.0) was added to pellets. Extracts were then stored at -20°C until processed for chitin measurements. Samples were thawed and subjected to two digestions with 4 μl of purified Streptomyces plicatus chitinase-63 (4 μg/ul in PBS) at 37°C for 36–40 h and then for 20–24 h. The amounts of chitin were then determined by using the modified Morgan-Elson procedure as described previously [27]. The levels of chitin, expressed as GlcNAc concentration, were then normalized to the dry weight of the sample. Of the 84 mutants whose chitin levels differed significantly from wild type (p < 0.01 in a Student's t-test, see Additional file 2), 54 with larger changes were further considered (see text).
Calcofluor white sensitivity/resistance
Sensitivity to Calcofluor white was assessed using a modified version of the method described by Ram et al. [21]. Cells were grown overnight, and then diluted to an optical density of OD600 nm = 0.5. Five μl of this suspension, as well as 1:10, 1:100, and 1:1000 dilutions of this suspension, were spotted on SD plates (buffered to pH 6.2 with 10 mM MES) containing 10 μg/ml or 50 μg/ml Calcofluor white (Fluorescent Brightener 28, Sigma), and control plates. Plates were incubated at 30°C for 48 hours, photographed, and then rechecked after 72 hours. A literature search indicated that the phenotype we found agreed with that previously reported for 29 mutants (22 interacting mutants + 7 query mutants). In 4 cases however (ARC18, PDE2, PEX6 and SPF1), we found a wild type sensitivity for mutants that had previously shown altered Calcofluor white sensitivity [22,42,58,59]. These discrepancies may be due to differences in Calcofluor white concentration or to allelic or strain variation.
Authors' contributions
GL participated in the design of the study and its coordination, collected and analyzed data, and drafted the manuscript. JS carried out confirmations of SGA screens and Calcofluor white sensitivity assays. CAS carried out chitin determinations and data analysis. AMS, PM, SH and AHYT carried out SGA screens. HB and CB conceived the study, and oversaw its design and coordination. HB participated in data analysis and manuscript writing. All authors read and approved the final manuscript.
Supplementary Material
Additional File 1
Title: Interactions reported previously and not included in our global analysis.
Click here for file
Additional File 2
Title: Sensitivity to Calcofluor white and chitin levels of mutants.
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Acknowledgements
This work was supported by Genome Canada (CB and HB), Génome Québec (HB), Genome Ontario (CB), NIH (grant RO1 A125780, CAS) and NSF (grant, IBN-0316963, CAS) We thank P. Robbins and A. Firon for their interest in this work.
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| 15715908 | PMC554099 | CC BY | 2021-01-04 16:38:18 | no | BMC Genet. 2005 Feb 16; 6:8 | utf-8 | BMC Genet | 2,005 | 10.1186/1471-2156-6-8 | oa_comm |
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BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-6-151571004110.1186/1471-2164-6-15Research ArticleComparative genome analysis of cortactin and HS1: the significance of the F-actin binding repeat domain van Rossum Agnes GSH [email protected] Ellen [email protected] Vera van Buuren-van [email protected] Philip M [email protected] Ed [email protected] Department of Pathology, Leiden University Medical Center, Albinusdreef 2, 2300 RC, Leiden, The Netherlands2 Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands3 Department of Pathology, University Medical Center Groningen, Hanzeplein 1, 9700 RB, Groningen, The Netherlands2005 14 2 2005 6 15 15 15 9 2004 14 2 2005 Copyright © 2005 van Rossum et al; licensee BioMed Central Ltd.2005van Rossum et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
In human carcinomas, overexpression of cortactin correlates with poor prognosis. Cortactin is an F-actin-binding protein involved in cytoskeletal rearrangements and cell migration by promoting actin-related protein (Arp)2/3 mediated actin polymerization. It shares a high amino acid sequence and structural similarity to hematopoietic lineage cell-specific protein 1 (HS1) although their functions differ considerable. In this manuscript we describe the genomic organization of these two genes in a variety of species by a combination of cloning and database searches. Based on our analysis, we predict the genesis of the actin-binding repeat domain during evolution.
Results
Cortactin homologues exist in sponges, worms, shrimps, insects, urochordates, fishes, amphibians, birds and mammalians, whereas HS1 exists in vertebrates only, suggesting that both genes have been derived from an ancestor cortactin gene by duplication. In agreement with this, comparative genome analysis revealed very similar exon-intron structures and sequence homologies, especially over the regions that encode the characteristic highly conserved F-actin-binding repeat domain. Cortactin splice variants affecting this F-actin-binding domain were identified not only in mammalians, but also in amphibians, fishes and birds. In mammalians, cortactin is ubiquitously expressed except in hematopoietic cells, whereas HS1 is mainly expressed in hematopoietic cells. In accordance with their distinct tissue specificity, the putative promoter region of cortactin is different from HS1.
Conclusions
Comparative analysis of the genomic organization and amino acid sequences of cortactin and HS1 provides inside into their origin and evolution. Our analysis shows that both genes originated from a gene duplication event and subsequently HS1 lost two repeats, whereas cortactin gained one repeat. Our analysis genetically underscores the significance of the F-actin binding domain in cytoskeletal remodeling, which is of importance for the major role of HS1 in apoptosis and for cortactin in cell migration.
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Background
Cortactin (also designated EMS1 , CTTN, cttn, Amplaxin, see Genecard [1]) was initially identified as one of the most prominent tyrosine phosphorylated proteins in v-Src infected chicken embryo fibroblasts [2]. Cortactin was independently isolated from mouse NIH3T3 cells [3] and human tumor cell lines [4]. Human cortactin is encoded by the EMS1 gene, which is located on chromosome 11q13 [4,5]. Gene amplification of 11q13 region and concomitant overexpression of cortactin frequently occurs in several human carcinomas [4,6-8] and correlates with lymph node metastasis and increased mortality [9-11]. Elevated expression of cortactin increases cell motility, invasion [12-14] and metastasis [15].
The deduced amino acid sequence of cortactin revealed three main distinguishable domains: the N-terminal acidic domain containing a DDW-Arp2/3 binding motif followed by a six and one-half 37-amino acid F-actin binding repeat domain, a central region and an SH3 domain at the very C-terminal. The DDW-Arp2/3 binding site and the actin-binding domain together regulate F-actin polymerization and dynamics by activating the Arp2/3 complex [16] and both are necessary for translocation of cortactin to sites of actin polymerization [17]. Recently, we reported the identification of two alternative splice variants of human cortactin lacking either 6th or the 5th /6th repeat, present in normal tissues as well as squamous cell carcinomas cell lines [14]. These splice variants differ significantly in their ability to (i) bind F-actin, (ii) cross-link F-actin (iii) activate Arp2/3 mediated actin polymerization and (iv) induce cell migration in vitro [14]. This indicates that also the number of repeats determines the affinity for F-actin and ability to regulate cell migration. Similar cortactin splice variants were also reported in the mouse [18], rat [19] and frog [20]. The SH3 domain is a conserved protein module found in various signal proteins and mediates the interaction with various proteins such as N-WASP involved in actin polymerization, dynamin-2 in endocytosis, ZO-1 in cell-cell interactions and SHANK-2 in neuronal growth cones (reviewed in [21]). The central part of the protein between the F-actin repeat domain and the SH3 domain contains an alpha-helix sequence and a proline-rich region with three c-Src tyrosine phosphorylation sites [22,23] and three serine/threonine phosphorylation sites [24]. Cortactin tyrosine phosphorylation occurs in response to growth factor treatment, integrin cross-linking, bacterial invasion and cell shrinkage (reviewed in [21]). Tyrosine phosphorylation of cortactin reduces its F-actin cross-linking activity and is required for its ability to stimulate cell migration [13]. Since cortactin operates mainly in cytoskeletal rearrangements, it may link other proteins via its SH3 domain to sites of actin polymerization. Alternatively, serine phosphorylation of cortactin by Erk enhances, whereas Src phosphorylation inhibits the activation of N-WASP by cortactin [25] and as a result affects actin polymerization. This suggests that cortactin at first instance may be directed to the site of actin polymerization by other proteins. Thus, changes in protein expression level, phosphorylation state, the relative expression of splice variants and interactions with other proteins can all influence cell migration.
Cortactin shows the highest similarity to the hematopoietic lineage cell-specific protein 1 (HS1). Human HS1 (also designated HCLS1 , see Genecard [26]) was originally isolated by its homology to the adenovirus E1A gene [27]. HS1 overall similarity to cortactin at the amino acid level is 51% but is highest at the SH3 domain (86%) and the 37-amino-acids repeat domain (86%), except that HS1 carries only three and one-half repeats. Despite this high homology, the function of HS1 differs considerable from cortactin. First, HS1 is mainly expressed in hematopoietic cells [27], whereas cortactin is widely expressed in all cell types except most hematopoietic cells [28]. Only in platelets and in megakaryocytes both genes are expressed [29,30]. Second, in concordance with this tissue distribution, HS1 is tyrosine phosphorylated after receptor cross-linking in B-cells [31], T-cells [32], mast cells [33] and erythroid cells [34], but at different residues compared to the functional phosphorylation residues in cortactin [13,23]. Third, HS1 is, like cortactin, a cytoplasmic protein, but after tyrosine phosphorylation HS1 translocates to the nucleus [35], whereas cortactin is never found in the nucleus. This is because HS1, but not cortactin, contains a nuclear localization signal (NLS) [36,37]. Fourth, HS1 plays an important role in the receptor-mediated apoptosis and proliferative responses as demonstrated by the analysis of HS1 deficient mice [38] and WEH1-231 B lymphoma cells [37,39]. An HS1 tyrosine mutant that could not translocate to the nucleus, also failed to induce apoptosis [37]. Consistent with its role in apoptosis, HS1 is able to bind to the mitochondrial protein HAX-1, a Bcl2 like protein [40]. Finally, the SH3 domain of HS1 at the C-terminus binds to other proteins (Ste20 related kinase HPK1 [41] and HS1-BP3 [42]) than those binding to cortactin, despite the very high amino acid sequence similarity of both SH3 domains (86%). This most probably reflects the different tissue-specific expression pattern.
Cortactin and HS1 share also remarkable similarities. First, HS1 binds with its DDW-motif directly to Arp2/3 and is involved in Arp2/3 mediated actin polymerization in vitro , although less efficient than cortactin [43]. Second, HS1 binds to F-actin with its 37-amino-acid repeat domain [36], however, it contains only three and one-half repeat in contrast to cortactin. Third, also HS1-splice variants have been detected such as a variant lacking the 3rd repeat of the F-actin binding domain in a systemic lupus erythematosus (SLE) patient resulting in increased apoptosis after B-cell receptor (BCR) stimulation [44]. Fourth, HS1 is sequentially phosphorylated on three tyrosine residues by various Src family tyrosine kinases [31,45] and two serine/threonine residues [30], although at different residues than cortactin [25]. Finally, both cortactin and HS1 can accumulate into podosomes, structures found in osteoclasts [46] and marcrophages [47], but also in RSV transformed cells [48] and carcinoma cells [49].
Although cortactin and HS1 share a high amino acid sequence and structural similarity, their functions differ considerable. In this paper, we compare their genomic organization in order to provide more insight into their evolution, which may form the basis towards understanding specific functions of both genes. We describe the genomic organization and the exon-intron boundaries for human cortactin. Both the genomic cDNA and deduced amino acid sequences of human cortactin were compared to cortactin and HS1 genes from other species. Genomic comparisons revealed the evolution and underscore the significance of the conserved F-actin binding repeat domain for HS1 and cortactin and the importance of alternative splicing for cortactin function.
Results and discussion
The genomic organization of cortactin homologues
We have previously described the isolation and sequencing of the EMS1 cDNA [28,49] (DDBJ/EMBL/GenBank Accession No. M98343) coding for the human cortactin protein. To evaluate the genomic structure, we determined the exon/intron-boundaries. Nucleotide sequence comparisons with human EMS1 cDNA sequence revealed homology with two human genomic clones (DDBJ/EMBL/GenBank Accession No. AP000487 and AP000405) (Table 1). The genomic structure of the EMS1/cortactin gene was determined by performing BLASTn comparisons of EMS1 cDNA against the genomic clones (Figure 1A). By amplifying the intron sequences (smaller than 2 Kb) using primers on adjacent exons followed by end-sequencing of these products, we confirmed the intron/exon boundaries of the human EMS1/cortactin gene. The EMS1 gene contains 18 exons spanning over about 38 Kb of genomic DNA. The length of the individual exons ranges from 55 to 178 bp, except the last exon (1564 bp). The splice donor and acceptor sequences, the sizes of the introns and exons of the human EMS1/ cortactin gene are provided in the supplementary materials [see Additional file 1]. The ATG is at position 169, at the first nucleotide of exon 3, indicating that the first two exons encode the 5' untranslated region (UTR). The F-actin-binding repeat domain is encoded by exon 5 to exon 12 with 5 exons of 111 nucleotides in length (exons 6, 8, 9, 10 and 11) (Figure 1A and [see Additional file 1]). The sequence encoding the DDW Arp2/3 binding site is located within exon 3 and the SH3 domain is encoded by exon 17 and 18. The 3' UTR is 1420 nucleotides in length with a polyadenylation signal AATAAA at position 3225.
Table 1 Accession numbers of cortactin and HS1 sequences
Gene mRNA/EST Protein Genomic DNA Chromosome
COMPLETE CORTACTIN AND HS1 SEQUENCES
Human (Homo sapiens, Hs)
wt-cortactin M98343a AAA58455 AP000487 11q13b
AP000405
SV1-cortactin c BC008799 AAH08799
BC033889 AAH33889
NM_138565 NP_612632
HS1 d X16663 CAA34651 NT_005612 3q13
BC016758 AAH16758
Chimpanzee (Pan troglodytes, Pt)
wt-cortactin AADA01305241e 9
HS1 AADA01307895e 2
Mouse (Mus musculus, Mm)
wt-cortactin U03184 AAA19689 NT_00336 7F5
SV1-cortactin f BC011434 AAH11434
XM_144788 XP_144788
AK084249 BAC39148
HS1 X84797 CAA59265 NW_006107 16B
BC007469 AAH07469
D42120
Rat (Rattus Norvegicus, Rn)
wt-cortactin NW_043405 1q41
SV1-cortactin (isoform B) AF054619 AAC08425
SV2-cortactin (isoform C) AF054618 AAC08424
HS1 XM_221421 XP_221421 NW_042728 11q11
Chicken (Gallus gallus, Gg), wt-cortactin M73705 AAA49031 AADN01110316g 5
SV1-cortactin BU109838g
HS1 ENSGALG00000009778e,p Un
Frog (Xenopus laevis, Xl), wt-cortactin AB027611h BAB79435
Frog (Xenopus tropicalis, Xt), wt-cortactin scaffold_32906I
Zebrafish (Danio rerio, Dr), wt-cortactin AF527956i AAQ09010
HS1 Finished_845o 4
Pufferfish (Takifugu rubripes, Tr), wt-cortactin SINFRUG00000156355e scaffold_853e
HS1 SINFRUG00000124755e scaffold_1329e
Pufferfish (Tetraodon nigroviridis, Tn), HS1 CAG04186 scaf14731 19
Fruit fly (Drosophila melanogaster, Dm) NM_079702 NP_524426 AE003733 3R
AB009998 BAA34397
AB030177 BAB01490
Mosquito (Anopheles gambiae, Ag) XM_315193 XP_315193 AAAB01008952i 2R
Sea urchin (Strongylocentrotus purpuratus, Sp) NM_214617 NP_999782 scaffold_101e
AF064260i AAD08655
Sponge (Suberites domuncula, Sd) Y18027 CAC38778
Y18860 CAC80140
INCOMPLETE CORTACTIN AND HS1 SEQUENCES
Cattle (Bos taurus, Bt), SV1-cortactin TC154749j,k
B222447k
Pig (Sus scrofa), wt-cortactin TC48123j,l
Frog (Xenopus laevis, Xl) HS1 BC060434 AAH60434
Sea squirt (Ciona intestinalis, Ci) TC32922j,m scaffold_101i
White shrimp (Litopenaeus setiferus, Ls) BE846976
White shrimp (Litopenaeus vannamei, Lv) BE188605
Root knot worm (Meloidogyne incognita, Mi) BE188583
BQ613692n
BQ625292n
Root knot worm (Meloidogyne chitwood, Mc) CB856307
BQ613692
Root knot worm (Meloidogyne javanica, Mj) BE578389
aAll accession numbers except as noted below, may be found in the mRNA, EST, protein of genomic databases of NCBI [65].
bChromosomal locations were obtained from UniGene, NCBI [65].
cSV, splice variant. Accession numbers from EST's BE714795, BE717740, BE717751, BE717765, BE717811, E717819, BE717829, BE717871, BE274120, BE728099 [65].
dHS1= Haematopoietic lineage cell-specific protein 1 = HCLS1= hematopoietic cell-specific Lyn substrate 1
eAccession number was obtained from the EnsEMBL [67].
fSV, splice variant. Accession numbers from EST's BE290787, BF321856, BG519413, BG174188, AA762862, AI099054, BF135250 [65].
gAccession numbers were obtained from the U.S. Poultry Gene Mapping Project [75].
hFrom 2500 bp untill 3578 bp of this mRNA is mRNA from another gene. Genomic DNA are pieces of sequences.
iAccession numbers were obtained from the DNA Data Bank of Japan [70].
jAccession numbers from EST's from the TIGR) [69].
kHomologue to actin binding domain of human SV1-cortactin.
lHomologue to C-terminal part of human cortactin incuding the SH3 domain.
mHomologue to repeat 1 to 5 of the actin binding domain of human cortactin.
nAccession numbers were obtained from the European Bioinformatics Institute. Homologue from 5' untills repeat 3 of the actin binding domain of human cortactin [73].
oThe deduced cDNA and protein sequence from the genomic zebrafish Finished_845 sequence is more related to human HS1, while the zebrafish mRNA/protein sequence (AF527956, AAQ09010) showed more homology to human cortactin.
pThe deduced cDNA and protein sequence from the genomic chicken ENSGALG00000009778 showed more homology to human HS1[67].
Figure 1 Exon map of cortactin and HS1 from different species. Exon/intron boundaries found in the genomic databases by performing BLAST searches with the cortactin cDNA of different species to their genomic DNA, are indicated as vertical boxes in different colors. A lack of boxes means that the boundaries were not found. The genomic organization of some species could not be fully elucidated, because cDNA/genomic sequences were not completely available. The actin binding repeat domain of the cortactin protein is represented by red boxes and the SH3 domain by the purple box. The vertical green stripe indicates the sequence coding for the Arp2/3 binding domain. Pro = proline rich region. The y in the proline rich region represents tyrosine phosphorylation sites. Hs, human; Pt, chimpanzee; Mm, mouse; Rn, rat.
Other cortactin homologues have been reported in mouse [3], rat [19], chicken [50], fruit fly (Drosophila melanogaster ) [51], and frog (Xenopus laevis ) [20]. We searched in numerous databases for all known cortactin genes in other species (listed in Table 1). The identification is based on overall amino acid sequence and overall structural homology with human cortactin. Cortactin homologues exist in mammalians (human, chimpanzee, cattle, pig, mouse, rat), birds (chicken), amphibians (frog), fishes (zebrafish, pufferfish), urochordates (sea squirt), invertebrates (sea urchin), insects (fruit fly, mosquito), shrimps, worms and sponges. To date, there is no evidence for the existence of cortactin in unicellular species, nor in plants. Thus, cortactin seems to be restricted to metazoans.
For several species, both cDNA and genomic sequences (total or partial) are available and therefore we were able to reveal their genomic organization using BLASTn. The exon/intron-boundaries were determined and compared to human cortactin [see Additional file 1]. As schematically presented in Figure 1, the genomic organization and the lengths of the exons as well as the locations of the exon/intron boundaries are highly conserved from urochordates to mammalians. Pufferfishes have the shortest known genome of all vertebrate species due to much shorter introns, nevertheless most exon/intron boundaries were conserved and similar to mammalian cortactin. Intriguingly, the number of repeats in the actin-binding domain differs between species (Figure 1A–G). The number of exons and the location of the intron/exon borders of insect cortactin (Drosophila and mosquito) differ considerably with mammalian cortactin, despite the proteins sequences are very similar. Drosophila and mosquito carry 4 repeats in the actin-binding domain. In both species, repeat 1-to-3 and 4 are on separate exons with in mosquito the 4th repeat of the actin binding domain to be encoded by a single 111 bp large exon 2 (Figure 1F,G). Both, sponge (the lowest metazoan) and sea squirt (urochordate) cortactin protein carry 5 repeats. During evolution, after creation of sponges and worms, the coelomata divided into insects and urochordates (that evolved later into vertebrates). The genomic organization of ancestors of the coelomata should reveal the roots of cortactin evolution. However, complete cDNA and/or genomic DNA of cortactin homologues in these species are not yet available.
The genomic organization HS1 homologues
Both nucleotide and amino acid sequence comparisons with cortactin revealed the highest similarity with the hematopoietic lineage cell-specific protein 1 (HS1). So far, HS1 homologues have been reported in human [27], mouse [33], rat and chimpanzee (NCBI database), suggesting that HS1 exists in mammalians only. We determined the intron/exon boundaries of mammalian HS1 genes by aligning the cDNA with the genomic DNA using BLASTn (Figure 1H and [see Additional file 2]). The number and lengths of the exons and the locations of the exon/intron boundaries were very similar to cortactin, especially in the exons that encode the actin-binding domain (compare [see Additional file 1] and [see Additional file 2]). The exons 10–13 of HS1 encoding the centre region between the actin-binding domain and the SH3 domain are longer (633 bp versus 489bp in cortactin) and more divergent compared to corresponding exons of cortactin.
In addition to a single cortactin homologue in all other species, nucleotide sequences comparisons using the mammalian HS1 mRNA and genomic DNA sequences revealed (incomplete) genomic sequences in chicken, pufferfish, zebrafish and frog (Table 1 and Figure 1I–M) that were more related to the HS1 protein (Figure 3 and [see Additional file 3]). Because no HS1 homologues for these species were present in the mRNA/dbEST database (except for X. laevis HS1), the cDNA (and corresponding protein) sequences were deduced from the genomic DNA with BLASTn or were predicted by Ensemble program. In these lower species, two cortactin related proteins exist. To distinguish between cortactin and HS1 variants, only the most conserved N-terminal part of cortactin and HS1 protein variants, including repeat 3 (corresponding to amino acid 1–190 of human cortactin) was used in BLASTp analysis. In each species, one protein variant turned out to be more homologous to human cortactin, and was called cortactin, whereas the other protein variant appeared to be more related to HS1 and was called HS1. This analysis unveiled HS1 proteins with more than 3 repeats in chicken and pufferfish Tetraodon nigroviridis (containing 4 1/2 repeats), pufferfish Takifugi rubripes and Xenopus laevis HS1 (5 1/2 repeats) and zebrafish HS1 (6 1/2 repeats) (Figure 1 I-M).
Moreover, alignments of the exon/intron boundaries of these HS1 genes to the mammalian HS1 genes [see Additional file 2] revealed that exon 7 (repeat 3) of HS1 was most similar to exon 10 (repeat 5) of cortactin suggesting that in mammalians exon 8 and 9 (repeat 3 and 4) of HS1 were lost during evolution. This is supported by the presence of at least one sequence of 111 nucleotides in the 5670 bp intron 6 of human HS1 (location 3271–3381) that is predicted by the program HMMER when performing alignments using a consensus sequence of the 37 amino acid repeats. However, this sequence is not functional because it does not represent an exon based on the consensus sequence of exon-intron junctions ('gt ... ag' rule of intron sequences) and no human transcripts or ESTs of HS1 including this sequence are present in the NCBI databases. In summary, HS1 is not restricted to mammalians only, but exist also in fishes, amphibians and birds and its genomic structure is very similar to that of cortactin.
Different promoter regions explain distinct tissue specificity of cortactin and HS1
Cortactin is widely expressed in most cell types suggesting to be important for vital functions, while HS1 expression is restricted to hematopoietic cells suggesting to be tailored later in evolution to serve a specific function in these cells. In concordance with their tissue-specific expression pattern, we suppose that their expression might be differently regulated. Therefore, we compared the upstream promoter regions of several cortactin and HS1 genes (Figure 2). The mammalian cortactin gene is very GC rich and contains putative SP-1 transcriptional factor binding sites that are common to many TATA-less promoters and typical for promoter regions in 'widely-expressed housekeeping genes'. Ets family transcription factors, found in the HS1 promoters, are specific for hematopoietic cells and involved in controlling the expression of many B cell- and macrophage-specific genes [52] and are critical for development of lymphoid and myeloid cell lineages. The promoter region of Drosophila and mosquito cortactin shares putative transcription factors found both in mammalian cortactin and HS1. Thus at least in mammalians, the nature of the promoters seemed to determine the broad distribution of cortactin expression in various tissues except most hematopoietic cells and the limited expression of HS1 to hematopoetic cells.
Figure 2 A schematic view over 800 bp of the proximal promoters. Distribution of putative binding sites where represented for the transcription factors SP1 (red), GATA1 or GATA2 (green), AP-1 (dark blue), E2F (yellow), cEts (purple), C/EBPa or C/EBPb (light blue) and the TATAA box (gray) and CCAAT box (white) in the promoter regions of cortactin, human (HsCort), chimpanzee (PtCort), mouse (MmCort), mosquito (AgCort), Drosophila (Dmcort), and HS1, human (HsHS1), chimpanzee (PtHS1), mouse (MmHS1) and rat (RnHS1). The mRNA starting point (assigned +1) is indicated by an arrow.
Figure 3 Phylogenetic relationship of cortactin and HS1 genes. Evolutionary comparison of the N-terminal of cortactin and HS1 proteins including repeat 3 (corresponding to nucleotide 1–190 of human cortactin), represented in a phylogenetic tree based on a cluster alogorithmic alignment generated using GeneBee ClustalW 1.83 program. The number of repeats in the full length actin binding domain for the indicated species are depicted between brackets. Hs, human; Pt, chimpanzee; Mm, mouse; Rn, rat; Gg, chicken; Xl, frog Xenopus laevis ; Dr, zebrafish; Tr, pufferfish Takifugu rubripes ; Tn, pufferfish Tetraodon nigroviridis ; Dm, fruit fly Drosophila ; Ag, mosquito; Sp, sea urchin; Sd, sponge.
The significance of the actin binding repeat domain in cortactin and HS1
We recently reported the identification of two alternative splice variants of human cortactin; SV1-cortactin lacking the 6th repeat and SV2 lacking the 5th and 6th repeat resulting in a different F-actin binding properties and decreased cell migration [14]. As shown in Table 1, cortactin splice variants exist in other mammalians as well as in chicken and frog. So far, splice variants in other species have not been identified, suggesting that alternative splicing of cortactin seems to be restricted to higher metazoans. All intron sequences of cortactin bordering the splice site junctions follow the general GT/AG rule [53] except for intron 11 (GC/AG) [see Additional file 1]. As has been shown for other genes, a GT-to-GC transition might be responsible for the generation of an alternatively mRNA transcript [54]. However, in frog (Xenopus laevis ), the SV1-cortactin variant exists despite the splice donor of intron 11 begins with a GT [20]. Thus, concerning the genome of these different species, alternative splicing of the actin-binding domain of cortactin seems to be facilitated during evolution by modulating the splicing machinery by a GT-to-GC transition to create cortactin related variants that influences cellular properties [14]. The relative expression of cortactin splice variants by tissue origin [14] suggested that splice variants might have tissue-specific functions such as fine-tuning the organization of the F-actin cytoskeleton and consequently regulating cell adhesion and migration.
Alternative splicing also occurs in human HS1. Recently a splice variant lacking the 3rd repeat (exon 7) has been found in an SLE patient [44], resulting in enhanced BCR-mediated cell death. This alternative splicing event was due to a germ line mutation. In contrast, the splice donor of HS1 intron 6 begins with a GC [see Additional file 2]. With respect to the similarities between cortactin and HS1, it might be of interest to investigate the occurrence of splicing of HS1 exon 6 and possible biological consequences. The 3rd repeat and its NLS links HS1 to a role in apoptosis, while such a role has not been described for cortactin lacking a NLS. Since the cytoskeleton architecture in hematopoietic lineage cells is very different from that in adherent cells, it is likely that HS1 plays an important role in the construction of tissue-type specific actin networks. Other types of actin cytoskeleton factors, such as the Arp2/3 complex activators of the WASP family have been reported to have distinct tissue specific expression profiles as well. Thus, the apparent role of HS1 in apoptosis is likely due to its actin remodeling related function. Additionally, our genomic comparisons revealed that the 3rd repeat of HS1 corresponds with the 5th repeat of cortactin, and therefore it might be of interest to investigate whether cortactin SV2 variant (lacking the 5th and 6th repeat) might be involved in apoptosis.
The 4th repeat of cortactin has been suggested to be required for F-actin-binding [17]. Genomic comparisons revealed that HS1 lacks this 4th repeat. Nonetheless, HS1 does bind to F-actin and activate the Arp2/3 complex, although at a lower efficiency than cortactin [43]. This suggests that not only a single repeat but the number of repeats is crucial for the F-actin-binding affinity [14,18]. In addition, HS1 contains a PIP2 binding site in each of its 3 repeats, whereas cortactin has only one in the 4th repeat. PIP2 reduces F-actin cross-linking by cortactin, probably due to competition for the same binding site. Due to its higher affinity for PIP2 [36], HS1 restores this cortactin/F-actin cross-linking process by trapping PIP2. This might be of importance in platelets and megakaryocytes where both, cortactin and HS1 are expressed. Taken together, the composition of the repeat domain is also involved in diverting the functions of both genes.
An elegant way to study the function of a protein is to perform loss-of-function experiments. So far, cortactin knock-out models have not yet been generated successfully, because deletion of one allele of cortactin leads to premature differentiation of embryonic stem cells (personal communication in [55]). However, complete loss-of-function mutants of the Drosophila cortactin gene were viable and fertile, except impaired border cell migration during oogenesis [56]. Down-regulation of cortactin by RNA interference, revealed an essential role for cortactin in dendritic spine morphogenesis [57] and in E-cadherin mediated contact formation in epithelial cells [58]. Mice lacking HS1, showed normal development of the lymphoid system [38], however, the antigen-receptor induced clonal expansion and deletion of B and T lymphocytes were impaired. Thus, loss of function studies underscores the divergent functions of HS1 and cortactin in different cell systems.
Cortactin and HS1 are derived from an ancestral vertebrate cortactin-gene by gene duplication
To examine the genesis of the cortactin family, we studied the relationship between the cortactin and HS1 homologues by generating a phylogenetic tree based on a multi-sequence alignment with the ClustalW 1,83 program [see Additional file 3]. We compared the N-terminal regions including repeat 3 (corresponding to nucleotide 1 to 190 of human cortactin), because this is the best-conserved region among all homologues (Figure 3). One cluster contains all known HS1 proteins and appeared to be closest related to a cluster composed by insects (Mosquito (Ag), Drosophila (Dm)), urochordate (sea urchin, (Sp)) and sponge (Sd) cortactin. In this last cluster all the species with only one gene (with the highest similarity with cortactin) are present. This suggests that with the appearance of the vertebrates, an ancestral gene became duplicated to create two genes, which later evolved into cortactin and HS1. This hypothesis is supported by the fact that many genes duplicated at this stage in the evolution, the overall amino-acid sequence in both genes is very similar and the introns are located at the same amino acid position. Furthermore, gene duplication often correlates with a tissue specific expression pattern of the duplicated genes, which is true for mammalian cortactin and HS1.
Figure 4 displays a hypothetical model for the origin of the cortactin and HS1 genes during evolution. The oldest ancestor is the sponge that, like sea squirt (urochordate), carries one cortactin protein with 5 c1/2 repeats. Insects have also one cortactin gene and evolved to 4 1/2 repeats. During evolution, after the creation of the sponge and the worms, the coelomata divided into insects and urochordates (that evolved later into vertebrates). This suggests that during the evolution, the number of repeats decreased in the insects. Unfortunately, no genomic sequences of ancestors of the coelomata that could reveal the roots of cortactin evolution are available yet to perform more detailed genomic analysis.
Figure 4 Model for the origin of cortactin and HS1 during evolution. Exon/intron boundaries from the exons encoding the actin binding repeat domain are represented in yellow. The actin binding repeat domain of the cortactin protein is represented by red boxes.
The genome of pufferfish Takifugu rubripes contains two cortactin-related genomic sequences both including 5 1/2 repeats. Most likely, an ancestor vertebrate cortactin gene underwent gene duplication. From this moment on during evolution, two cortactin/HS1-releated genes are present in all higher species. One gene evolved to mammalian HS1 with a specific function in apoptosis in hematopoietic cells. For its function, exon 8 and 9 (encoding repeat 3 and 4) were not useful and lost during evolution. However, the HS1 protein in pufferfish Takifugu rubripes and frog Xenopus laevis contains 5 1/2 repeats, while chicken and pufferfish Tetraodon nigroviridis HS1 carries 4 1/2 repeats. It might be of interest to investigate the function of these HS1 proteins and their functional differences to mammalian HS1. The other gene evolved to a ubiquitously expressed mammalian cortactin protein with a vital function in the organization of the cytoskeleton and cell migration. The 6th repeat of cortactin most likely originated from a duplication event of the 5th repeat, since the 6th repeat is most similar to the 5th repeat in all species with 6 1/2 repeats. We recently demonstrated that 6 1/2 repeats are necessary for optimal F-actin cross-linking activity and cell migration, while the splice variant lacking both the 5th and 6th repeats (SV2) was less efficient [14]. Thus, the number of repeats in the F-actin binding domain of cortactin fine-tunes its function in cytoskeletal remodeling. For that reason, in higher metazoans, alternative splicing of the F-actin binding domain is most likely facilitated by a GT-GC transition in the splice donor. Alternatively, we can not exclude that gene duplication might have taken place after duplicated of the 5th repeat (dotted arrows), since both zebrafish cortactin and HS1 contain 6 1/2 repeats.
Conclusions
We report the genomic organization of cortactin and HS1 genes of several species. These genes display a conserved genomic organization as the coding regions have almost identical exon/intron structure. Comparison of 5' sequences allows possible regulatory elements that stress their specific tissue distribution. Comparative analysis of the genomic organization and amino acid sequences of cortactin and HS1 provides insight into the evolution of the conserved actin-binding repeat domain, which forms the basis towards understanding specific functions of both genes. Most likely, both genes originated from a gene duplication event and subsequently HS1 lost two repeats, whereas cortactin gained one repeat. Our analysis genetically underscores the significance of the F-actin binding domain in cytoskeletal remodeling, which is of importance for the major role of HS1 in apoptosis and for cortactin in cell migration.
Methods
The genomic structure of human cortactin
To determine the genomic structure of the human cortactin gene, an algorithm was applied based on the consensus sequence of exon-intron junctions ('gt ... ag' rule of intronic sequence) as well as on the codon usage within ORF. Nucleotide sequence comparisons with human cortactin sequences (NCBI, GenBank accession no. M98343) using BLASTn [59] revealed homology with two genomic clones (GenBank accession no. AP000487 and AP000405). With these clones, we determined all exon/intron boundaries and size of all introns and exons (Table 2A) of the human cortactin gene by (1) performing BLAST comparisons with the cDNA against the genomic DNA and (2) using the GeneFinder program [60] based on the consensus sequence of exon-intron junctions ('gt ... ag' rule of intronic sequence) as well as on the codon usage within ORF [61].
To confirm the predicted genomic structure, we determined the intron/exon boundaries using a cloning procedure as described [62]. Genomic DNA of two cosmid clones COS-7.12 and COS-3.72 covering the cortactin gene as determined by the full-length cDNA [5], was amplified with randomly selected primers from the cDNA sequence (GeneBank accession no. M98343). All PCR products that were larger than the cDNA control sample were considered to be caused by intron sequences and compared to genomic sequence (accession number AP000487 and AP000405) using BLASTn [59]. The size of intron 1, 5, 8, 12 and 13 was too large to obtain a reliable sequence.
Because no overlapping genomic sequences immediately 5' of the first exon were present in the database, we performed sequence analysis of a 2.7-kb HincII-HincII fragment representing the first exon and its 5'-flanking sequences from cosmid COS-7.12 cloned into pUC18 (p5'EMS_3135). In addition, we sequenced a 5-kb PCR product using a 5'-primer in the vector (within the TET gene) and 3'-primer (p3135p601: 5'-ccgggtcggccctggattcc-3') within exon 1, subcloned in pUC18 (p5'EMS_4911). Nucleotide sequences of both products were compared with the genomic clones representing the cortactin gene present in the NCBI database (Accession number AP000487 (GI 8118774 and GI 6277297) and AP000405 (GI 8118742)) and used to define the 7.4 kb 5'-flanking region. The PROSCAN program [63] from BIMAS was used to define the 316 bp promoter region preceding exon 1. Putative transcription factor binding sites where determined by the TFSEARCH program [64] and graphically represented in figure 2. Sequences from human cortactin were submitted to NCBI GenBank [65] as accession No. M98343 (cDNA) and AJ288897 (promoter).
Database searching
The (deduced) protein and genomic sequences of all cortactin and HS1 genes were retrieved from various WEB-sites and their available sequence data are summarized in Table 1. In addition, partial cortactin sequences (ESTs and/or genomic) of various organisms were identified based on amino acid sequence homology with existing cortactin proteins. The genomic organization of the sea squirt and Takifugu rubripes could not be completely elucidated, because cDNA/genomic sequences were only partially available. All data were compiled using BLAST searches of the following databases: National Center for Biotechnology Information (NCBI) (Bethesda, MD, USA) [65]; The Wellcome Trust Sanger Institute (Cambridge, UK) [66]; EnsEMBL of The Wellcome Trust Sanger Institute (Cambridge, UK) [67]; DOE Joint Genome Institute (Walnut Creek, CA, USA) [68]; TIGR: The Institute for Genomic Research (Rockville, MD, USA) [69]; DNA Data Bank of Japan (Mishima, Shizuoka, Japan) [70]; Nematode.net Genome Sequencing Center (St. Louis, MO, USA) [71]; Wormbase (NY, USA) [72]; European Bioinformatics Institute (EBI) (Cambridge, UK) [73]; Genoscope National Sequencing Center (Evry, France) [74]; The U.S. Poultry Gene Mapping Project (MI, USA) [75] and UCSC Genome Bioinformatics (Santa Cruz, CA, USA) [76].
To determine the exon/intron boundaries of all cortactin and HS1 genes, available genomic sequences were subjected to sequence alignments of each species-specific cDNA sequence using the BLAST program of NCBI. Using the same algorithms, as described for human cortactin, the exon/intron-boundaries could be predicted. The complete genomic sequences of the 5' flanking region of cortactin of human, chimpanzee, mouse, rat, fruit fly, and mosquito were determined using the various accession numbers of genomic DNA in Table 1. Putative transcription factor binding sites of 800 bp of the 5' flanking regions where determined by the TFSEARCH program (Figure 2). The predicted exon in intron 6 of HS1 was predicted by the bio-informatics program HMMER [77]) The human cortactin 6 1/2 repeats of the actin-binding domain were aligned, resulting in a consensus sequence: (kfGvqkdrvDksAvGfdyqekvekhesqkDysk). With HMMER this consensus sequence was 'tBLASTn' to intron 6 of human HS1. With an acceptable probability (E-value 0.095), the program predicted an exon in this intron 6 (at location 3271–3381).
Amino acid sequence comparisons
Sequence alignments were carried out using the BLAST program of NCBI. The multiple sequence alignments of various cortactin proteins were constructed using Basic GeneBee ClustalW 1.83 [78]. The genome, cDNA or protein was completed for all cortactin homologues and the number of repeats differs across species and between HS1 and cortactin. Only the N-terminal of cortactin and HS1 proteins including repeat 3 (corresponding to amino acid 1–190 of human cortactin) was used to generate a phylogenetic tree, because this is the most conserved part. Predicted nuclear localization signals sequences were obtained using Predict NLS program [79].
List of abbreviations
aa, amino acid(s); bp, base pair(s); BCR, B-cell receptor; EST, expressed sequence tag; HS1, hematopoietic lineage cell-specific protein 1; NLS, nuclear localization signal; RT-PCR, reverse transcriptase polymerase chain reaction; SH3, Src homology; UTR, untranslated region.
Authors' contributions
AGSHvR designed the study on comparative genome analysis, performed database searches, sequence alignments and gene structure prediction and drafted the manuscript. ESS designed, conducted and analyzed the cloning and sequencing of the promoter of human cortactin. VvBvS conducted and analyzed the PCR and sequencing experiments of the exon-intron boundaries of human cortactin and its splice variants. PMK read the manuscript and provided comments. ES helped with writing the paper, provided overall technical guidance and coordination. All authors read and approved the final manuscript.
Supplementary Material
Additional File 1
Splice donor and acceptor sequences of cortactin in different species.
Click here for file
Additional File 2
Splice donor and acceptor sequences of HS1 in different species.
Click here for file
Additional File 3
Multiple amino acid sequence alignment of cortactin and HS1 homologues.
Click here for file
Acknowledgements
This work was supported by grant NKB-RUL 98–1647 of the Dutch Cancer Society. We thank Berend Snel for general discussions and for critical reading of the manuscript.
==== Refs
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Alexander H Alexander S Identification of introns by reverse-transcription PCR Biotechniques 1996 20 778 780 8723915
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| 15710041 | PMC554100 | CC BY | 2021-01-04 16:39:33 | no | BMC Genomics. 2005 Feb 14; 6:15 | utf-8 | BMC Genomics | 2,005 | 10.1186/1471-2164-6-15 | oa_comm |
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BMC GastroenterolBMC Gastroenterology1471-230XBioMed Central London 1471-230X-5-51572071010.1186/1471-230X-5-5Research ArticleCentral neuropeptide Y receptors are involved in 3rd ventricular ghrelin induced alteration of colonic transit time in conscious fed rats Tebbe Johannes J [email protected] Clemens G [email protected] Silke [email protected] Michael [email protected]äfer Martin KH [email protected] Department of Internal Medicine, Division Gastroenterology – Endocrinology, Philipps Universität Marburg, 35033 Marburg, Germany2 Department of Internal Medicine, Division Cardiology, Philipps Universität Marburg, 35033 Marburg, Germany3 Department of Molecular Neuroscience, Institute of Anatomy and Cell Biology, Philipps Universität Marburg, 35033 Marburg, Germany2005 18 2 2005 5 5 5 25 8 2004 18 2 2005 Copyright © 2005 Tebbe et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Feeding related peptides have been shown to be additionally involved in the central autonomic control of gastrointestinal functions. Recent studies have shown that ghrelin, a stomach-derived orexigenic peptide, is involved in the autonomic regulation of GI function besides feeding behavior. Pharmacological evidence indicates that ghrelin effects on food intake are mediated by neuropeptide Y in the central nervous system.
Methods
In the present study we examine the role of ghrelin in the central autonomic control of GI motility using intracerobroventricular and IP microinjections in a freely moving conscious rat model. Further the hypothesis that a functional relationship between NPY and ghrelin within the CNS exists was addressed.
Results
ICV injections of ghrelin (0.03 nmol, 0.3 nmol and 3.0 nmol/5 μl and saline controls) decreased the colonic transit time up to 43%. IP injections of ghrelin (0.3 nmol – 3.0 nmol kg-1 BW and saline controls) decreased colonic transit time dose related. Central administration of the NPY1 receptor antagonist, BIBP-3226, prior to centrally or peripherally administration of ghrelin antagonized the ghrelin induced stimulation of colonic transit. On the contrary ICV-pretreatment with the NPY2 receptor antagonist, BIIE-0246, failed to modulate the ghrelin induced stimulation of colonic motility.
Conclusion
The results suggest that ghrelin acts in the central nervous system to modulate gastrointestinal motor function utilizing NPY1 receptor dependent mechanisms.
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Background
The presence or absence of food in the gut stimulates the release of several regulatory peptides. These orexigenic (NPY, AGRP, ghrelin, MCH, Orexin-A, ...) and anorexigenic (CRF, CCK, CART, GLP-1, leptin, insulin, ...) peptides participating in the hypothalamic control of feeding behavior and satiety have been shown to be additionally involved in the autonomic control of gastointestinal (GI) functions like secretion and motility. For example fasted motor activity of the GI tract, e.g. the colon, is observed after intracerebroventricular (ICV) injection of neuropeptide Y whereas CRF ICV-treatment cause the disruption of fasted colonic motor activity [1]. Stomach-derived ghrelin is the first peripheral orexigenic peptide identified [2-6]. There is convincing evidence from several groups of investigators that ghrelin acts in the CNS and the periphery to simulate not only feeding but also GI function such as gastric acid secretion and gastric motility in rodents [7,9-11]. However, it is still unknown whether ghrelin is involved in the CNS control of other digestive functions besides gastric acid secretion and motility. Recent studies suggest that CNS-signaling by circulating ghrelin is mediated downstream by neurons of arcuate nucleus and the paraventricular nucleus of the hypothalamus, in particular, neurons expressing neuropeptide Y and agouti-related protein (AGRP) [12-14]. Furthermore it has been demonstrated that there is an anatomical interaction and functional relationship between ghrelin and neuropeptide Y. Using electrophysiological recordings Cowley et al have found that ghrelin stimulated the activity of arcuate NPYergic neurons and mimicked the effect of NPY in the paraventricular nucleus of the hypothalamus [15]. In addition ghrelin simulates food intake through hypothalamic NPY1 receptors [1,16,17]. Thus, the question came up "are NPY receptors involved in the ghrelin effect on GI function"? Among others, neuropeptide Y plays a role in the CNS control of gastrointestinal function [1,18]. NPY activates at least six receptor subtypes, NPY1 to NPY6. NPY binds preferentially with high affinity to Y1 and Y2 receptors, and there is evidence suggesting that these two receptor subtypes are involved in CNS regulation of digestive function by NPY action in arcuate nucleus and the paraventricular nucleus of the hypothalamus [18].
Taken together there is overwhelming evidence that ghrelin, beside its satiety modulatory capacity, is involved in the CNS control of digestive function of the upper gastrointestinal tract. In the CNS ghrelin and NPY, the most potent orexigenic neuropeptides known, are anatomical associated and functionally related. Moreover hypothalamic NPYergic neurons are downstream mediators of feeding related ghrelin action.
In the present study we scrutinize the hypothesis that central neuropeptide Y receptor activation is involved in the ghrelin induced modulation of gastrointestinal motility using a microinjection-model in conscious fed and freely moving rats.
Methods
Animals
All experimental components described were performed in accordance with the requirements of German legislation for the protection of animals and were licensed and supervised by the appropriate government body.
Male Sprague-Dawley rats with a mean body weight of 350 ± 50 g were maintained on a 12 : 12 h photoperiod. They were housed in colony cages under conditions of controlled humidity and temperature (22 ± 2°C) for at least 7 days prior to the surgical procedure. The animals were fed a standard rat diet (Altromin®, Lage, Germany) an tap water ad libitum. After surgical procedures, rats were housed individually. During experimental procedure the animals had continuous access to food and water.
Drugs
Ghrelin (Bachem, Heidelberg, Germany) doses of 0.03 nmol (100 ng), 0.3 nmol (1 μg) and 3 nmol (10 μg)/5 μl were dissolved in 0.15 M sterile saline (B. Braun, Melsungen, Germany). The NPY1 receptor antagonist, BIBP-3226 (200 nmol/5 μl; Sigma-RBI, Natrick, MA, USA) [see Ref.: [19]] and the NPY2 receptor antagonist, BIIE-0246 (120 nmol/5 μl; Boehringer-Ingelheim, Biberach, Germany) [see Ref.: [21]] were dissolved in sterile 0.15 M saline. The NPY receptor antagonists were used in similar equipotent nanomolar concentrations. The used intracerebroventricular concentrations of the receptor antagonists were comparable with the ICV-dosages used by other groups in rodents [18,20]. Probes were aliquoted and frozen (-80°C). Fresh aliquots were thawed on each experimental day before injections. Any excess was discarded. In our hands nanomolar concentrations of BIBP-3226 and BIIE-0246 were effective in antagonization of NPY receptor subtypes without any side effects. In particular no central depressive effects or conspicuous behavior was observed after BIBP-3226 treatment [18].
Cerebral cannulas
For surgical procedures, rats were anesthetized with a mixture of ketamine (75 mg kg-1 i.p., Parke-Davis, Freiburg, Germany) and xylazine (5 mg kg-1 i.p., Bayer AG, Leverkusen, Germany). Animals were positioned in a stereotactic apparatus (David Kopf Instruments, Tujunga, CA). The head was fixed in a nose-down-position (-3 mm) and the skull exposed. Then trepanation of the skullcap was performed according to coordinates obtained from Paxinos and Watson [22] (mm from bregma: anterior-posterior = -3.30; lateral = ± 0.0; dorsoventral = -3.8). According to these coordinates a 22-gauge guide cannula (Bilarney / Plastic one, Düsseldorf, Germany) was implanted into the third ventricle. The cannula was anchored by dental cement and stainless steel screws affixed to the skull. Dummy cannulas (28 G), extending 2 mm beyond the guide cannula tips, were inserted to prevent blockage. After cerebral surgery, animals were individually housed. The animals were allowed 4 days recovery after guide cannula surgeries before the abdominal surgical procedures were performed.
Colonic catheter
This method was performed as described elsewhere [1]. Prior to all abdominal surgeries, the animals were food deprived overnight. Four days after cerebral surgery, rats were anaesthetized with a mixture of ketamine (75 mg kg-1) and xylazine (5 mg kg-1). After laparatomy a polyethylene microcatheter (inside diameter, 1.2 mm; outside diameter 1.7 mm; Becton Dickinson, New Jersy, USA) was chronically implanted into the proximal colon 1 cm distal from the caecocolonic junction. The catheter was fixed at the colonic wall by a purse-string suture and routed subcutaneously to the interscapular region, where it was exteriorized through the skin and secured. The animals were allowed 7 days recovery after abdominal surgeries before the beginning of habituation training sessions. Experiments were performed in fed, conscious rats.
Intraperitoneal (IP) and intracerebroventricular (ICV) microinjection
The doses of ghrelin were calculated according to the lowest effective doses to stimulate food intake [see Ref.: [23]]. For IP injection a 1 ml syringe (Hamilton, Reno, NV, USA) was used. Ghrelin and vehicle were injected IP after central administration of vehicle or NPY receptor antagonist. For IP injections the low dose of ghrelin administered peripherally was 0.3 nmol kg-1/1 ml 0.15 M saline and the high dose of ghrelin was 3 nmol kg-1/1 ml saline. NPY receptor antagonists were injected ICV 15 min before ghrelin was given peripherally at doses of 200 nmol/rat (BIBP-3226) and 120 nmol/rat (BIIE-0246) respectively.
For ICV injections a 1 μl micro syringe (Hamilton, Reno, NV, USA), attached to a 32 G injection needle via a PE-50 tube-catheter was used. The stainless steel injection cannulas (32 G) were cut to protrude 2 mm beyond the tips of the guide cannulas. The conscious animals were gently restrained by hand, the injection needle was inserted through the guide cannula, and vehicle (5 μl 0.15 M saline) or NPY receptor antagonists (BIBP-3226 200 nmol/5 μl; BIIE-0246 120 nmol/5 μl) and ghrelin (0.03 nmol, 0.3 nmol or 3 nmol/5 μl), were consecutively injected ICV slowly over a 60 s period. We used a 15 min time interval between ICV injection of receptor antagonist or vehicle and ICV ghrelin administration. The injection needle was left in place for 2 min after injection to allow diffusion of the solution and to prevent back flow. Then dummy cannulas were reinserted into the guide cannulas. After the last experimental testing session, the rats were anesthetized and 5 μl of alcian blue 8GX were injected ICV.
Colonic transit time measurement
Colonic transit time was calculated by using an enteral dye-marker. Trypan blue, a non-absorbable dye, was injected in 0.2 ml volume through the catheter positioned in the proximal colon and followed by a 0.2 ml saline flush immediately after the ICV or IP microinjection. Colonic transit time was evaluated as the time interval between dye injection and the discharge of the first blue pellet. Faecal pellet output was monitored continuously by a self-developed, automated observation system that mechanically registers the time of all bowel movements for 24 h. The device consists of a conveyor belt placed under the mesh bottom cage, which transports faecal pellets with defined velocity to a collector.
Brain histology
The methods were performed as described in previous studies [17]. When experiments were completed, rats were anaesthetized with ketamine (75 mg kg -1 i.p.) and xylazine (5 mg kg -1 i.p.), and 0.05% alcian blue 8GX was microinjected intracerebroventricular under the same conditions as vehicle or peptide. The anaesthetized animals were transcardially perfused with phosphate buffered saline (PBS) buffer (0.1 M, pH 7.4) followed by Zamboni's fixative (2% formaldehyde and 2% picric acid in 0.1 M PBS buffer, pH 7.4). The brains were removed and cryoprotected in 25% sucrose. The site of injection was confirmed by inspection of intracerebroventricular dye distribution. Animals that received injections outside of the 3rd ventricle were excluded from data analysis.
Experimental design
Experiment I: Effect on colonic motorfunction of peripheral (IP) and central (ICV) ghrelin administration
The aim of the first experiment was to determine whether exogenous ghrelin would alter colonic motor function. Thus in experiment I, dose response effects of ghrelin in the cerebrospinal fluid (ICV) and the periphery (IP) on colonic transit time were examined. Ghrelin or saline as a vehicle was administered ICV or IP in conscious lightly restrained rats as previously described. For IP injection the low dose of ghrelin administered peripherally was 0.3 nmol kg-1 BW and the high dose of ghrelin was 3.0 nmol kg-1 BW. After injections, rats were subsequently returned to their home cages and maintained in a non-stressful environment to monitor colonic transit time. In order to minimize interindividual variation, and to reduce the number of animals needed to perform this study, animals were tested twice in this study. In randomized order, each rat received vehicle and a single dose of ghrelin or vehicle ICV or IP. The time interval between the experiments performed on the same animal was at least 4 days.
Experiment II: Effect of BIBP-3226 and BIIE-0246 pretreatment on centrally and peripherally injected ghrelin induced modulation of colonic transit
In experiment II the hypothesis that ghrelin acts at the CNS to modulate colonic motor function via a NPY receptor dependent pathway was addressed. Therefore, we determined if a pretreatment with selective NPY1- (BIBP-3226) and NPY2 (BIIE-0246) receptor antagonists administered into the cerebrospinal fluid would block the alterations of colonic motor activity induced by centrally and peripherally administered ghrelin. The animals were pretreated with the NPY-Y1 and -Y2 receptor antagonists, injected ICV or vehicle (0.15 M saline), 15 min prior to ICV or IP ghrelin injections. Thereafter colonic transit time was assessed as described above.
Data analysis
The criterion used to include results in the data analysis of the ICV-injected group was the correct placement of the ICV cannulas.
Results are expressed as mean ± SEM. The data of all studies were analyzed by ANOVA and differences between groups were evaluated by the Student-Newmann-Keuls test. P < 0.05 was considered significant.
Results
Effect of peripherally (IP) and centrally (ICV) administered ghrelin on colonic motor function
In experiment I, dose response effect of peripherally and centrally administered ghrelin on colon transit time in fed and freely moving rats were examined. As demonstrated in Fig. 1, ghrelin injected into the cerebrospinal fluid (CSF) stimulates colonic transit time dose dependently. In rats microinjected with vehicle into the CSF or IP, the average colonic transit time was 322 ± 8 min. As demonstrated in Fig. 1, 0.03 nmol, 0.3 nmol and 3 nmol/5 μl ghrelin injected ICV dose-dependently decreased transit time by 24%, 34% and 43% respectively in conscious fed rats. Peripherally administered ghrelin accelerated transit time up to 36% (Fig. 1).
Effect of ICV NPY receptor antagonist pretreatment on 3rd ventricular and peripherally ghrelin induced stimulation of colonic transit
The hypothesis that ghrelin acts in the brain to stimulate colonic transit via NPY receptor dependent mechanisms was addressed. As shown in Fig. 2, pre-treatment with BIBP-3226 (200 nmol) which is a selective NPY1 receptor antagonist 15 min prior to ICV application of 0.3 nmol ghrelin totally blocked the ghrelin induced effect on colonic transit. Application of BIBP-3226 into the CSF of the control group that was microinjected with vehicle, had no effect. Changes in colonic transit time induced by IP injection of ghrelin (3.0 nmol kg-1 BW) were canceled by ICV injection of NPY1 receptor antagonist, BIBP-3226. (Fig.: 2). Pretreatment with the selective NPY2 receptor antagonist, BIIE-0246, failed to affect the ghrelin induced alteration of colonic transit time. (Fig.: 2)
Discussion
The present experiments using a freely moving conscious rat model permit the measurement of colonic motility in rats in the physiological fed status. The results demonstrated that ghrelin given ICV and IP stimulates gastrointestinal motility indicated by shortened colonic transit time. In addition we found that the NPY type 1 receptor is primarily involved in the ghrelin induced modulation of fasted motor activity of the colon.
There is convincing evidence that the most effective appetite stimulating peptides, NPY and ghrelin, act in the CNS and the periphery to simulate not only feeding but also GI function such as gastric acid secretion and gastric motility [1,8-10]. Stomach derived ghrelin, first described in 1999 by Kojima et al., is the first peripheral orexigenic peptide identified [4]. Ghrelin was identified as endogenous ligand for the GH secretagogue receptor (GHS R) and a peripheral metabolic signal informing the brain about stomach nutrient load [17,24]. Physiological studies suggest a functional relationship of ghrelin and NPY within the brain. It has been demonstrated that peripherally (i.v.) and central (ICV) administered ghrelin increases the expression of the immediate early gene c-fos, a marker of neuronal activity, in the arcuate nucleus and the PVN in awake fed rats [25]. Furthermore, exogenous ghrelin increases mRNA levels for NPY into the arcuate nucleus and simulates food intake through hypothalamic NPY1 receptors [14,16,26]. Further Fujino et al. have demonstrated that ICV pretreatment with neuropeptide Y antiserum completely blocked ghrelin induced gastric and duodenal motoractivity [9]. Taken together these data suggest that there is an anatomical interaction and functional relationship between ghrelin and NPY within the brain. Six recognized subtypes of neuropeptide Y receptors have been described (NPY1 to NPY6). Two of these, NPY1- and NPY2 receptors, are found in high density in the hypothalamus. There is compelling evidence that, in particular, NPY1 and NPY2 receptors are involved in the CNS regulation of gastrointestinal function. [1,8,27] For this reason, we focused on neuropeptide Y1 and Y2 receptor pathways in the present study and did not investigate the role of neuropeptide Y receptor subtypes Y4 and Y5 which are also expressed in the hypothalamus and are also involved in the autonomic control of feeding behavior and GI function. In the present study pretreatment with the NPY1 receptor antagonist, BIBP-3226, blocked stimulation of colonic motility induced by systemic microinjection of exogenous ghrelin (ICV and IP). In our hands BIIE-2046, which is a selective antagonist of the NPY Y2 receptor, failed to affect the ghrelin induced induction of fasted motor activity. It was previously described that knocking out NPY significantly decreases ghrelin stimulated feeding [17,28]. In this context Fujino et al. have recently demonstrated that the ghrelin induced fasted gastroduodenal motor activity in rats is blocked by ICV injection of GHS-R antagonist as well as NPY antiserum [9]. The results presented by Fujino et al. also suggest that the vagal pathway may mediate the action of centrally administered ghrelin on gastroduodenal motility [9]. Thus we can speculate that central NPY pathways, e.g. centrally NPY receptor activation, are the primary downstream mediator of circulating ghrelin. This interpretation is consistent with neuroanatomical and physiological facts: Neuropeptide Y works at two sites, locally within the arcuate nucleus to inhibit POMC neuronal activity and at afferent-terminal sites, in particular the paraventricular nucleus of the hypothalamus. Guan et al. have shown that neuropeptide Y- and ghrelin like immunoreactive (LI) neurons within the arcuate nucleus could influence each other by complex synaptic transmissions [29]. Furthermore Cowley et al. have demonstrated that ghrelin stimulated the activity of arcuate neuropeptide Y-LI neurons and mimicked the effect of neuropeptide Y in the PVN [15]. Compelling evidence showed that NPY projections from the arcuate nucleus (ARC) to the PVN are involved in the CNS regulation of food intake and other physiological functions of the organism, e.g. digestive function, by neuroendocrine and autonomic pathways [17,18]. For example NPY released from ARC neurons activates NPY-Y1 receptors in the hypothalamus, e.g. the PVN, and results in the stimulation of GI motor function [18]. Furthermore arcuate NPYergic neurons have been thought to regulate feeding behavior by NPY receptor subtypes Y1 and Y5 in the PVN and adjacent areas [17]. Pretreatment with a Y1, but not other receptor antagonist markedly inhibited ghrelin-induced feeding, pointing to NPY receptor Y1 as one of the downstream pathways [9,17]. With regard to the characteristic physiological feature that peripheral ghrelin does not cross the blood-brain barrier in rodents it is important to note that the arcuate nucleus is the only hypothalamic structure located outside the brain-blood barrier [30]. Thus we can speculate that circulating ghrelin modulates gastrointestinal motility via activation of hypothalamic, in particular by using NPYergic pathways via activation of NPY-Y1 receptors, in the arcuate nucleus. This hypothesis is in good agreement with our observation that the effect of peripherally (IP) administered ghrelin on colonic motility is blocked by ICV pretreatment with the specific NPY1 receptor antagonist, BIBP-3226. The NPY2 receptor antagonist BIIE-0246 injected in the 3rd ventricle at the equipotent dose as BIBP-3226 was not effective to antagonize the ghrelin effect on GI motility significantly. This data suggests that ghrelin unfolds a stimulatory effect on colonic motility primarily by acting on central NPY1 and not via NPY2 receptors. This interpretation is confirmed by the observation that Y1 receptors acts rather postsynaptically and the Y2 receptor rather presynaptically [31,32] The question of whether neuropeptide Y4 or Y5 receptors in the CNS are involved in the CNS control of gastrointestinal function should be examined in future studies.
Conclusion
We hypothesize that circulating ghrelin exhibits its effect by activating hypthalamic neurons, in particular neurons in the arcuate nucleus bearing GHS- and NPY-Y1 receptors. Further this ghrelin induced neuronal activation leads to stimulation of GI motor function by activation of higher hypothalamic brain sites, e.g. activation of neuronal projections within the paraventricular nucleus of the hypothalamus. On the other hand it is possible that the site of action of circulating ghrelin is not the hypothalamus but other brain sites. In our model using 3rd ventricular injection of ghrelin this could simply mean that the peptide gained access to the 4th ventricle and reached further caudal brain sites, e.g. NTS, DVC and medulla oblongata. With respect to the distribution of GHS- and NPY receptors in the CNS this hypothesis is possible, but how ghrelin action on any of these brain sites would modulate digestive function is not known. This question should be examined in future studies
In summary, we presented evidence that ghrelin is involved in the CNS control of GI function. Apart from humoral pathways ghrelin acts into the CNS to control GI function by a mechanism of action involving neuropeptide Y1 receptor pathways. Further this study support the hypothesis giving by Chen et al. that ghrelin has an absolute requirement for neuropeptide Y pathways to unfold its physiological effects [28].
List of abbreviations
AGRP agouti-related peptide
ARC arcuate nucleus
CART cocain- and amphetamine-regulated transcript
CCK cholecystokinin
CNS central nervous system
CSF cerebrospinal fluid
CRF corticotropin releasing factor
DVC dorsal motor nucleus of vagus
GHS-R growth hormone secretagogue receptor
GLP-1 glucagon like peptide-1
ICV intracerebroventricular
MCH melanocortin hormone
MI microinjection
NPY neuropeptide Y
NTS nucleus of the solitary tract
POMC proopiomelanocortin
PVN paraventricular nucleus of the hypothalamus
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
JJT participated in the design and coordination of the study, performed the microinjection studies and drafted the manuscript. CGT was the surgeon in charge and participated in the animal experiments. M-KHS and SM were involved in the design and coordination of the study. MR participated in the analysis and interpretation of data and revised the manuscript critically.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We are grateful to Boehringer-Ingelheim, Biberach, Germany, for the generous donation of BIIE-0246. This work was supported by a grant from the German Research Foundation (DFG) to J.J.T. and M. K.-H. S. (DFG: TE 307 1-2). Part of this study contained in the medical thesis of CG Tebbe. We thank R. Weber for excellent technical support.
Figures and Tables
Figure 1 Effect of ghrelin injected into the 3rd ventricle (ICV) and intraperitoneally (IP) on colonic transit time. Ghrelin injected ICV as well as IP induced a dosed-related stimulation of propulsive colonic motor activity. MI = microinjection The bars represent the mean ± SEM. * P < 0.05 vs. vehicle. ■ = vehicle-group; A = 0.03 nmol ghrelin /5 μl 0.15 M saline; B = 0.3 nmol ghrelin /5 μl 0.15 M saline; C = 3 nmol ghrelin /5 μl 0.15 M saline; D = 0.3 nmol ghrelin kg-1 BW; E = 3.0 nmol ghrelin kg-1 BW
Figure 2 Effect of pretreatment with NPY receptor antagonists on fasted motor activity of the colon induced by centrally (ICV) and peripherally (IP) administered ghrelin. BIBP-3226, which is a selective NPY Y1 receptor antagonist, injected ICV antagonizes the stimulation of colonic transit induced by ghrelin injected in the same route and IP. The NPY Y2 receptor antagonist BIIE-2046 injected ICV fails to affect the stimulated colonic motoractivity induced by ICV or IP injection of ghrelin. MI = microinjection The bars represent the mean ± SEM. * P < 0.05 vs. vehicle-group; #P < 0.05 vs. ghrelin-group
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Nakazato M Murakami N Date Y Kojima M Matsuo H Kangawa K Matsukura S A role for ghrelin in the central regulation of feeding Nature 2001 409 194 198 11196643 10.1038/35051587
Tomasetto C Wendling C Rio MC Poitras P Identification of cDNA encoding motilin-related-peptide/ghrelin precursor from dog fundus Peptides 2001 12 2055 2059 10.1016/S0196-9781(01)00557-5
Date Y Nakazato M Murakami N Kojima M Kangawa K Matsukura S Ghrelin acts in the central nervous system to stimulate gastric acid secretion Biochem Biophys Res Commun 2001 280 904 907 11162609 10.1006/bbrc.2000.4212
Fujimiya M Itoh E Kihara N Yamamoto I Fujimura M Inui A Neuropeptide Y induces fasted pattern of duodenal motility via Y2 receptors in conscious fed rats Am J Physiol Gastrointest Liver Physiol 2000 279 G32 38
Fujino K Inui A Asakawa A Kihara N Fujimura M Fujimia M Ghrelin induces fasted motor activity of the gastrointestinal tract in conscious fed rats J Physiol 2003 550 227 240 12837928 10.1113/jphysiol.2003.040600
Masuda Y Tanaka T Inomata N Ohnuma N Tanaka S Itoh Z Hosoda H Kojima M Kangawa K Ghrelin stimulates gastric acid secretion and motility in rats Biochem Biophys Res Commun 2000 276 905 908 11027567 10.1006/bbrc.2000.3568
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Seoane LM Lopez M Tovar S Casanueva FF Senaris R Dieguez C Agouti-related peptide, neuropeptide Y, and somatostatin-producing neurons are targets for ghrelin actions in the rat hypothalamus Endocrinology 2003 144 544 551 12538615 10.1210/en.2002-220795
Cowley MA Smith RG Diano S Tschop M Pronchuk N Grove KL Strasburger CJ Bidlingmaier M Esterman M Heiman ML Garcia-Segura LM Nillni EA Mendez P Low MJ Sotonyi P Friedman JM Liu H Pinto S Colmers WF Cone RD Horvath TL The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis Neuron 2003 37 649 61 12597862 10.1016/S0896-6273(03)00063-1
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| 15720710 | PMC554101 | CC BY | 2021-01-04 16:03:27 | no | BMC Gastroenterol. 2005 Feb 18; 5:5 | utf-8 | BMC Gastroenterol | 2,005 | 10.1186/1471-230X-5-5 | oa_comm |
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BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-5-181571793310.1186/1471-2407-5-18Research ArticleAccess to communication technologies in a sample of cancer patients: an urban and rural survey Abdullah Ma'n [email protected] Dale E [email protected] Donna [email protected] Kurt [email protected] Anthony [email protected] Sara [email protected] William M [email protected] Regenstrief Institute for Health Care, Department of Medicine, Indiana University School of Medicine and the Richard L. Roudebush VA Medical Center, Indianapolis, IN, USA2 Community Cancer Care, Community Health Network, Indianapolis, IN, USA2005 17 2 2005 5 18 18 17 9 2004 17 2 2005 Copyright © 2005 Abdullah et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
There is a growing awareness among providers of the symptom burden experienced by cancer patients. Systematic symptom screening is difficult. Our plan was to evaluate a technology-based symptom screening process using touch-tone telephone and Internet in our rural outreach cancer program in Indiana. Would rural patients have adequate access to technologies for home-based symptom reporting?
Objectives
1) To determine access to touch-tone telephone service and Internet for patients in urban and rural clinics; 2) to determine barriers to access; 3) to determine willingness to use technology for home-based symptom reporting.
Methods
Patients from representative clinics (seven rural and three urban) in our network were surveyed. Inclusion criteria were age greater than 18, able to read, and diagnosis of malignancy.
Results
The response rate was 97%. Of 416 patients completing the survey (230 rural, 186 urban), 95% had access to touch-tone telephone service, while 46% had Internet access (56% of urban patients, 38% of rural patients). Higher rates of Internet access were related to younger patient age, current employment, and higher education and income. The primary barrier to Internet access was lack of interest. Use of the Internet for health related activities was less than 50%. The preferred means of symptom reporting in patients with internet access were the touch-tone telephone (70%), compared to reporting by the Internet (28%).
Conclusion
Access to communication technologies appears adequate for home-based symptom reporting. The use of touch-tone telephone and Internet reporting, based upon patient preference, has the potential of enhancing symptom detection among cancer patients that is not dependent solely upon clinic visits and clinician inquiry.
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Background
In recent years awareness of the symptom burden experienced by many cancer patients has grown [1,2]. At some time in their illness, symptoms such as fatigue, pain, nausea, depression, and hopelessness are very likely to occur. These symptoms can be disabling and they can even limit treatment. There is a growing body of literature demonstrating that interventions for these troubling symptoms are effective [3,4]. These interventions can improve the patient's quality of life by enabling the patient to function better at home and at work.
While there is awareness among providers of symptom distress experienced by patients and there are effective symptom interventions, the problem in the day-to-day care of cancer patients is symptom identification [5]. At a recent meeting convened by the National Institute of Health, it was concluded that little is known about the actual frequency and validity of symptom screening for common cancer and cancer treatment related symptoms. In the summary statement there was expert consensus about the need for routine screening for symptoms from the point of diagnosis. Assessments should be repeated during the course of treatment. Symptom data should be integrated into routine care of cancer patients.
Community Cancer Care (CCC) is an organization with home offices in Indianapolis, Indiana, that provides professional services and program development services to 23 hospitals throughout the state of Indiana. Professional oncology services are provided by 18 medical oncologists-hematologists who are employed by CCC or serve under contract. One psychiatrist, an advanced-practice nurse, and a certified nurse are dedicated to quality-of-life efforts. Each year, an average of 2500 new patients are seen in the network of clinics. At any given time, approximately 16,000 patients are receiving care in the CCC network. While the CCC has clinics in metropolitan Indianapolis, rural outreach and program development in rural hospitals have been a major focus of CCC since its inception in 1983. Twenty-one clinics are located in Indiana towns with populations less that 16,000. Twenty counties served by CCC have populations less than 45,000.
Using paper and pencil scales we unsuccessfully tried to install a symptom screening process into the daily clinic workflow. The clinic process was slowed. Some patients could not complete the instruments. Patients' report of their symptoms could not be analyzed quickly and placed on the chart for the provider to use. Symptom screening was limited to the day of the clinic visit. We could not easily evaluate a patient's status between office visits. Trends in symptom occurrence were difficult to identify. With pencil and paper instruments it was a laborious and expensive process to establish a database for our patients' symptom reports, a necessary step in program evaluation.
Because of these limitations, our goal is to develop a technology solution to gather, analyze, and present symptom reports to physicians and nurses. Several feasible options for reporting symptoms would include either a touch-tone telephone or an Internet connected computer. Because of well-documented differences between access to the telephone service and the Internet [7], we conducted a survey in urban and rural oncology clinics to determine how many of our network patients had access to the required communication technology. For patients who had access to the Internet we were interested in identifying predictors of access as well as patients' willingness to use the Internet for symptom reporting and other cancer-related reasons.
Methods
Procedures
The study design and survey instrument were reviewed and approved by the Institutional Review Board of Community Medical Research Institute in Indianapolis.
A convenience sample of cancer patients was gathered from the clinic network of CCC. Three urban clinics and seven rural clinics were conveniently selected for data collection. All of these sites had concentrated, busy clinic days during which patients could be recruited. Clinics were designated "urban" or "rural" based on their zip code being categorized urban or rural by the U.S. Department of Health and Human Services, Office of Rural Health Policy [6].
Staff members at clinic sites were instructed to offer the survey instrument to all patients attending clinic during selected weeks of March and April 2003. All patients were volunteers. All patients had to be at least 18 years of age, be able to read, and have a diagnosis of malignancy (either solid tumor or blood). The number of patients who refused to complete the survey was recorded.
The survey instrument
The survey instrument included nine items about demographics and access to touch tone telephone service and the Internet. If patients indicated they did not have access to the Internet the survey instrument directed them to questions about reasons they did not have access. If patients indicated they did have access to the Internet, the survey instrument directed them to seven additional questions about how they use or might use the Internet.
Statistical analysis
We used two-sample t-tests to test for mean differences and chi-square tests to test for differences in proportions of demographic characteristics across clinic setting and access to the Internet. Logistic regression models were used to evaluate access to the Internet as a function of clinic setting adjusting for demographic characteristics.
Results
Four hundred and sixteen patients completed the survey (230 rural, 186 urban). The response rate was 97%. Thirteen patients refused to complete the survey stating they were too ill or too tired. Table 1 summarizes characteristics of the sample, comparing patients in urban vs. rural settings. Patients in the rural sample were significantly older, had lower education levels, and were more likely to be Caucasian than patients in the urban sample. Touch-tone telephone service was available to most (95%) respondents, while 46% (95% [CI] 0.41–0.51), had access to the Internet. Compared to urban patients, those in rural settings had comparable telephone access but were less likely to have Internet access (38% vs. 56%, p < .001). Most patients (> 80%) reported accessing e-mail and Internet from home. As shown in Table 2, patients with Internet access were significantly younger and had higher education and income levels than patients without Internet access. Additionally, patients with Internet access were more likely to be currently employed and from an urban clinic. Table 3 summarizes the results of a logistic regression model for Internet access. Higher income and current employment increased the likelihood of having Internet access while older age and less education decreased the likelihood.
Two-thirds (67%) of people cited lack of interest for not having Internet access. Other common reasons were unfamiliarity with the Internet (21%), cost (20%), and hesitation to use a computer (13%). There were no significant differences between the urban and rural patients regarding why they did not access the Internet.
Fifty percent of patients with Internet access reported using it for health care purposes in both rural and urban clinics, and nearly 60% reported having used the Internet to seek information about their cancer. Among the 169 patients with Internet access who indicated their preferred method(s) for symptom reporting, the telephone was identified as the most popular method (70.4% of respondents) followed by Internet-based symptom reporting (28%) and touch-screen computer in the clinic waiting room (15%). Compared to urban patients, rural patients were somewhat more likely to prefer telephone symptom reporting (79% vs. 63%, P = .02) and less likely to prefer Internet bases reporting (20% vs. 35%, P = .02).
Finally, 137 respondents indicated the different ways they might use the Internet for their health care. Requesting information from a physician or nurse was the most frequently cited potential use (77% of respondents). Other reasons included, submitting information about their own condition (59%), identifying and managing symptoms (54%), scheduling appointments (52%), and obtaining prescriptions (50%).
Discussion
The high rate (95%) of access to touch-tone telephone service among cancer patients in our network is comparable to data from other government surveys [7,8]. Internet access in both our urban sample (56%) and our rural sample (38%) are below general population estimates for the United States [9], but equal to the data generated for Indiana in a 2000 survey [7]. In a more recent survey, 63% of Indiana residents reported access to the Internet [10]. While the proportion reporting access in our sample was less, this may in part be due to the over-sampling of rural subjects as well as certain demographic characteristics. Age, education level, income, and employment status were major variables influencing Internet access. While fewer individuals in rural seetings reported having internet access, the rural-urban differences were no longer significant when adjusting for age, educational level, annual income and employment status. Thus rural-urban differences may be due to socio-demographic factors more than to a higher presence of technology barriers in rural settings.
Barriers to Internet use identified by patients and limited use of the Internet offer opportunities for better patient communication and education. Over half of the patients without Internet access reported they were not interested. Perhaps waiting room computers with links to cancer-related web sites with good educational and problem-solving content could spur interest. Educational programs for our cancer patients about the Internet and its use may also be helpful. Cost of Internet services did not seem to be a significant factor.
These data suggest that a very significant proportion of cancer patients (more that half of those with Internet access) were willing to use this modality to communicate with their cancer clinic for multiple tasks. While email may offer a convenient means of communication with a physician's office, there are many barriers to its use. Eysenbach has written a thorough review of the potential problems of liability and time pressures [11]. While Katz and his colleagues found no time-savings when email was used as a communication tool, it may well be that it could be an effective tool in some rural settings [12]. Other researchers have also suggested that patient satisfaction and participation in their health care can be increased by use of the Internet by patients [13].
The findings of this survey must be interpreted with caution. While very few patients refused to complete the survey, the patient sample is a convenience sample not a total sample and not a random sample of our patients. With only 46% of our sample (191 patients) having access to the Internet, generalization should be cautious pending replication in a larger sample. The survey instrument did not include questions about readiness to use a touch tone phone for completing a symptom questionnaire by patients, and this is a limitation to the study.
Conclusion
Our findings suggest that either touch-tone telephone or Internet-based computer methods might be used to collect home-based symptom ratings for cancer patients in both urban and rural centers. While access to technologies is adequate, acceptance and usability of such a system remains to be demonstrated. Patient preference for a telephone-based or Internet-based system can be definitively ascertained only after patients use both systems. With lack of interest being the most common barrier to Internet access, education and "get acquainted" programs for patients who do not have Internet access may be warranted. Alternatively, since many patients prefer touch tone telephone for symptom reporting, the use of IVR (Interactive Voice Recording) technology provides another way for symptom reporting, coupled with centralized nurse care management of cancer-related symptoms. Indeed, we are proceeding to test this in a study in which cancer patients will have an option of home-based symptom monitoring by either IVR or the internet coupled with centralized nurse care management of cancer-related symptoms. Patient resource centers with Internet access in outpatient clinics may be another mechanism to consider.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
MA: Assisted with data analysis and interpretation of results. Reviewed, corrected and submitted the final manuscript.
DET: Designed the project including the questionnaire, obtained IRB approval, supervised completion of the survey, assisted with data analysis and writing the final manuscript.
DB: Coordinated data collection from clinic sites and compiled survey results.
KK: Assisted with data analysis, interpretation of results, in addition to review, preparation and submission of the final manuscript.
AP: Carried out the data analysis and assisted with interpretation of results in addition to preparation of the methods and results sections of the manuscript.
SE: Assisted in site recruitment and review of the manuscript.
WMD: Assisted in site recruitment and review of the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The authors thank Pamela Dickerson and Robert Nelson, MD for assistance in completion of this study. This study was supported by the Quality of Life Foundation.
Figures and Tables
Table 1 Patient characteristics by urban vs. rural setting
Total Sample (n = 416) Urban (n = 186) Rural (n = 230) P-value
Mean Age 62.7 60.7 64.3 0.013
Gender 0.924
% Female 56.7 57.0 56.5
% Male 43.3 43 43.5
Ethnicity <0.001
% Caucasian 88.6 76.2 98.7
% Non-Caucasian 11.4 23.8 1.3
Employment 0.154
% Currently employed 29.1 32.6 26.2
% Unemployed 70.9 67.4 73.8
Annual Income 0.155
% < 20 K 31.3 27.3 35.1
% 20 – 50 K 42.5 42.2 42.7
% > 50 K 26.2 30.4 22.2
Education <0.001
% 8th grade or less 6.3 4.3 7.8
% Some high school 14.5 11.9 16.5
% High school or GED 41.5 32.4 48.7
% Any college 37.8 51.4 27.0
Table 2 Comparison of patient characteristics by E-mail / Internet access
Patient Characteristic No Email / Internet (n = 219) Email / Internet (n = 191) P-value
Mean Age 69.1 55.3 0.013
Gender 0.351
% Female 59.0 54.5
% Male 41 45.5
Ethnicity 0.150
% Caucasian 91.6 87.2
%Non-Caucasian 8.4 12.8
Employment <0.001
% Currently employed 10.5 50.8
% Unemployed 89.5 49.2
Setting <0.001
% Urban 36.5 53.9
% Rural 63.5 46.1
Annual Income <0.001
% < 20 K 52.4 9.8
% 20 – 50 K 38.0 47.0
% > 50 K 9.6 43.3
Education <0.001
% 8th grade or less 10.4 1.1
% Some high school 20.8 6.8
% High school or GED 49.3 33.0
% Any college 19.5 59.2
Table 3 Logistic regression results for E-mail / Internet access1
Patient Characteristic Odds Ratio 95% CI
Rural 0.64 (0.35, 1.19)
Age 0.95 (0.93, 0.97)
Female 0.65 (0.37, 1.17)
Caucasian 1.70 (0.65, 4.40)
Currently employed 2.19 (1.06, 4.53)
Annual Income
< 20 K 1.00
20 – 50 K 4.54 (2.15, 9.61)
> 50 K 5.98 (2.43, 14.75)
Education
8th grade or less 0.17 (0.02, 1.52)
Some high school 0.21 (0.07, 0.59)
High school or GED 0.46 (0.25, 0.87)
Any college 1.00
1 All other variables significant at the univariate level were included in the model and found not to be significant
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Patrick DL Ferketich SL Frame PS Harris JJ Hendricks CB Levin B Link MP Lustig C McLaughlin J Reid LD Turrisi AT 3rdUnutzer J Vernon SW National Institutes of Health State-of-the-Science Panel National Institute of Health State-of-the-Science Conference Statement: Symptom Management in Cancer: Pain, Depression, and Fatigue, July 15–17 2002 J Natl Cancer Instit 2003 95 1110 1117
U.S. Department of Health and Human Services, Office of Rural Health Policy
Falling through the net: toward digital inclusion A report on Americans' access to technology tools, US Dept of Commerce Washington, DC 2000
Anderson JE Nelson DE Wilson RW Telephone coverage and measurement of health risk indicators: data from the National Health Interview Survey Am J Public Health 1998 88 1392 1395 9736886
The Pew Internet and American life project
Incontext Kelley School of Business, Indiana University 2003
Eysenbach G The impact of the internet on cancer outcomes CA Cancer J Clin 2003 53 356 371 15224975
Katz SJ Stern DT Dobias K Moyer CA Cox D Effect of a triage-based email system on clinic resource use in primary care [Abstract] J Gen Intern Med 2002 17 199
Murray E Lo B Pollack L Donelan K Catania J White M Zapert K Turner R The impact of health information on the Internet on the physician-patient relationship Arch Intern Med 2003 163 1727 1734 12885689 10.1001/archinte.163.14.1727
| 15717933 | PMC554102 | CC BY | 2021-01-04 16:03:07 | no | BMC Cancer. 2005 Feb 17; 5:18 | utf-8 | BMC Cancer | 2,005 | 10.1186/1471-2407-5-18 | oa_comm |
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BMC BiolBMC Biology1741-7007BioMed Central London 1741-7007-3-51573724010.1186/1741-7007-3-5Research ArticleNegative density-distribution relationship in butterflies Päivinen Jussi [email protected] Alessandro [email protected] Veijo [email protected] Atte [email protected] Janne S [email protected] Kimmo [email protected] Niklas [email protected] Metsähallitus, Natural Heritage Services, P.O. Box 36, 40101 Jyväskylä, Finland2 Department of Biological and Environmental Science, P.O. Box 35, 40014 University of Jyväskylä, Finland3 Department of Biological and Environmental Science, P.O. Box 65, 00014 University of Helsinki, Finland4 Faculty of Forest Sciences, University of Joensuu, P.O. Box 111, 80101, Joensuu, Finland5 Section of Natural Sciences, Jyväskylä University Museum, P.O. Box 35, 40014 University of Jyväskylä, Finland6 South Karelia Allergy and Environment Institute, Lääkäritie 15, 55330, Tiuruniemi, Finland7 Department of Zoology, Stockholm University, 106 91 Stockholm, Sweden2005 1 3 2005 3 5 5 10 1 2005 1 3 2005 Copyright © 2005 Päivinen et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Because "laws of nature" do not exist in ecology, much of the foundations of community ecology rely on broad statistical generalisations. One of the strongest generalisations is the positive relationship between density and distribution within a given taxonomic assemblage; that is, locally abundant species are more widespread than locally sparse species. Several mechanisms have been proposed to create this positive relationship, and the testing of these mechanisms is attracting increasing attention.
Results
We report a strong, but counterintuitive, negative relationship between density and distribution in the butterfly fauna of Finland. With an exceptionally comprehensive data set (data includes all 95 resident species in Finland and over 1.5 million individuals), we have been able to submit several of the mechanisms to powerful direct empirical testing. Without exception, we failed to find evidence for the proposed mechanisms creating a positive density-distribution relationship. On the contrary, we found that many of the mechanisms are equally able to generate a negative relationship.
Conclusion
We suggest that one important determinant of density-distribution relationships is the geographical location of the study: on the edge of a distribution range, suitable habitat patches are likely to be more isolated than in the core of the range. In such a situation, only the largest and best quality patches are likely to be occupied, and these by definition can support a relatively dense population leading to a negative density-distribution relationship. Finally, we conclude that generalizations about the positive density-distribution relationship should be made more cautiously.
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Background
Species that are locally abundant tend to be more widespread than species that are locally rare [1,2]. This positive relationship between density and distribution of species has been observed in a variety of species assemblages over a spectrum of spatial scales, and it has been suggested that it may be almost an universal pattern in ecology [3-6]. However, a few studies document a negative relationship between density and distribution [7-14] (but see [15]). Only recently Gaston et al. [5] encouraged ecologists to pay more attention to the possibility of a negative relationship.
Nine mechanisms have been proposed to explain the positive relationship between density and distribution [2,3,14,16-25]. Of these, two are artefactual (sampling artefact, phylogenetic non-independence) and seven are ecological (pattern of aggregation, range position, niche breadth, resource availability, density dependent habitat selection, dispersal ability and vital rates). Negative relationship between density and distribution can be generated by similar mechanisms that give rise to a positive relationship, but for substantially different circumstances and parameter values [5,6]. Below we discuss briefly all of the mechanisms that can possibly explain the positive relationship between density and distribution, and evaluate whether they could also generate a negative relationship.
Sampling artefact
Since low density species are less likely to be detected in surveys, a positive density-distribution relationship may result from systematic under-recording of the distribution of species that occur at low density [2,3,20]. It follows logically that sampling artefact is not expected to generate a negative density-distribution relationship [5]. However, there is a possibility that rare species with limited, but known, distribution face proportionally higher sampling effort resulting in inflated estimates of their density. When this is the case, a false negative relationship between density and distribution may be generated.
Phylogenetic non-independence
Both positive and negative density-distribution relationship may result from related species being considered as independent data points [19,26,27]. However, phylogenetic non-independence may be the causative factor of a density-distribution relationship only if species density and distribution are determined by species specific life history characteristics affected by the common ancestry, and if there are differences between taxa in their density and distribution. Phylogenetic non-independence has been rejected as an explanation for the positive density-distribution relationship in all previous studies that have controlled its effects [5,14,25,28,29].
Patterns of aggregation
A positive density-distribution relationship may be generated as a result of an underlying theoretical spatial distribution of individuals. For a given level of aggregation, a species with more individuals in a given area is expected to occur in more locations than a species with fewer individuals in the same area [20,24]. However, whether this purely statistical mechanism can actually cause a positive density-distribution relationship rather than serves as a restatement of the relationship in another form, is questionable [29]. This mechanism is unable to generate a negative density – distribution relationship [5].
Range position
Empirical observations suggest that habitat occupancy and the density of individuals decline when moving along a gradient from the centre of the species geographical distribution range toward its edge [2,30-32]. Therefore, a positive density-distribution relationship in any particular region may result when species are at different positions relative to the centre of their geographical range [16]. Gaston et al. [5] argued that this mechanism cannot generate negative density-distribution relationship. More recently, Hanski [33] has pointed out that if patches are more isolated toward range edges, only the largest or best quality patches, i.e. those patches that are able to support the most dense populations, will be occupied. We note that this may lead into a negative density-distribution relationship.
Niche breadth
Brown [2] hypothesized that a positive density-distribution relationship arises because species which have an ability to use a broader range of resources are assumed to be widespread and more abundant. However, while some evidence exists that species with greater niche breadth are more widespread [3,5,34-40], virtually all published studies fail to document a positive interspecific relationship between niche breadth and abundance (see the reviews in [5,25]). On the contrary, many species with wide niche breadth are widely distributed but locally rare [5,25]. However, even if the relationship is commonly and almost predominantly negative, it is not often significantly different from zero. Nevertheless, it seems that the relationship between niche breadth and abundance may in fact generate a negative rather than a positive density-distribution relationship.
Resource availability
On the assumption that density and distribution of resources determine the density and distribution of the species utilizing them, a positive or a negative density-distribution relationship in the resource will generate the same relationship in the consumer [3,22]. Most of the density-distribution relationships of the resource are reported to be positive [2,41].
Density dependent habitat selection
If a species tends to inhabit more habitats when density is high and fewer when density is low, then locally abundant species will tend to occupy more habitats and have wider distributions [17]. At present, there is little evidence for positive density-dependent habitat selection [5,25]. Instead, some evidence exists for negative density-dependent habitat selection [42-44]. A negative density-distribution relationship arises when a species has a wider distribution, i.e. inhabits more habitats, when the density is low.
Dispersal ability
Metapopulation theory may explain both positive and negative density-distribution relationships [21]. A positive relationship may arise where a species that has high density is less likely to go extinct in a given patch than a species that has lower density, or where dispersal increases with density, thereby promoting colonization of empty patches [3,18]. Conversely, a negative relationship may emerge when the species differ in dispersal ability, since dispersal can reduce density but increase distribution [21,33].
Vital rates
If a set of species differs only in respect to its mortality rate, then species with higher mortality will have a lower density. Moreover, the species will inhabit fewer patches than a species with low mortality, leading to a positive density-distribution [23]. Under rather restricted conditions, the vital rates mechanism could also generate a negative density-distribution relationship [23]; however, to test this mechanism, population level data on density-dependent birth and death rates are necessary [5].
Here, we will examine the relationship between density and distribution of Finnish butterflies (Hesperioidea and Papilionoidea), and test the possible mechanisms affecting this relationship. The density-distribution patterns of butterflies have been examined in many previous studies, and if a relationship was found, it was generally positive [3,14,25,38,45-47]. However, most of the studies were conducted in the British Isles, thus representing a sort of pseudoreplication in terms of range of environments and selection of butterfly species studied. Using density and distribution data from two extensive butterfly censuses, we will assess which of the four mechanisms – phylogenetic non-independence, range position, niche breadth and dispersal ability – are best at explaining the density-distribution relationship of Finnish butterflies. We will also discuss the effects of sampling artefact on the density-distribution relationship.
Results
Density-distribution relationships
A strong negative relationship was detected between the density and distribution of Finnish butterflies (linear regression; F 1,93 = 229.97, P < 0.001, r2 = 0.71) (Fig. 1).
Distribution of butterflies
To understand the effects of range position and dispersal ability on the distribution of the butterflies, we analysed the data using simple linear regression and with a multiple linear regression to even out correlated effects. In simple linear regressions, both variables had a significant positive effect on butterfly distribution (Table 1). The overall multiple linear regression was highly significant (F 2,76 = 121.90, P < 0.001, r2 = 0.76), and both of the predictor variables had an independent positive effect on the distribution of the butterflies (Table 2). The relationship between distribution of the butterflies and both of the predictor variables are shown in figures 2 and 3.
To study the effect of niche breadth on the distribution of the butterflies, we analysed the effects of larval specificity and adult habitat breadth on butterfly distribution using analysis of variance (ANOVA) and Tukey's multiple comparisons test. There was no significant interaction between larval specificity and habitat breadth on butterfly distribution (ANOVA F 4,72 = 1.32, P = 0.270), and thus the interaction term was removed from the analysis. However, both larval specificity and habitat breadth had a significant effect on butterfly distribution (F 2,76 = 8.20, P < 0.001 and F 2,76 = 12.53, P < 0.001, respectively). A significant difference was found in the distribution of butterfly species with different habitat breadths. Specialist butterfly species had smaller distribution than generalist species (Tukey MD, mean difference = -2.03, SE = 0.40, P < 0.001), and intermediate species had smaller distribution than generalist species (Tukey MD = -1.24, SE = 0.44, P = 0.018). Specialist species tended to have smaller distributions than intermediate species, but the difference was not significant (Tukey MD = -0.79, SE = 0.35, P = 0.063; Fig. 4). No significant difference was found in the distribution of monophagous and oligophagous butterfly species (MD = -0.441, SE = 0.46, P = 0.611). However, the distribution of the polyphagous butterfly species was greater than mono- or oligo-phagous species (Tukey MD = 1.44, SE = 0.36, P < 0.001 and Tukey MD = 1.00, SE = 0.42, P = 0.051, respectively) (Fig. 5).
Density of butterflies
In simple linear regressions, both range position and dispersal ability had significant negative effects on the density of the butterflies (Table 3). The overall multiple regression of the density of the butterflies on their range position and dispersal ability was highly significant (F 2,76 = 38.84, P < 0.001; r2 = 0.51), and both the predictor variables had independently negative effects on butterfly density (Table 4). The relationships between the density and the predictor variables are depicted in Figures 6 and 7.
To study the effect of niche breadth on the density of the butterflies, we analysed the effects of larval specificity and adult habitat breadth by ANOVA and Tukey's multiple comparisons test. No significant interaction was observed between larval specificity and habitat breadth on butterfly density (ANOVA F 4,72 = 0.84, P = 0.505), and thus the interaction term was removed. However, habitat breadth had a significant main effect on butterfly density (F 2,76 = 8.49, P < 0.001). Specialist species had a greater density than intermediate (Tukey MD = 1.53, SE = 0.40, P < 0.001) or generalist species (Tukey MD = 1.62, SE = 0.49, P = 0.003), but there was no significant difference between intermediate and generalist species (Tukey MD = 0.09, SE = 0.52, P = 0.984; Fig. 8). Larval specificity also had an overall effect on butterfly density (F 2,76 = 5.63, P = 0.005). Monophagous species had significantly greater density than polyphagous species (Tukey MD = 1.54, SE = 0.40, P < 0.001). However, Tukey test detected no differences between monophagous and oligophagous, or between oligophagous and polyphagous species (Tukey MD = 1.03, SE = 0.52, P = 0.124 and Tukey MD = 0.51, SE = 0.47, P = 0.526, respectively) (Fig. 9).
Phylogenetic non-independence
Controlling for the phylogenetic non-independence by using the method of phylogenetically independent contrasts (CAIC) verified that none of the results reported above were artefacts of treating species as independent data points. The results with the phylogenetically independent contrasts have been tabulated in Table 5 and clearly support the previous studies on distribution, abundance or distribution-abundance relationships in which the phylogenetic non-independence not a causative factor in any of the results [5,14,25,28,29,39,40,48].
Discussion
Density and distribution of butterflies
A positive relationship between the density and distribution of species is expected to be an almost universal pattern in ecology [3-6]. In contrast to this, we found a strong negative relationship between density and distribution in Finnish butterflies. Here we will discuss three mechanisms-range position, niche breadth and dispersal ability – that could be responsible for generating this negative relationship.
Range position
According to the range position hypothesis, species are expected to inhabit increasingly fewer localities when moving along a gradient from the centre of the species' geographical distribution range toward the edges of the range [2,31,32]. In addition, density should also decline along this gradient [2,30]. If these two assumptions are correct, a positive density-distribution relationship in any particular region will result when species are at different positions relative to the centre of their geographical ranges [16]. However, there is little evidence for the latter of the two assumptions [31,32]. Moreover, metapopulation theory suggests that if patches are more isolated toward range edges, only the largest or best quality patches will be occupied, i.e. those patches that are able to support the most dense populations [33]. If this is the case, a negative density-distribution relationship will be observed. In our study, we found a strong negative relationship between range position and density indicating that species on the edge of their geographical distribution are indeed more abundant on the patches they occupy. To illustrate the effect of this relationship on the density-distribution relationship, we divided the range position of the butterflies into three classes (1155 km / 3 = 1 – 385 km, 386 – 770 km and 771 – 1155 km) and plotted these on top of the density-distribution relationship (Fig. 10). Species on the edge of their geographical distribution (stars) had the smallest distribution and highest density, whereas the species furthest from the edge of their geographical distribution (filled boxes) had the largest distribution and lowest density. Consequently, being at different positions relative to the centre of the geographical ranges may indeed cause a negative density-distribution relationship.
Niche breadth
The niche breadth hypothesis predicts that a positive density-distribution relationship arises because species that are able to use a broader range of resources are widespread and also have high density [2]. There is evidence of a positive relationship between niche breadth and distribution [3,5,34-40], but it is difficult to see why wider niche breadth should lead to higher density [5]. Many studies have failed to document a positive interspecific relationship between niche breadth and density (see [5,25]. In fact, most studies summarized in Gaston et al. [5] gave negative (although not statistically significant) relationships between niche breadth and density (see also [25]. In our study, we analysed niche breadth with two variables, adult habitat breadth and larval feeding specificity. We found that butterfly distribution was strongly positively related to adult habitat breadth and to larval feeding specificity, but more interestingly, both variables were significantly negatively related to density. To illustrate how these relationships may affect the density-distribution relationship, we plotted both habitat breadth (Fig. 11) and larval feeding specificity (Fig. 12) on the density-distribution relationship. These figures illustrate that the negative density-distribution relationship may be caused by differences in the niche breadth: habitat specialist species and monophagous species have higher abundance, but simultaneously have smaller distribution, than habitat generalist or polyphagous species. Based on the available empirical evidence, it seems that the relationship between distribution and niche breadth is generally positive [3,5,34-40]. If we accept this to be the case, and the density-distribution relationship is determined by niche breadth, then the form of the relationship is caused by the relationship between density and niche breadth.
Dispersal ability
According to the metapopulation theory, differences in dispersal ability of species may generate negative density-distribution relationships [3,21,33]. If, for any reason, species differ in their dispersal ability and dispersal ability has a positive effect on distribution, but a negative effect on density, a negative density-distribution relationship will result. We found a strong positive relationship between dispersal ability and distribution and a strong negative relationship between dispersal ability and density, which support earlier studies in butterflies [14,25,40]. We divided the dispersal ability of the butterflies into three classes (10/3 = 0 – 3.33, 3.34 – 6.66 and 6.67 – 10) and plotted the classes on the density-distribution relationship. Fig. 13 illustrates that species with the lowest dispersal ability have the highest density but lowest distribution, while species with the highest dispersal ability have the lowest density and highest distribution, leading into the observed negative relationship between density and distribution.
Conclusion
We conclude that many of the ecological mechanisms proposed to create a positive density-distribution relationship are also able to generate a negative relationship. Indeed, it is intriguing that many studies, which have found a positive density – distribution relationship have failed to find an ecological mechanism that would explain the observed pattern. This may not be surprising if the ecological mechanisms are more likely to generate a negative rather than positive density – distribution relationship. In support of this view we found a strong negative density-distribution relationship but what is more important also empirical support for ecological mechanisms that are able to explain the observed pattern.
Our study area (Finland) is a long, northern country extending more than 1100 km from south to north. Many of the studied butterfly species meet the edge of their distributional range in Finland. On the edge of a distribution range suitable habitat patches are likely to be more isolated than in the core of the range. In such a case, only the largest and best quality patches are likely to be occupied, but because these are the best quality patches, they may support a relatively dense population [33], leading into a negative density – distribution relationship. We suggest that one important determinant of the density-distribution relationships is the geographical position of the study area and, therefore, future studies should place greater emphasis on the comparison of the relationship in different geographical areas.
Methods
Data set
Our data is predominantly based on published literature [49,50] and the results of The National Butterfly Scheme in Finland (NAFI). We included all 95 butterfly species that are classified as resident or fluctuating in Finland [50] and excluded 21 species classified as migratory, irruptive or extinct [50].
Distribution of butterflies
The distribution of butterfly species is based on the "Atlas of Finnish Macrolepidoptera" [50]. This atlas contains extensive and detailed distribution data of butterflies in Finland, covering all reliable records and observations of butterflies from 1747 to 1997 [50]. The data are compiled from many different sources including c. 1500 literature references and information extracted from several large museum and private collections [50]. The distribution data in the atlas are divided into old observations (before 1988) and new observations (1988 – 1997). We chose to describe the distribution of butterflies by the new observations, which represent the current distribution more accurately and also correspond more favourably with our density data. Moreover, there was a very strong positive correlation between distribution of butterflies based on old and new observations (Pearson correlation: r = 0.966, n = 95, P < 0.001). We describe the distribution for a given species as the number of 10 km × 10 km grid squares on the Finnish national coordinate system from which the species was recorded during the period 1988 – 1997 [50].
Density of butterflies
The National Butterfly Scheme in Finland (NAFI) is a formal study that was established in 1991 for the purpose of monitoring the population trends of the butterflies across the country [51,52]. Monitoring studies may be liable to sampling biases [53], therefore explicit instructions were drafted in order to avoid any bias in sampling or reporting the abundance of butterflies [52]. NAFI provides quantitative abundance data including the 10 km × 10 km grid square of the Finnish national coordinate system in which the observation was made, the year, the number of individuals of each species observed and the number of observation days [52]. During the first ten-year period (1991–2000) of NAFI, 432 lepidopterologists participated in the study, providing data on 1 523 989 individuals, representing a total of 106 butterfly species.
To obtain density for each butterfly species per 10 km × 10 km grid square, we divided the total number of individuals of each butterfly species by the number of 10 km × 10 km squares occupied by the species. Some rare butterfly species with known occurrence sites may face proportionally higher sampling effort than common species [53]. To remove the effect of sampling effort on density, we divided the density by the number of observation days. Note that observation days are the days when the observer was observing at a given square, and thus include also days when a given species was not observed. The number of observation days for each species averaged 20 815, varying between 53 and 50 595 days.
It is possible that size confounds the density and distribution data if larger species are more visible and thus reported more often than smaller species. We analysed the possibility that the size of the butterfly species is a confounding factor. There is practically no dimorphism between the sizes of male and female butterflies. Therefore, as a size measure we used only the female wing span reported in Marttila et al. [49], in which the mean of a sample of 20 females was reported with an exception of some rare species with fewer individuals measured. We found no evidence of any size bias: there was no relationship between the female wing span and the density or distribution of the species (linear regression F 1,93 = 0.04; P = 0.835, r2 = 0.00 and F 1,93 = 0.99; P = 0.987, r2 = 0.00, respectively).
Range position
To determine the range position, we measured the distance (km) between the northernmost distribution record and the southernmost point (Hankoniemi) of Finland from the maps in Huldén et al. (2000). Note that the longest possible range was 1155 km. In analysis including range position, we only included species, the distribution range of which begins from southern Finland [50]. Thus, we excluded the species which occur only in Northern Finland (n = 14, [49]), and two species (Lycaena helle and Clossiana thore) which do not range to southern Finland.
Niche breadth
We describe the niche breadth for each butterfly species using two different measures: 1) larval host plant specificity, and 2) the number of habitat types occupied by the adults. We excluded from this analysis the species which occur only in Northern Finland (n = 14, [49]). This was done because their larval host plants are unknown or are not confirmed and because the habitat types of Northern Finland are unique compared to other Finnish habitat types.
The data on larval host plant specificity in Finland are based on Huldén et al. [50] and Wahlberg [54]. We classified the larval host plant specificity into three classes: (1) monophages, i.e. species that feed on a single plant species; (2) oligophages, i.e. species that are restricted to one genus of food plants; and (3) polyphages, i.e., species that feed on one or more than one family of food plants.
The main habitats that Finnish butterflies occupy have been categorised into four types [49]: (1) uncultivable lands (e.g. edge zones beside industrial area, harbour and storage areas, loading places, uncropped fields, and other unbuilt areas which have been exposed to human impact), (2) meadows (many kinds of non-cultivated open grasslands), (3) forest edges (e.g. road sides), and (4) bogs. Adult niche breadth is the number of habitat types in which the adults typically are found. As there were only two species (Pieris napi and Gonepteryx rhamni) that occupied all four main habitat types, habitat types three and four were combined. Value one represents specialist species that are limited to one habitat type, value two represents intermediate species that occur in two habitat types, and value three represents generalist species that occur in three or four habitat types.
Dispersal ability
Metapopulation theory predicts that species with greater dispersal ability are likely to occupy more habitat patches than species with lower dispersal ability [3]. To describe the relative dispersal ability of butterfly species, we adopted the method described in Cowley et al. [14]. We sent a questionnaire to experienced lepidopterists in Finland and asked them to give a "dispersal ability index" (0–10) for each butterfly species. In the questionnaire, a zero value indicates that a given butterfly species is extremely sedentary and a value of ten means that it is extremely mobile. To obtain a relative dispersal ability for each butterfly species, we calculated the average dispersal ability from returned questionnaires (n = 13).
To ensure our measure of dispersal ability is reliable, we compared our measure with the dispersal ability estimated previously by Bink [55] (nine dispersal ability classes), Pollard and Yates [56] (three dispersal ability classes based on mark-release-recapture studies), Cowley et al. [14] (continuous variable based on questionnaires) and Cook, Dennis & Hardy [57] (continuous variable based on vagrancy in grids). The correlations between our measure and those of Bink [55], Pollard and Yates [56], Cowley et al. [14] and Cook et al. [57] were all strongly positive and significant (Pearson correlation; r = 0.672, n = 73, P < 0.001, ANOVA; F3,27 = 8.74, r = 0.567, P < 0.001, Pearson correlation; r = 0.703, n = 31, P < 0.001 and Pearson correlation r = 0.602, n = 11, P = 0.050, respectively), indicating that our dispersal ability is in line with other independent estimates and thus reliable (see also [58]).
Phylogenetic non-independence
Lack of statistical independence among species for the traits of interest was tested using the method of phylogenetically independent contrasts [19] as implemented in the CAIC program [59]. Statistical control of phylogenetic non-independence requires knowledge of the phylogeny [19,27]. However, knowledge of the general phylogenetic relationships among butterfly species is still in a state of flux [60], and there are no studies available that look explicitly at the relationships of species in Finland. However, the recent surge of published studies on various groups of butterflies allows us to compile a likely phylogeny for Finnish butterflies (Fig. 14). We took the relationships of the butterfly families from [60], the relationships within Papilionidae from [61], and the relationships within Nymphalidae from various sources [62-65]. Relationships within Pieridae, Lycaenidae and Hesperiidae are based on current taxonomy, with morphologically well-defined groups shown as monophyletic. In the analysis all branch length were assumed equal because no estimate of evolutionary distance exist for the entire data set. However this option is justified under the assumption of punctuated evolution. Regression analysis was used to investigate the standardized linear contrasts calculated by CAIC [19]. Note that the regression lines must pass though the origin [66,67].
Authors' contributions
JP, VK, AK and JSK compiled and analysed most of the data, and wrote the manuscript. NW constructed the phylogeny and AG conducted the phylogenetic analyses. KS provided the density data.
Acknowledgements
We thank Andrew Warren for discussing the systematics of Hesperiidae with us. We also thank all of the lepidopterologists who lent us their expertise by replying to our dispersal ability questionnaire. We are grateful to Jari Niemelä, Marko Nieminen, Stuart Pimm, Tomas Roslin and Teija Virola for comments on the manuscript. We thank Tapani Lahti for help in compiling the data. The study was supported by Ella and Georg Ehrnrooth Foundation and the Centre of Excellence Programme in Evolutionary Ecology to J.P., and Academy of Finland to V.K and J.S.K.
Figures and Tables
Figure 1 Distribution in relation to density of the Finnish butterflies. X- and Y-axes are in log10 scale. Examples of species: 1 = Agriades glandon, 2 = Argynnis laodice and 3 = Papilio machaon.
Figure 2 Range position in relation to distribution of the Finnish butterflies. Y-axis is in log10 scale.
Figure 3 Dispersal ability in relation to distribution of the Finnish butterflies. Y-axis is in log10 scale.
Figure 4 Habitat breadth in relation to distribution of the Finnish butterflies. Error bars represent mean ± 1 SE.
Figure 5 Larval specificity in relation to distribution of the Finnish butterflies. Y-axes are in log10 scale. Error bars represent mean ± 1 SE.
Figure 6 Range position in relation to density of the Finnish butterflies. Y-axis is in log10 scale.
Figure 7 Dispersal ability in relation to density of the Finnish butterflies. Y-axis is in log10 scale.
Figure 8 Habitat breadth in relation to density of the Finnish butterflies. Y-axes are in log10 scale.
Figure 9 Larval specificity in relation to density of the Finnish butterflies. Y-axes are in log10 scale.
Figure 10 Distribution in relation to density of the Finnish butterflies. Three range position categories are separated with different symbols. The distance between the southernmost point of Finland and the northernmost observation is 1–385 km in category one, 386–770 in category two and 771–1155 in category three.
Figure 11 Distribution in relation to density of the Finnish butterflies. Species are classified in three categories according to their habitat breadth.
Figure 12 Distribution in relation to density of the Finnish butterflies. Species are divided in three categories according to their larval specificity.
Figure 13 Distribution in relation to density of the Finnish butterflies. (d) Species are divided in three categories according the dispersal ability of the butterflies (1 = 0 – 3.33, 2 = 3.34 – 6.66 and 3 = 6.67 – 10). Note that X- and Y-axes are in log10 scale.
Figure 14 The phylogeny of butterflies used in this study. The relationships of groups are based on a number of published studies (see text for details).
Table 1 Simple linear regressions for the distribution of butterflies.
Slope SE t df P
Range position 0.00 0.00 11.29 77 < 0.001
Dispersal ability 0.65 0.06 10.15 93 < 0.001
Table 2 Multiple linear regression for the distribution of butterflies.
Slope SE t df P
Range position 0.00 0.00 10.04 76 < 0.001
Dispersal ability 0.33 0.05 6.66 76 < 0.001
Table 3 Simple linear regressions for the density of butterflies.
Slope SE t df P
Range position -0.00 0.00 -7.51 77 < 0.001
Dispersal ability -0.73 0.10 -7.47 93 < 0.001
Table 4 Multiple linear regression for the density of butterflies.
Slope SE t df P
Range position -0.00 0.00 -5.83 76 <0.001
Dispersal ability -0.28 0.08 -3.56 76 <0.001
Table 5 Linear regression results between all variables after controlling for the phylogenetic non-independence. N is the number of independent contrasts.
Dependent variable Independent variable N Slope r2 P
Density Distribution 45 -1.13 0.67 0.000
Distribution Body size 45 -0.72 0.01 0.513
Distribution Range position 42 1.62 0.58 0.000
Distribution Dispersal ability 45 0.80 0.76 0.000
Distribution Larval specificity 14 0.46 0.55 0.002
Distribution Habitat breadth 17 0.47 0.48 0.002
Density Body size 45 1.92 0.03 0.232
Density Range position 42 -1.90 0.30 0.000
Density Dispersal ability 45 -1.04 0.54 0.000
Density Larval specificity 14 -0.51 0.59 0.000
Density Habitat breadth 17 -0.71 0.45 0.002
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| 15737240 | PMC554103 | CC BY | 2021-01-04 16:02:56 | no | BMC Biol. 2005 Mar 1; 3:5 | utf-8 | BMC Biol | 2,005 | 10.1186/1741-7007-3-5 | oa_comm |
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BMC MedBMC Medicine1741-7015BioMed Central London 1741-7015-3-51570750210.1186/1741-7015-3-5Research ArticleKhat use as risk factor for psychotic disorders: A cross-sectional and case-control study in Somalia Odenwald Michael [email protected] Frank [email protected] Maggie [email protected] Thomas [email protected] Claudia [email protected] Birke [email protected] Harald [email protected]äfner Heinz [email protected] Brigitte [email protected] Department of Psychology, University of Konstanz, Fach D25, D-78476 Konstanz, Germany2 Outpatient Clinic for Refugees, University of Konstanz, Feursteinstr. 55, Haus 22, D-78479 Reichenau, Germany3 Ctr. for Psychiatry Reichenau (ZPR), Feursteinstr.55, D-78479 Reichenau, Germany4 Worldbank, Multi-Country Demobilization and Reintegration Program in the greater Great Lakes Region of Africa, Goma, Democratic Republic of Congo5 AG Schizophrenieforschung, Zentralinstitut für Seelische Gesundheit, J5, D-68159 Mannheim, Germany2005 12 2 2005 3 5 5 15 10 2004 12 2 2005 Copyright © 2005 Odenwald et al; licensee BioMed Central Ltd.2005Odenwald et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Little is known about the prevalence of khat-induced psychotic disorders in East African countries, where the chewing of khat leaves is common. Its main psycho-active component cathinone produces effects similar to those of amphetamine. We aimed to explore the prevalence of psychotic disorders among the general population and the association between khat use and psychotic symptoms.
Methods
In an epidemiological household assessment in the city of Hargeisa, North-West Somalia, trained local interviewers screened 4,854 randomly selected persons from among the general population for disability due to severe mental problems. The identified cases were interviewed based on a structured interview and compared to healthy matched controls. Psychotic symptoms were assessed using the items of the WHO Composite International Diagnostic Interview and quantified with the Positive and Negative Symptoms Scale. Statistical testing included Student's t-test and ANOVA.
Results
Local interviewers found that rates of severe disability due to mental disorders were 8.4% among males (above the age of 12) and differed according to war experiences (no war experience: 3.2%; civilian war survivors: 8.0%; ex-combatants: 15.9%). The clinical interview verified that in 83% of positive screening cases psychotic symptoms were the most prominent manifestations of psychiatric illness. On average, cases with psychotic symptoms had started to use khat earlier in life than matched controls and had been using khat 8.6 years before positive symptoms emerged. In most cases with psychotic symptoms, a pattern of binge use (> two 'bundles' per day) preceded the onset of psychotic symptoms, in contrast to controls of the same age. We found significant correlations between variables of khat consumption and clinical scales (0.35 to 0.50; p < 0.05), and between the age of onset of khat chewing and symptom onset (0.70; p <0.001).
Conclusion
Evidence indicates a relationship between the consumption of khat and the onset of psychotic symptoms among the male population, whereby not the khat intake per se but rather early onset and excessive khat chewing seemed to be related to psychotic symptoms. The khat problem must be addressed by means other than prohibition, given the widespread use and its role in Somali culture.
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Background
The present study investigated the relationship between psychotic symptoms and the abuse of khat in the Horn of Africa. The leaves of the khat shrub (catha edulis) are traditionally chewed in Arab countries, the Horn of Africa and East Africa [1] and recently this habit has spread to Western countries including the USA [2]. Due to improved transportation facilities, khat consumption has substantially increased during recent decades. This is reflected in the most recent issue of the World Drug Report: in 2001 five countries reported an increase in khat use and none a decrease; in 2002 an increase was reported in four, a decrease, again, in none [3]. Kalix (1996) [4] estimates that about 6 million individual portions are consumed each day worldwide. During our field work in the city of Hargeisa, North-West Somalia (Somaliland), where khat use is not restricted by law, we observed that current ways of intake do not correspond to the documented traditional use in the region. The traditional way of consumption was socially highly regulated: adult males (more seldom females) would gather and chew khat together at a so-called 'khat party', usually on weekends and afternoons until the time of the evening prayer [5,6]. Contrary to this formerly restricted use, current habits involve use by adolescents, chewing khat in tea-shops that operate day and night, early morning use, as well as "binging" and "speed runs" that may last for more than 24 hours. Our study was based on observations by social workers of collaborating non-governmental organizations and by our team during field studies on war-related trauma. It was shown that the widespread use of khat is related to the large number of individuals with visible signs of psychosis who are either homeless or kept in hiding, e.g. in physical chains, by family members who afraid to expose them to the general public.
The main psycho-active component of khat leaves is cathinone (S(-)alpha-aminopropiophenone) [7]. Cathinone resembles amphetamine in chemical structure and affects the central and peripheral nervous system [8,9] and behavior [10,11] similarly. Amphetamines and some of its derivatives have been shown to induce psychotic symptoms in experimental settings in humans [12,13] and animals [14] and have been known to exacerbate psychotic states in psychiatric patients [15,16]. Similarly, khat-induced psychotic states have been described in several case studies [17-20]. However, the number of group, community and population-based studies on khat use and psychiatric symptoms [21-23] is still limited. Despite the ongoing scientific debate about amphetamine-induced psychosis it remains unclear whether the use of amphetamine-like substances including khat may actually cause a psychotic disorder in an otherwise healthy individual, or trigger the onset of schizophrenia in an individual with high vulnerability to the disease [24,25]. The fact that presumed amphetamine psychoses do not fully remit within weeks of abstinence in a substantial percentage of individuals [26] may also suggest that those individuals actually had a amphetamine-triggered schizophrenia. Increased drug use among psychotic patients may also come from an attempt to counteract nonspecific physical symptoms or side effects of neuroleptics [27].
The first goal of this study was to verify the impression of an unusually high prevalence of psychotic disorders in the city of Hargeisa via an epidemiological survey carried out in a representative sample of households. In addition, we wished to study the association between khat abuse and psychotic disorders. If khat abuse does induce psychotic disorders, a higher prevalence of psychotic diagnoses, mainly in men (as women rarely consume khat), was to be expected in Hargeisa compared to localities with less khat use. In addition, a case-control study served to examine whether individuals identified in the survey suffering from psychotic symptoms presented a pattern of khat consumption different from matched controls.
Methods
Sample
In order to screen for households with mentally ill members, 612 households with 4,854 members were randomly selected as representative of the city of Hargeisa (approximately 400,000 inhabitants). For household selection the town was first subdivided into 30 sections of approximately equal populations. For this purpose we used the same sections as UNICEF in their assessment of vaccination coverage for children in October 2002, which were defined in a joint approach by UNICEF and the municipality of Hargeisa. A section had to be subdivided into square-shaped clusters if it was L-shaped, had a natural boundary within its limits (e.g. a steep hill) or had a much greater length than depth. A map for each section or cluster was produced using an aerial photograph. For random selection the geographical center of each cluster was determined and a random direction was chosen using a compass, a watch and a list of random numbers between 1 and 12 (a watch was oriented according to the compass, whereby 12 o'clock was adjusted to North; the random number defined the direction according to the hours on the face of the clock). Following the random direction until the border of the cluster was reached, all houses on the right side within a range of 15 meters were numbered. The first household to be approached was selected by a second set of random numbers. The next houses were selected by door-to-door distance until the predefined number was reached. Trained local staff interviewed the heads of the 612 households. If the head of household was not available, another adult member of the household was asked to answer to the questions. If no household member could be interviewed, the next house was selected according to the selection procedure.
The following question was used to determine whether a person severely disabled due to mental health problems resided in the household: "Are there any members of your household who currently have mental problems that are so severe that the person has been unable to provide income or has been unable to help in the household for at least four weeks?" This criterion was fulfilled for 169 (137 male, 32 female) cases. These individuals will be referred to as 'positive screening cases'. A subsample of 52 'positive screening cases' was then randomly selected and examined in a clinical interview. In this group there were 44 males and 8 females. These individuals will be referred to as 'examined cases'.
For each 'examined case' a matched control, who did not meet the criteria for disability due to mental health problems, was identified. Controls were matched by gender, age, and educational level. These 'case controls' were subjected to the same clinical interview as the examined cases. Forty-nine of the 52 examined cases and one control were diagnosed with psychiatric or neurological disorders (on this basis we estimated a sensitivity of 0.98 and a specificity of 0.94 for the screening procedure). We included only those forty-three examined cases (82.7%) in the further analysis who – in addition to the impairment of function – showed as main manifestations of mental problems at least one very severe or two moderate positive psychotic symptoms in the absence of organic somatic damages that might explain them; from hereon these are termed 'cases with psychotic symptoms'. The disorders of other examined cases were stroke (2), traumatic brain injury (1), mental retardation (2), and dementia (1). The case control with a positive diagnosis reported hallucinations with intact reality testing during khat intoxication and was replaced by a healthy individual. Socio-demographic characteristics for the two studied groups are summarized in Tables 1 and 2.
Table 1 Socio-demographic characteristics of the sample of N = 4,854. For socio-demographic data, mean ± standard deviation and percentages and absolute numbers (in parenthesis) respectively are reported. P-values refer to differences between ex-combatants, civilian war survivors, and persons without any war experience tested by Kruskal-Wallis1 and chi 2 2.
Whole group Ex-combatants Civilian war survivors No war experience p
Total Number 4,854 250 2,667 1,937 -
Male 2,449 240 1,201 1,008
Female 2,405 10 1,466 929 < 0.0012
Age 22.2 ± 17.8 42.8 ± 13.5 28.7 ± 16.6 10.7 ± 12.1 < 0.0011
Age range 0 – 102 12 – 102 0 – 101 0 – 90 -
Years of formal education 2.6 ± 4.1 5.2 ± 5.6 3.3 ± 4.3 1.4 ± 3.0 < 0.0011
Economic situation of household* 2.8 ± 1.8 2.5 ± 1.7 2.9 ± 1.7 2.8 ± 1.8 0.0051
Percentage of khat users in week before interview among men > 12 yrs 31.3% (495 of 1,581) 60.3% (144 of 239) 28.1% (306 of 1,090) 17.9% (45 of 252) < 0.0012
Khat bundles/week before interview among khat chewing men > 12 yrs 7.6 ± 4.4 7.9 ± 4.8 7.6 ± 4.4 6.7 ± 3.2 0.5441
'Screening cases' among men > 12 yrs 8.4% (133 of 1,581) 15.9% (38 of 239) 8.0% (87 of 1,090) 3.2% (8 of 252) < 0.0012
'Screening cases' among women > 12 yrs 1.9% (30 of 1,600) 20% (1 of 5) 1.6% (22 of 1,372) 2.8% (7 of 248) 0.0662
* The household economic situation was approximated as the sum of four significant assets (water tap, electrical power, TV set, car) and type of housing (hut = 1, house = 2, closed compound = 3). The mean of the two ratings is presented. For the combined rating Cronbach's Alpha = 0.78; correlation with income = 0.73, p < 0.001.
Table 2 Socio-demographic characteristics of the 43 cases with psychotic symptoms and 43 'case controls.' Mean ± standard deviation and percentages and absolute numbers (in parenthesis) respectively are reported. P-values refer to group differences assessed by t-test/Wilcoxon test.
Patients with psychotic symptoms Matched controls Sig.
total male female total male female p
N 43 38 5 43 38 5 1.0
age 33.9 ± 10.5 34.6 ± 10.0 28.6 ± 13.8 34.7 ± 10.3 35.3 ± 10.0 30.4 ± 12.8 0.718
Education (yrs in school) 5.6 ± 4.5 6.4 ± 4.3 0.0 6.3 ± 4.9 7.1 ± 4.6 0.0 0.511
Martial status (% married) 16.3% (7) 18.4 % (7) 0 % (0) 44.2% (19) 50.0% (19) 0% (0) 0.005
Employed or at school 0% (0) 0% (0) 0% (0) 41.9% (18) 47.4% (18) 0% (0) < 0.001
Procedures and materials
All interviews were carried out in October and November 2002. Prior to the interview, participants were informed about the background of the study and the survey procedure. All participants signed a written informed consent, which was first read and explained to them (as most of the participants were illiterate). In the case of mentally challenged participants, background and procedure were also explained to the responsible caretakers whose written informed consent was a prerequisite for their participation. Interviews were approved by the local authorities and the National Demobilization Committee of Somaliland. Representatives of the different communities were informed and invited to observe the assessment in the field. Newspaper advertisements, daily radio programs and flyers informed the population about and helped them to understand the real purpose of the assessment (at first rumors had spread that the research team would provide free medication for mentally disturbed individuals and created unreal expectations). Consequently, the level of cooperation was extremely high. Out of 73 households approached under the supervision of the first author only three refused their participation. Therefore, we estimate that in total less than 5% of all households refused participation.
Local interviewers were recruited among NGO and hospital staff who already had experience in working with mentally disabled persons. They participated in a two-week training course, which involved teaching the basic psychiatric concepts (e.g. psychotic symptoms), interviewing skills, training on the screening-instrument and field work under close supervision of experts. After the end of the course, fourteen of the seventeen trainees were employed for the duration of the study. Five interviewer teams, each comprising one male and one female interviewer, and four local supervisors did the field work. The local supervisors received additional training on the random sampling method. During the first weeks of interviewing the first author closely supervised the teams in the field.
The screening interview assessed individual socio-demographic information, khat consumption and experiences in the civil war (subjects were grouped as either active war participants, civilian war survivors, or without any war experiences). Khat consumption (number of bundles/week) was assessed for the week prior to the interview. The household economic situation was approximated as the sum of four significant assets (water tap, electrical power, TV set, car) and type of housing (Table 1).
Clinical interviews were carried out by some of the authors (MO, MS, CC, BL), all trained in the assessment of psychotic disorders and PANSS rating. Socio-demographic information and war-history were detailed. A standard event list was used to quantify the number of traumatic event types a person had experienced; the list included 11 types of events: active combat experience, accident, natural disaster, witness murder or killing, rape, sexual molestation, physical assault, being kidnapped or captured, torture, other, or suffered shock because a close person had experienced a traumatic event. In a short semi-structured qualitative interview the main areas of psychological condition and functioning were assessed. Additionally, psychotic symptoms were assessed using the items of the Composite International Diagnostic Interview (CIDI), Section G [28]. The individual's age at which the family first noticed positive psychotic symptoms that prevailed over six months was taken as indication of the onset of a psychotic disorder. Khat consumption (average number of bundles/day) was assessed for the week before the interview and for the week prior to the onset of symptoms in examined cases. Case controls were asked about consumption in the week prior to the interview and for consumption at the age of symptom onset reported by his/her examined case-pair (Table 2). As many examined cases were not able to give valid information, the primary care taker and other family members were also interviewed.
Clinical interviews were administered in the English language with the help of trained local interpreters, who had participated in the same training course as the interviewers. After the interview, the interviewing psychologist rated the current psychopathology using the Positive and Negative Syndrome Scale, PANSS [29].
All items of the standardized interviews were translated from English into Af Somali by mixed teams of clinical experts and bilingual staff. Several steps of independent back-translations and corrections were necessary until a satisfactory translation was achieved. Content validity was assured by the involvement of clinical experts at all levels of the translation process. Also, interpreters were introduced to the concepts targeted by the respective questions.
Data analysis
Differences between cases and controls were confirmed using chi2-tests (or Fisher's exact tests), binomial testing, Student's t-test or Wilcoxon, and ANOVA or Krsucal-Wallis test (all tests two-tailed). Univariate Analysis of Variance (ANOVA) was used to explore the effects of war-trauma on the khat intake. Means and standard deviations are reported.
Results
Of the sample of 4,584 inhabitants of Hargeisa the screening interview disclosed 169 positive screening cases (i.e. 3.5%). Positive screening cases were more often male than female (133 of 1,581 men, i.e. 8.4%, and 30 of 1,600 women above the age of twelve years, i.e. 1.9%; p < 0.001; Table 1). Khat chewing was more frequent among male positive than among negative screening cases above the age of 12 years: 46.6% of the 133 positive screening cases had chewed khat in the week before the interview in contrast to 29.9% of the 1,448 negative screening cases (p < 0.001). Consumers among positive screening cases had also chewed a greater amount of khat in the week preceding the interview (positive screening cases: 4.1 ± 6.3 bundles; negative screening cases: 2.2 ± 4.0 bundles; p = 0.001).
The proportion of positive screening cases was substantially higher among males above the age of 12 who had active war experience (ex-combatants) than in male civilian war survivors of the same age (p < 0.001, Table 1). The latter proportion was significantly higher than in men without any war experience (p = 0.007).
Psychotic symptoms meeting our criteria were determined for 83% (43) of the examined cases. Retrospective investigation suggested that the onset of psychotic signs occurred at the age of 23.4 ± 9.9 years (N = 38 men: 24.1 ± 9.8 years; N = 5 women: 18.4 ± 9.6 years; p = 0.230).
PANSS ratings of these 43 cases showed a substantially higher magnitude of current psychotic symptoms compared to a sample of 240 medicated schizophrenic patients [30] in the following subscales: Positive 27.1 ± 7.3, Negative 32.0 ± 8.9, Composite -4.9 ± 10.6, General Psychopathology 52.7 ± 12.7, Anergia 16.6 ± 5.5, Thought Disturbance 16.4 ± 5.3, Activation 7.8 ± 3.0, Paranoia/Belligerance 10.8 ± 4.1, Depression 9.5 ± 3.9, and Supplemental Scale 17.4 ± 7.4. In further exploratory observations, we noticed a high tendency towards aggressive and hyperactive behavior. During interviews, most patients reported that they were in contact with a ghost ('djin'), often associated with auditory, visual and somatosensory hallucinations.
Fifteen of the 43 cases with psychotic symptoms (35%) were under current medication at the time of assessment; another nine (21%) had received medication in the past. A variety of drugs had been given, ranging from neuroleptics (12 patients) to prometazine (6), benzodiazepines (3), tricyclic antidepressants (3), carbamazepine (1) and other unknown drugs (10).
Uncontrollable (disruptive, violent) behavior had led to restraint of cases with psychotic symptoms by chaining them to an object in 28 of the 38 men (i.e. 74%) and 3 of the 5 women (i.e. 60%) (p = 0.608). Additionally, 9 men (24%) and 3 women (60%) had been locked up to control their behavior (p = 0.589). On average, the 31 cases with psychotic symptoms who had ever been chained had spent 4.2 ± 5.2 years in chains (men: 4.5 ± 5.4 years; women: 1.8 ± 1.9 years; p = 0.410) and the 10 cases who had ever been locked up spent on average additional 5.2 ± 6.5 years restrained (men: 4.0 ± 6.6 years; women: 10.0 ± 4.2 years; p = 0.264).
The proportion of khat user was higher among cases with psychotic symptoms than among matched controls: all except one of the 38 male psychotic cases, in contrast to 25 of the 38 male controls, had used khat (p < 0.001). In the cases with psychotic symptoms, regular khat consumption had started at an earlier age (16.6 ± 4.8 years) than among their matched controls (20.7 ± 7.0 years, p = 0.010; Figure 2). All except one case with psychotic symptoms (compared to 61% of controls, i.e. 14 of 23) had started to chew before the age of 23 years (p = 0.004). None of the women admitted to having ever chewed.
Figure 2 Khat intake and traumatic experiences. Average daily khat intake in bundles (at the age when the case with psychotic symptoms showed onset of symptoms) according to number of traumatic life events. We divided the cases with psychotic symptoms and case controls into three groups according to the number of event types experienced as follows: none or one: 11 cases and 13 controls; two or three: 9 cases and 15 controls; more than three: 11 cases and 14 controls. Bars indicate mean and standard error.
In the weeks preceding the onset of psychotic symptoms, cases with psychotic symptoms had chewed an average of 2.5 ± 2.0 bundles/day compared to 0.5 ± 0.6 bundles/day in controls (p < 0.001; Figure 1). Excessive khat intake (> two bundles/day) in this period was found in 78% of chewers among male cases with psychotic symptoms (i.e. 29 of 37) but in only 4% of chewers among controls (i.e. 1 of 25; p < 0.001). For the cases with psychotic symptoms, the age of onset of khat chewing correlated significantly with symptom onset (r = 0.70, p < 0.001). The lapse of time between first use of khat and onset of symptoms was greater than one year in 31 of the 38 male cases with psychotic symptoms (i.e. 82%); it varied around a mean of 8.6 ± 6.6 years (median 7 yrs). In the week before the interview 54% of the lifetime khat chewers among male cases with psychotic symptoms (i.e. 20 of 37) and 36% of the controls (i.e. 9 of 25) had chewed khat (p = 0.162). Furthermore, psychotic patients consumed an average of 1.5 ± 1.0 bundles/day, with controls consuming an average of 0.9 ± 0.7 (p = 0.172; Figure 1).
Figure 1 Patterns of khat consumption. Left: age of first khat intake among lifetime chewers in patients with psychotic symptoms (35) and case controls (23); right: amount of khat use (in 'bundles' per day) in the week before the onset of first positive symptoms (25 cases with psychotic symptoms, 24 controls) and in the week before the clinical interview (16 cases with psychotic symptoms, 9 controls). Bars indicate mean and standard error.
Khat use correlated with severity of symptoms: In cases with psychotic symptoms onset of khat use correlated significantly with higher scores on the PANSS subscales Negative Symptoms (r = - 0.36, p = 0.039, N = 34), General Psychopathology (r = - 0.44, p = 0.009; N = 34), Anergia (r = - 0.39, p = 0.022, N = 34) and Thought Disorder (r = - 0.42, p = 0.014, N = 34), while the amount of khat consumed prior to onset of psychotic symptoms correlated significantly with the subscale Paranoia/Belligerence (r = 0.49, p = 0.006, N = 30) and the Supplemental scale (r = 0.50, p = 0.005, N = 30). The amount of khat consumed in the week before the interview correlated with the PANSS subscale Anergia (r = - 0.35, p= 0.029, N = 38).
The number of traumatic event types experienced did not differ between cases with psychotic symptoms and matched controls (cases with psychotic symptoms: 3.1 ± 2.3 events; matched controls: 2.9 ± 2.4 events; p = 0.749). Also, the age when the first traumatic event was experienced did not differ between them (cases with psychotic symptoms: 20.2 ± 9.6 years; matched controls: 20.6 ± 9.2 years; p = 0.853). In order to estimate the relationship between number of experienced traumatic events and khat intake we used univariate ANOVA with khat intake before onset of psychotic symptoms (for controls at the age of symptom onset of matched pair) as dependent variable, and number of traumatic events in three categories (no or one event, two or three, four or more) and being a case or a control as independent variable. We found significant effects for the number of traumatic events (Sum of squares 36.6; df 2; F = 17.028; p < 0.001) and for the variable 'case or control' (Sum of squares 53.9; df 1; F = 50.136; p. < 0.001) and a significant effect of their interaction (Sum of squares 31.0; df 2; F = 14.404; p < 0.001) (total R2 = 0.620). In Figure 2 the results are displayed graphically.
Discussion
The present study revealed a number of interesting and relevant findings.
(1) In a representative subsample of male inhabitants of Hargeisa (older than 12 years) we found 8.4% severely disabled due to mental problems; of these, 83% had severe psychotic symptoms. Compared to the prevalence rates of psychotic disorders reported for male samples elsewhere [31,32] this is higher than expected. Furthermore the gender ratio among the mostly psychotic positive screening cases was unusual (1 women to 7.7 men).
(2) Khat abuse showed a significantly earlier onset and was more frequently excessive in male cases displaying psychotic symptoms than in matched controls.
(3) There is a higher vulnerability to disability due to mental disorders in those groups of society who were directly involved in combat in the past.
(4) Among the positive screening cases examined and their matched controls we found that khat intake before the onset of psychotic symptoms (respectively at the same age for matched controls) was higher when more traumatic events had previously been experienced.
These results are intriguing, as we may have detected only the very tip of the iceberg with our sub-optimal screening procedure, leaving less severe cases of mental and neurological disorders undiscovered.
The findings of the present study suggest that there is an association between khat consumption and psychosis; however the existence of a causative relationship and its direction between the two remains unclear. Furthermore, it seems that it is not the consumption of khat per se but rather specific patterns of it that are related to psychosis, especially early onset in life and the excessive intake (> two bundles per day). Both seem to be related to active participation in war, e.g. child ex-combatants used khat while fighting in order to counteract fear and enhance performance [33]. Excessive khat consumption has already been reported to be associated with higher severity of psychiatric symptoms [21]. Our data also suggest that there might be a moderating effect of the number of traumatic experiences on subsequent khat intake. This would be in line with the self-medication hypothesis.
Khat consumption might be related to the outbreak of psychosis in various ways. Considering psychoses as the result of genetic and acquired vulnerability and additional stress factors, the early onset of khat abuse may have substantially increased the risk in already vulnerable individuals. In animal studies, damage imposed on the developing brain, e.g. by drugs, increases the potential of amphetamine-like agents to change neuro-chemical systems and to induce psychotic-like behavior [34]. In humans, drug abuse during puberty has been found to precede the onset of psychotic symptoms and prodromi [35] and to be related to poorer treatment outcome [36]. Additional risk factors and particular stressors, such as war experiences, may contribute to an increased vulnerability. It is presently unclear, however, whether traumatic experiences act indirectly through higher drug consumption in traumatized men or whether they exert a direct effect on the brain, increasing the risk of developing psychosis. The temporarily higher khat abuse preceding first symptoms in cases with psychotic symptoms compared to case-controls may suggest that khat consumption is the primary agent, causing the onset of psychoses. This assumption is further strengthened by the inequality in the gender ratio, as women are generally denied access to the drug.
Moreover, khat consumption may affect the course of a psychotic disorder and the maintenance of symptoms. In contrast to the high khat consumption prior to the onset of psychotic symptoms, the amount of current khat consumption did not differ between cases with psychotic symptoms and controls and a significant number of patients did not have current access to the drug. Drug-effects on the course of illness may be attributed to sensitization [37] or to lasting neurotoxic effects of prolonged stimulant intake [14]. To clarify the question of how continued khat consumption might affect the further development of psychotic symptoms, the number of relapses related to khat intake would be of tremendous importance. Our observation was that once a patient has developed severe psychotic symptoms he is restrained (e.g. chained) and kept away from the drug until symptoms remit. However, as soon as he is released the patient engages again in (often excessive) khat consumption until symptoms relapse and he is restrained again. Many patients had lived through this circle several times. In Western samples of amphetamine-induced psychosis, first relapses have mostly been studied [37]. Furthermore, it seems plausible that the conditions under which cases with psychotic symptoms were frequently kept may have infuenced the course of any psychopathological development.
Some methodological shortcomings of the study are related to the nature of field work in this specific post-conflict setting, which involved restricted freedom of movement due to the security situation and limited resources. However, although a perfect design is not possible in a rather complex field situation, the importance of such studies is evident from the fact that little or no information from post-war Somalia is available. First, we could not control whether our sample was comparable to the population from which it was drawn due to the lack of recent statistics in Somalia.
Second, the validity of our screening and clinical interview can be questioned because of various points, e.g. whether the descriptions of symptoms and disability might be adequate for the Somali culture, or whether factors such as interpretation or the interviewing of a whole family rather than a single patient might distort the information. In our approach we decided to use disability as a selection criterion and a descriptive rather than a normative approach to identify the reasons for it. Thus, we decided to use the more unspecific terms 'cases with psychotic symptoms' or 'psychotic symptoms' rather than 'schizophrenia' or 'drug-induced psychosis'. However, the fact that our local interviewers found 123 of the 169 positive screening cases (72.8%) restrained, i.e. chained or locked up, and the overall high average PANSS scores of the examined cases, show that there are 'hard' criteria, which justify the use of the term 'psychosis' and validate our screening. Nevertheless, we recognize that with our method we must have failed to discover persons suffering from psychotic and other mental disorders (false positives). But assuming that this error is somehow the same for the whole sample, our finding that the group of society that engages most in khat chewing (males above the age of 12) most often suffers severe impairment due to mental disorders shows evidence that khat is related to mental health problems.
Thirdly, the fact that we could not choose our matched controls from the pool of negative screening cases – the resources that would have been necessary to (repeatedly) contact and arrange appointments with them exceeded our capacities – leads without any doubt to an overestimation of the specifity of our detection procedure.
Furthermore, the retrospective third-person assessment of the onset of psychotic signs, as well as the retrospective and subjective assessment of khat use, will have introduced some error variance in the data. However, we believe that especially in the Somali culture khat intake cannot be compared to any food intake, of which the retrospective assessment is problematic. In the Somali culture khat has a special significance, which also comes from existing traditional knowledge about its dangers. Therefore we suppose that retrospective assessment of khat intake is more valid than that of food intake. Also, the effects of social desirability might have affected both the detection of positive screening cases and the assessment of khat use. The fact that dozens of families approached the compound of our collaborating partner agencies in order to find help for their mentally ill members (often they brought them in chains to the compound) shows the great need for mental health services. Thus, the tendency to hide family members from our interviewer teams can be estimated as marginal. On the contrary, during the interviews our greater fear was that households would over-report the presence of mentally challenged family members. For this reason, we counted only those persons as positive screening cases when their existence in the household could be validated (e.g. by their physical presence). The assessment of khat consumption is another point where effects of social desirability can be assumed. Whereas the information given by examined cases could be validated by the information of family members, we were well aware of the danger that the matched controls might have under-reported their khat intake. In order to allow for this in the interview we spent much time and effort to identify the real khat intake (e.g. often we had to 'negotiate' about the answer to the khat questions – a habit inherent in Somali culture). Nevertheless, the high correlations validate the assessment procedures.
Also, the sample size was too small to answer some of the questions in detail, especially the small number of female examined cases. However, the results were nonetheless statistically very robust.
In sum, our design could not determine the existence of a causal relationship between khat and psychosis – this would have been too ambitious a goal. However, our main findings do not contradict the hypothesis that khat might cause psychosis.
Future research should focus on the question of causality. Unfortunately, the Horn of Africa is probably the region where stimulant abuse currently reaches highest levels on a world-wide scale. This circumstance offers a unique research opportunity for cross-sectional and repeated measurement studies, which would enable us better to understand the relationship between schizophrenia and drug effects and to fill the gap in knowledge related to khat use. At the same time, the alarmingly high prevalence of khat chewing among persons who suffer from psychotic symptoms in one of the countries of its highest use, and the magnitude of human suffering associated with it, demands the development of adequate community-based prevention and treatment interventions. The usefulness of standard procedures derived from developed countries and brought to the Horn of Africa must be viewed in a "service-research attitude"[38]. We believe that khat abuse has become a tragic obstacle for the reconstruction of this war-torn society; consequently, there is an urgent need to address this mental health issue with means other than prohibition and regulation of the demand side through taxation, as khat is integral to the Somali culture.
Conclusion
Not khat intake per se but rather specific patterns of it are linked to the development of psychotic symptoms, like early onset in life, excessive chewing (> 2 bundles/day) and use as self-medication for war trauma related symptoms. Although we cannot establish a causal relationship between khat and psychosis, we find some evidence that the prevalence of psychotic disorders is increased among the male adult population of Hargeisa. Ex-combatants are the group in society who are most affected by severe mental disorders, among them we find the highest abuse of khat. The way of khat use in Hargeisa is changing, indicated by e.g. the development of a group of heavy users who show consumption patterns similar to amphetamine addicts in Western countries. Measures of prevention and treatment of and further research on khat-related severe mental disorders have to be undertaken, however, taking into consideration that khat is an integral part of the Somali culture, which cannot simply be prohibited.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
Development of the study design and selection of instruments were accomplished by MO, FN, TE, MS, BR and HeH. MS, TE and MO performed pilot studies. Training and supervision of local interviewers was provided by MO, HaH, FN and BL. Clinical interviews were conducted by MO, MS, CC and BL. Statistical analysis was performed by MO and TE. The article was composed and revised by MO, TE, BR, FN and HeH.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We would like to thank Mrs. Christine Klaschik and Dr. Rebecca Horn for their participation in the data collection and training of the interviewers.
The study was supported by the Deutsche Forschungsgemeinschaft (DFG), the European Community (Project DECOSAC Sektor Project und EC DRP NW Somalia) and the German Technical Cooperation (GTZ). Neither funding organizations influenced the design or conduct of the study, the collection, management, analysis and interpretation of the data, or the preparation, review and approval of the manuscript.
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| 15707502 | PMC554104 | CC BY | 2021-01-04 16:27:56 | no | BMC Med. 2005 Feb 12; 3:5 | utf-8 | BMC Med | 2,005 | 10.1186/1741-7015-3-5 | oa_comm |
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BMC Infect DisBMC Infectious Diseases1471-2334BioMed Central London 1471-2334-5-91572534510.1186/1471-2334-5-9Research ArticleCNS activity of Pokeweed Anti-viral Protein (PAP) in mice infected with Lymphocytic Choriomeningitis Virus (LCMV) Uckun Fatih M [email protected] Larisa [email protected] Alexei O [email protected] Heather E [email protected] Alexander S [email protected] Parker Hughes Center for Clinical Immunology, St. Paul, MN 55113, USA2 Research Institute for Epidemiology and Microbiology, 220050 MINSK, Belarus2005 22 2 2005 5 9 9 25 6 2004 22 2 2005 Copyright © 2005 Uckun et al; licensee BioMed Central Ltd.2005Uckun et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Others and we have previously described the potent in vivo and in vitro activity of the broad-spectrum antiviral agent PAP (Pokeweed antiviral protein) against a wide range of viruses. The purpose of the present study was to further elucidate the anti-viral spectrum of PAP by examining its effects on the survival of mice challenged with lymphocytic choriomeningitis virus (LCMV).
Methods
We examined the therapeutic effect of PAP in CBA mice inoculated with intracerebral injections of the WE54 strain of LCMV at a 1000 PFU dose level that is lethal to 100% of mice within 7–9 days. Mice were treated either with vehicle or PAP administered intraperitoneally 24 hours prior to, 1 hour prior to and 24 hours, 48 hours 72 hours and 96 hours after virus inoculation.
Results
PAP exhibits significant in vivo anti- LCMV activity in mice challenged intracerebrally with an otherwise invariably fatal dose of LCMV. At non-toxic dose levels, PAP significantly prolonged survival in the absence of the majority of disease-associated symptoms. The median survival time of PAP-treated mice was >21 days as opposed to 7 days median survival for the control (p = 0.0069).
Conclusion
Our results presented herein provide unprecedented experimental evidence that PAP exhibits antiviral activity in the CNS of LCMV-infected mice.
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Background
The broad-spectrum anti-viral agent PAP (Pokeweed antiviral protein) [1] is a well-characterized 29-kDa plant-derived ribosome-inactivating protein (RIP) isolated from Phytolacca americana [2]. The anti-viral activity of PAP has been described against a wide range of viruses, including HIV-1, herpes simplex virus, cytomegalovirus, influenza virus and polio virus [2] (For the most recent review, please see: [3]). The activity of PAP is attributed to its ability to inhibit protein synthesis by catalytically cleaving a specific adenine base from the highly conserved alpha-sarcin/ricin loop (SRL) of the large ribosomal RNA [4,5] as well as from viral RNA[3]. The potent anti-HIV activity of PAP at nanomolar ranges taken together with the relative ease of large scale purification has led to the clinical use of PAP [2]. Since 1985, our group has studied the multifunctional efficacy of this potent agent [1,2,4-42].
The purpose of the present study was to further elucidate the anti-viral spectrum of PAP by examining its effects on survival of mice challenged with intracerebral inoculations of lymphocytic choriomeningitis virus (LCMV). LCMV is a rodent-borne arena virus that can result in persistent neuronal infection on mice [43,44]. Alpha-Dystroglycan (alpha-DG) was recently identified as a receptor for LCMV as well as for several other arena viruses including Lassa fever virus [45]. The binding affinity of LCMV to alpha-DG determines viral tropism and the outcome of infection in mice [46]. LCMV has also been associated with both postnatal and intrauterine human disease. Infection in man is acquired after inhalation, ingestion or direct contact with virus found in the urine, feces and saliva of infected mice, hamsters and guinea pigs. Congenital LCMV infection is a significant, often unrecognized cause of chorioretinitis, hydrocephalus, microcephaly or macrocelphaly as well as mental retardation. Acquired LCMV infection, asymptomatic in approximately one third of individuals, is productive of central nervous system manifestations in one half of the remaining cases. Aseptic meningitis or meningoenceophalitis are the predominant syndromes, although transverse myelitis, a Guillain-Barre-type syndrome, as well as transient and permanent acquired hydrocephalus have also been reported [47].
In the present study, we describe the significant efficacy of the highly stable, potent and broad-spectrum anti-viral agent PAP in a murine model of LCMV. Our results presented herein provide unprecedented experimental evidence that PAP exhibits antiviral activity in the CNS of LCMV-infected mice.
Methods
Purification of PAP
PAP was purified from spring leaves of the pokeweed plant, P. americanca, in four steps [22,38]. Briefly, spring leaves were homogenized in a neutral pH buffer and centrifuged to sediment the remaining cellular fragments. The supernatant was fractionated between 60–90% saturation of ammonium sulfate and the precipitate dialyzed against a low-ionic strength pH 7.5 buffer. The solution was passed through a DEAE cellulose column and the PAP-containing flow-through fraction was then applied to a cation exchange resin S-Sepharose column. The adsorbed PAP was eluted in a linear KCl gradient. The protein peak which eluted at 0.12 KCl was taken as PAP. This fraction was dialyzed extensively against water and lyophilized for storage at -20°C. The procedure resulted in homogeneous PAP, with a purity of >99% as measured by both SDS -12% PAGE and analytical cation- exchange high-performance liquid chromatography. Purified PAP induced concentration-dependent inhibition of HIV-1 replication in normal human peripheral blood mononuclear cells infected with the HIV-1 strain HTLVIIIB with an IC50 p24 of 14 ± 2 nM.
Animal infection
Animal infection was performed in an appropriate Animal BioSafety Level-3 Laboratory (ABL-3) at BRIEM (Research Institute for Epidemiology and Microbiology, MINSK, Belarus) with the technician wearing appropriate facility clothing. The culture was thawed in a water bath at 37°C and then diluted in normal saline to achieve the required concentration. In this study, all mice were challenged with 1000 PFU which is 100-times higher than the LD50 dose. Each group of animals was placed in a separate cage.
LCMV model
Three week old CBA mice were intracerebrally infected with 1000 PFU of WE54 strain of LCMV that resulted in lethality of 100% of control (non-treated) animals in 6–8 days after infection. Control animals were given physiologic salt solution as a placebo instead of the compound. In general, for non-treated animals, clinical signs of the disease manifested on the 5th and 7th days by presenting: weight loss, immobility, disheveled hair, convulsions, severe decubitus paralysis and death. All subjective measurement of decreased mobility and scruffy fur were done in a blinded fashion as not to influence the results. The protective properties of the experimental anti-viral drugs were assessed by using the following treatment-preventative regimen: Mice were treated either with vehicle (n = 20) or PAP (n = 10) (0.25 mg/kg) administered intraperitoneally 24 hours prior to, 1 hour prior to, and 24 hours, 48 hours, 72 hours, and 96 hours after virus inoculation. Mice were then observed for 21-days post infection. The protective effect of the experimental anti-viral drugs was evaluated according to the rise of the survival rate and prolongation of mean life of the experimental animals as compared with the control animals.
Statistical analysis
Statistical significance was determined using the Kaplan Meier Log-Rank test.
Results
In order to evaluate the anti-LCMV activity of PAP, mice were inoculated with intracerebral injections of LCMV at a dose level that is lethal to 100% of mice within 6–8 days. Mice were treated either with vehicle or PAP as described in the methods section. Of the 20 control mice, 3 died on day 1 immediately after intracerebral injection due to accidental brain injury and are excluded from the data analysis. All 17 of the remaining control mice developed clinical signs of LCMV infection between day 4 and 6, including weight loss, disheveled hair, decreased mobility and paralysis (Table 1). All control mice developed seizures between day 5 and day7 and died between day 6 and day 8 with a median survival of 7 days (Table 1, Figure 1).
Table 1 Anti-LCMV activity of PAP in CBA mice
Disease Unset
Days after inoculation with LCM Virus
Survival (days) Weight loss (grams) Decreased mobility Convulsions Hair disheveled
Vehicle
Mouse #1* <<1 NA 0 NA NA
Mouse #2* <<1 NA 0 NA NA
Mouse #3* <<1 NA 0 NA NA
Mouse #4 6 1.5 4.5 5.5 4.5
Mouse #5 6 2 4.5 5.5 4.5
Mouse #6 7 2 5 6 5
Mouse #7 7 1.5 5 6 5
Mouse #8 7 2.5 5 6 5
Mouse #9 7 4.5 5 6 5
Mouse #10 7 4.5 5 6 5
Mouse #11 7 4.5 5 6 5
Mouse #12 8 2 6 7 6
Mouse #13 8 2.5 6 7 6
Mouse #14 8 1 6 7 6
Mouse #15 8 1.5 6 7 6
Mouse #16 8 2 6 7 6
Mouse #17 8 2 6 7 6
Mouse #18 7 1.5 5 6 5
Mouse #19 8 2 6 7 6
Mouse #20 8 1.5 6 7 6
PAP 0.25 mg/kg
Mouse #1 <<1 NA NA NA NA
Mouse #2 <<1 NA NA NA NA
Mouse #3 6 1 4.5 5.5 5
Mouse #4 7 1.5 5 6 5
Mouse #5 8 1.5 6 7 5.5
Mouse #6 21 0 10 NO NO
Mouse #7 21 0 10 NO NO
Mouse #8 21 0 10 NO NO
Mouse #9 21 0 10 NO NO
Mouse #10 21 0 10 NO NO
*Mouse died after intracerebral injection; NA = not applicable, NO = not observed
Figure 1 Protective Activity of PAP in CBA Mice Challenged with LCM Virus. CBA mice were inoculated with intracerebral injections of the WE54 strain of LCMV at a 1000 PFU dose level that is lethal to 100% of mice within 7–9 days. Mice were treated either with vehicle or PAP administered intraperitoneally 24 hours prior to, 1 hour prior to and 24 hours, 48 hours 72 hours and 96 hours after virus inoculation. Mice were then observed twice daily for 21 days for morbidity and mortality. (Top) Shown are representative survival curves detailing the cumulative proportions (%) of mice surviving after virus inoculation. See Table 1 for more detailed information of the treatment outcome. (Bottom) Shown is Kaplan-Meir life-table analysis and statistical comparison using the log-rank test.
Of the 10 mice treated with the broad-spectrum antiviral agent pokeweed antiviral protein (PAP), 2 died on day 1 immediately after intracerebral injection due to accidental brain injury and are invaluable. Five of the remaining 8 mice (62.5%) treated with PAP remained alive > 21 days post-LCMV inoculation (median survival >21 days, p = 0.0069) (Table 1, Figure 1). These mice exhibited no LCMV infection-related weight loss or convulsions and showed no signs of scruffy fur. A significant improvement in mobility was also noted. Thus, PAP exhibited significant in vivo anti-LCMV activity in mice challenged intracerebrally with an otherwise invariably fatal dose of LCMV.
Discussion
In the present study, we describe the significant efficacy of the highly stable, potent and broad-spectrum anti-viral agent PAP in a murine model of LCMV. Our results presented herein provide unprecedented experimental evidence that PAP exhibits antiviral activity in the CNS of LCMV-infected mice. Future studies will examine the therapeutic activity of PAP against LCMV in post-challenge settings. As PAP is well described as an HIV agent, this observation is also relevant as HIV-1 infects the central nervous system (CNS) and it has been feared that the CNS may be a sanctuary site where HIV-1 could hide and continue to replicate despite otherwise effective antiretroviral treatment [48,49]. Antiretroviral therapy of HIV-infected children is associated with a decline in CSF HIV RNA and an improvement in neurological status. The development of genotypic mutations was different in CSF and plasma, suggesting discordant viral evolution. These results suggest that antiretroviral treatment in children should include agents with activity in the CNS [50]. While these results suggest that PAP crosses the blood brain barrier and may therefore be beneficial in other viral infections affecting CNS, they need to be interpreted with due caution for HIV infections involving the CNS since LCMV affects leptomeninges while HIV affects neurons. In future studies, we must also consider the possibility that PAP penetrates the blood brain barrier only when the latter is impaired by the intracerebral administration of the virus. If this is the case, PAP may not be useful in pre-challenge prophylaxis against LCMV-mediated CNS infection. While these results support the notion that the antiviral activity spectrum of PAP covers LCMV as well, an immunomodulatory effect of PAP may also contribute to the observed prophylactic efficacy of PAP against LCMV.
Cases of LCMV infections have been reported in Europe, the Americas, Australia, and Japan. According to the Center for Disease Control, currently there is no specific drug therapy for LCMV. Although the anti-viral agent ribavirin is effective against LCMV in vitro, there is no established evidence to support its use for treatment of LCMV in humans [51].
The anti-viral activity of PAP has been described against numerous pathogenic viruses, which included poliovirus, HIV-1, herpes simplex virus, cytomegalovirus, influenza virus and now, the negative-strand RNA virus Lymphocytic choriomeningitis virus. The ability of PAP to inhibit viral protein synthesis and depurinate viral RNA and DNA [15,16] as well as capped rRNA and mRNAs [52] and its ability to inhibit ribosomal frame shifting and retransposition, make it and ideal candidate for anti-viral strategies.
Conclusion
Treatment of CBA mice with the broad-spectrum anti-viral agent, PAP significantly improved the probability of survival following the LCMV challenge and decreased overall LCMV-related symptoms. The ability of PAP to exhibit anti-viral activity within the central nervous system is also encouraging within the framework of potential HIV-treatment. We have recently described the rational design and engineering of several recombinant PAP mutants with superior anti-HIV activity [11,17]. In our future studies, we plan to describe the potential anti-LCMV activity of these PAP mutants as well as optimizing the prophylactic and post-exposure treatment regimens. In addition, it will be important to determine if PAP or PAP mutants have activity against other viruses associated with lethal viral hemorrhagic fevers and/or encephalomyelitis, such as the Ebola viruses of the Filoviridae family [53-56].
List of abbreviations
LCMV: Lymphocytic choriomeningitis virus
PAP: Pokeweed anti-viral protein
HIV and FIV: Human and Feline immunodeficiency virus, respectively
Competing interests
FMU is listed as an inventor on a number of PAP patents which are owned by PHI. FMU, AV and HET are salaried employees at PHI. PHI is a non-profit public charity.
Authors' contributions
FMU designed the research project. LR, AP & LT conducted the Lassa experiments. FMU & AV provided PAP and coordinated the Lassa experiments with LR, AP and LT. FMU and HT wrote the manuscript. All authors read and approved manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
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| 15725345 | PMC554105 | CC BY | 2021-01-04 16:28:14 | no | BMC Infect Dis. 2005 Feb 22; 5:9 | utf-8 | BMC Infect Dis | 2,005 | 10.1186/1471-2334-5-9 | oa_comm |
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BMC Health Serv ResBMC Health Services Research1472-6963BioMed Central London 1472-6963-5-161572370110.1186/1472-6963-5-16Research ArticleWhat is important in evaluating health care quality? An international comparison of user views Groenewegen Peter P [email protected] Jan J [email protected] Herman J [email protected] der Eijk Ingrid [email protected] Wienke GW [email protected] NIVEL, Netherlands Institute for Health Services Research, PO Box 1568, 3500 BN, The Netherlands2 At the time of this research: department of Gastroenterology & Hepatology, University Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands2005 21 2 2005 5 16 16 5 9 2004 21 2 2005 Copyright © 2005 Groenewegen et al; licensee BioMed Central Ltd.2005Groenewegen et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Quality of care from the perspective of users is increasingly used in evaluating health care performance. Going beyond satisfaction studies, quality of care from the users' perspective is conceptualised in two dimensions: the importance users attach to aspects of care and their actual experience with these aspects. It is well established that health care systems differ in performance. The question in this article is whether there are also differences in what people in different health care systems view as important aspects of health care quality. The aim is to describe and explain international differences in the importance that health care users attach to different aspects of health care.
Methods
Data were used from different studies that all used a version of the QUOTE-questionnaire that measures user views of health care quality in two dimensions: the importance that users attach to aspects of care and their actual experience. Data from 12 European countries and 5133 individuals were used. They were analysed using multi-level analysis.
Results
Although most of the variations in importance people attach to aspects of health care is located at the individual level, there are also differences between countries. The ranking of aspects shows similarities. 'My GP should always take me seriously' was in nearly all countries ranked first, while an item about waiting time in the GP's office was always ranked lowest.
Conclusion
Differences between countries in how health care users value different aspects of care are difficult to explain. Further theorising should take into account that importance and performance ratings are positively related, that people compare their experiences with those of others, and that general and instrumental values might be related through the institutions of the health care system.
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Background
Large differences between countries exist in the use, costs, quality, and accessibility of health services [1]. Also large differences exist between countries in health care performance and in people's evaluations of their health care system [2-7]. But do these differences also exist in what people from different countries view as important in evaluating health care quality? The World Health Report 2000 has been criticized on its assumption of a universal value base to all health care systems; values such as responsiveness may be valued differently in different countries [8]. In this article we address this issue by comparing what people find important in general practice care in different countries. Grol et al studied patients' priorities in general practice [9]. They found both many similarities and differences between countries. Particularly, doctor-patient communication and accessibility of services were common priorities among general practice visitors in different countries. Service aspects, such as waiting times, were considered less important.
In this study we did a secondary analysis on surveys of patient views on quality of health care. Patients' views were measured using the QUOTE-questionnaire – with the acronym QUOTE standing for QUality Of care Through the patients' Eyes – that distinguishes two quality of care dimensions: performance and importance [10]. Performance relates to the actual experience of the use of health care services (rather than a patient satisfaction judgement), which is in line with recent developments within health services research. Importance refers to the fact that people see some features of health services as more significant than others. They reflect what people see as desired qualities in health care. This approach avoids problems with conceptualising people's evaluations of health care in terms of satisfaction (usually high levels of satisfaction, not specific enough to be used in quality improvement) and expectations (ambiguous relations between expectations and actual experiences).
To select relevant quality of care aspects, a general and a disease-specific approach was followed, using focus group discussions [10]. With this procedure a series of QUOTE-questionnaires has been tailored to the needs of various patient groups. In these QUOTE-questionnaires the expectations of people are reflected in the statements included in the instruments. These questionnaires have been used in several studies in different countries.
Research questions
In this article we first compare the importance dimension of QUOTE across several European countries and Israel to gain insight in the similarities and differences in people's views on quality of care. Secondly, we will look at the relationship between importance and performance ratings as part of an explorative analysis to explain differences in importance ratings between patients and/or countries. The general research question is:
Do patients in different European countries think differently about the relative importance of various aspects of quality of care, and if so, how can these differences be explained?
This general question is divided into the following separate questions:
a. To what extent do the importance judgements of patients cluster within countries when individual characteristics of patients are taken into account?
b. Does the ranking of importance judgements vary between countries?
c. What is the relationship between the average performance of health care systems, as judged by patients, and the individual importance judgements?
When we compare the importance judgements of patients between countries, we will take into account individual characteristics of respondents to rule out differences in the composition of the groups of respondents. With respect to the relationship between importance and performances scores it might be anticipated that in general people attach more importance to those aspects that they actually experience less often. Analogous to the economic mechanism of decreasing marginal utility, e.g. quick service without waiting time in the doctor's office might be valued as less important, if in general services are quick and people don't have to wait long. However, at the same time it can be hypothesized that it's no use aspiring to something that nobody has. If quality of care ratings, as seen through the eyes of the patient, are low on the average and if there is small variation in these performance ratings between individuals, people will probably not find these aspects important. This idea is based on a mechanism of social comparison [11].
Methods
Material
The First Dutch QUOTE-questionnaires (for disabled persons, COPD, arthritis and frail elderly people) served as a starting point for our database [10,12]. These questionnaires contain 16 general importance and performance items. In the SCOPE-project (Supporting Clinical Outcomes in Primary Care for the Elderly) the QUOTE-elderly was used in Finland, Ireland and the Netherlands [13]. A large contribution to the database comes from an international study of patients with inflammatory bowel disease (IBD) in eight countries [14]. This study used ten generic questions (of the original 16) relating to GPs. Additional material was obtained from the UK (QUOTE-disabled) and from Belarus and the Ukraine [15,16].
The QUOTE-questionnaires have been translated in the context of different projects. In all but two cases backward-forward translations have been used. Answering formats of importance items were: Not important (1); Fairly important (2); Important (3); and Extremely important (4). The answering formats for the performance items were: No (1); Not really (2); On the whole, yes (3); and Yes (4). The equivalence of the answering formats in different languages has not been assessed. The wording of the importance items that were used in the QUOTE-questionnaires are presented in tables 2 and 5 and throughout the result section of the article. The performance items ask for the actual experience of respondents. One of the importance items is, e.g., 'my GP should always take me seriously'. The corresponding performance item is: my GP always takes me seriously'. QUOTE-items included in the analysis refer to the organisation of health care services and the care giving process.
Table 1 Number of respondents in health care user groups and countries
Country User group Selection of users N per user group N per country
Belarus GP patients GP office 500 500
Denmark IBDa hospital files 102 102
Finland Elderly PHC files 143 143
Greece IBD hospital files 96 96
Ireland IBD hospital files 57 73
Elderly Homecare Organisation files 16
Israel IBD hospital files 46 46
Italy IBD hospital files 201 201
Netherlands Migrants Snowball method 152 2873
IBD hospital files 192
Elderly GP files 338
Disabled GP files/patient organisation 334
Diabetes GP files/patient organisation 681
COPD GP files/patient organisation 604
Arthritis GP files/patient organisation 572
Norway IBD hospital files 93 93
Portugal IBD hospital files 36 36
UK Disabled GP files 480 480
Ukraine GP patients GP office 490 490
Total 5133 5133
a IBD inflammatory bowel disease
Table 2 Descriptive statistics for importance items: mean, variance at user level, variance at country level, intra-class correlation coefficient, uncorrected (ICCu) and corrected for age and sex (ICCc)
Item My GP should Mean Var Users Var Country ICCu ICCc
3 always take me seriously 3.45 .377 .044 .105 .092
6 inform me, in understandable language, about the medicines that are prescribed for me 3.35 .532 .059 .100 .107
1 have a good understanding of my problems 3.24 .615 .147 .193 .087
9 make sure that I can see a specialist within 2 weeks after being referred to him/her 3.13 .591 .198 .251 .256
4 always keep appointments punctually 3.10 .545 .074 .120 .113
2 allow me to have an input into the decisions regarding the treatment or help I receive 3.07 .648 .071 .099 .063
7 prescribe medicines which are fully covered by the National Health System or social services 3.05 .896 .100 .100 .113
8 always be easy to reach by telephone 3.02 .509 .060 .092 .111
10 always communicate with other health and social care providers about the services I require 2.90 .650 .084 .114 .116
5 not keep me in the waiting room for more than 15 minutes 2.54 1.016 .062 .058 .087
Table 3 Mean scores of ten importance items per country
1* 2* 3* 4* 5* 6* 7* 8* 9* 10*
Belarus** 2.42 2.61 3.21 3.34 2.59 3.04 2.98 2.68
Denmark 3.23 3.22 3.47 2.75 2.37 3.57 2.43 2.62 3.10 2.57
Finland** 2.91 3.03 2.84 2.87 2.86 3.00
Greece 3.46 3.10 3.51 3.25 2.64 3.34 3.09 3.23 3.03 2.93
Ireland 3.47 3.15 3.60 2.90 2.42 3.55 3.02 3.01 3.21 2.96
Israel 3.71 3.70 3.84 3.52 3.07 3.75 3.40 3.14 3.51 3.12
Italy 3.10 2.89 3.23 2.63 1.95 3.12 2.79 2.70 2.03 2.39
Netherlands 3.21 3.28 3.59 3.24 2.50 3.42 3.01 3.33 3.27 3.29
Norway 3.42 3.14 3.60 3.08 2.46 3.57 3.00 2.81 3.17 2.56
Portugal 3.61 2.94 3.61 3.19 2.78 3.42 3.64 3.42 3.44 3.28
UK 3.37 3.20 3.44 3.02 2.57 3.40 3.21 3.27 3.46 3.20
Ukraine** 2.68 2.77 3.37 3.46 2.71 3.21 2.88 2.92
Overall 3.24 3.07 3.45 3.10 2.54 3.35 3.05 3.02 3.13 2.90
* See table 2 for the wording of the items.
** Not all ten items were used in these countries.
Table 4 Ranking of items by mean importance within each country; r1-r10 is the ranking, cell entries contain the item number corresponding to that rank.
Country r1 r2 r3 r4 r5 r6 r7 r8 r9 r10
Denmark 6 3 1 2 9 4 8 10 7 5
Greece 3 1 6 4 8 2 7 9 10 5
Ireland 3 6 1 9 2 7 8 10 4 5
Israel 3 6 1 2 4 9 7 8 10 5
Italy 3 6 1 2 7 8 4 10 9 5
Netherlands 3 6 8 10 2 9 4 1 7 5
Norway 3 6 1 9 2 4 7 8 10 5
Portugal 7 1 3 9 6 8 10 4 2 5
UK 9 3 6 1 8 7 10 2 4 5
Table 5 Pearson correlation coefficients between importance- and performance items on individual level and between average performance items in each country and the individual importance scores (mixed level)
Importance/performance items Individual level Mixed level
have a good understanding of my problems .346 ** .322
allow me to have an input into the decisions regarding the treatment or help I receive .277 ** .263
always take me seriously .126 ** .167
always keep appointments punctually .140 ** .081
not keep me in the waiting room for more than 15 minutes .150 ** .054
inform me, in understandable language, about the medicines that are prescribed for me .179 ** .170
prescribe medicines which are fully covered by the National Health System or social services .080 ** -.057
always be easy to reach by telephone .163 ** .184
make sure that I can see a specialist within 2 weeks after being referred to him/her .135 ** .119
always communicate with other health and social care providers about the services I require .281 ** .287
** p < .01
Table 1 gives the number of respondents in each user group and country and the selection of respondents. In the case of Belarus and Ukraine respondents were selected by distributing questionnaires to people who visited general practices. Response rates are not available for these two countries. In all other countries but one addresses were randomly selected from the files of health care (and in one case home care) institutions and (in The Netherlands) from membership lists of voluntary patient organisations, irrespective of actual visits to a GP. In the case of the QUOTE-Migrants, respondents from ethnic groups in the Netherlands were selected using a snowball sampling method; data for these respondents were collected through oral interviews in the respondents' mother language. In all other cases postal questionnaires were used, followed by one or two reminders. Response rates vary between 35% (elderly in the Netherlands) and 78% (the average of the IBD samples).
Statistical analysis
All 5133 health care users reported for each of (maximum) ten items their importance and performance ratings. The importance ratings are dependent variables in a series of statistical analyses with patients hierarchically nested in countries. In contrast to traditional forms of analysis of variance in which factors have 'fixed' effects, countries are considered to have 'random' effects. Such a variance component model is preferred over traditional analysis if the number of categories exceeds ten [17,18]. The degree of resemblance between patients belonging to the same country can be expressed by the intraclass correlation coefficient (ICC). If there is no resemblance between patients within countries, the ICC is zero or near zero. An ICC of .15 is considered quite high [19]. Most commonly ICCs are lower. For instance, the median ICC calculated for more that 1000 primary care variables, was .01 [20].
The ICC is statistically defined as the variance between countries divided by the total variance. An ICC of zero therefore implies no variance between countries, indicating the absence of differences between countries in patients' importance ratings. Age and sex were included as covariates to take into account differences in the composition of responder groups in the different countries, because of their association with importance scores [9,10,21-23]. Correction for different user groups turned out to be impossible due to the small number of countries for some user groups. Differences in number of cases between countries were taken into account in the statistical analysis. The estimates of country parameters are more precise with larger numbers per country.
In order to explore the relationship between importance and performance ratings Pearson correlations were calculated between the performance items, both at the individual level and aggregated to country level, and the importance rating on user level. In the introduction we have put forward two hypothetical relations between variation of performance within the countries and the importance ratings. To look at the relation between variation of performance and importance ratings, a distinction was made between countries with large variation in experienced health care quality and countries with smaller variation. Based on the mean standard deviation (SD) of all ten performance items, the countries were equally split into: Denmark, Italy, Belarus, Ireland, Ukraine and Portugal (mean SDs ranging from 1.17 to 1.40) indicating countries with high variation and Greece, Finland, Israel, Norway, Netherlands and UK (mean SDs ranging from .82 to 1.08) indicating countries with low variation. Furthermore, explore the relationship between variation in performance at country level, individual respondents performance experience and their importance ratings, we have divided the individual respondents into those who experienced high performance and those who experienced low performance. To keep these distinctions conveniently arranged in one table, we recoded the individual performance scores from the four point scale to a two point scale, with the combination of 'No' and 'No, not really', indicating poor quality, versus 'On the whole, yes' and 'Yes'.
Results
We start the presentation of the results with a description of the overall importance that respondents in all twelve countries attached to the different aspects and the clustering of their answers within countries. We then move to the differences in the ranking of aspects between countries. Finally, we will present results for the relationship between the actual experiences of respondents, both individually and aggregated to an average for each country, and the importance they attach to the different aspects.
Importance judgements and clustering
As shown in table 2, 'The GP should always take me seriously' is seen as most important, halfway between 'important' and 'extremely important' on the Likert scale. Less than 1% of users rated this item as 'not important' (not in table). The differences between users as well as countries for this aspect are the smallest of all aspects (smallest variance, both on user- and country level). 'The GP should not keep me in the waiting room for more than 15 minutes' is seen as least important, halfway between 'important' and 'fairly important'. About 20% of users rated this aspect as 'not important' (not in table). The differences between users for this aspect are largest (highest variance on user level). The importance of 'The GP should make sure that I can see a specialist within 2 weeks' shows the biggest differences between countries. The uncorrected ICCs vary from low (.058 aspect 5) to high (.251 aspect 9). The sex-age adjusted ICCs are a little (7%) lower on average, but still range to high.
In order to explore differences between user groups, we have computed intra-class correlation coefficients for user groups within the Netherlands only. These coefficients are on average higher than those regarding countries. We also analysed the differences between countries within the IBD patient groups. These differences are much like the figures of table 2. So the estimated intra-class coefficients of table 2 seem to refer more to differences between countries than differences between user groups.
Differences between countries and ranking
The variation between the countries for each aspect is illustrated in table 3 by mean importance scores for all aspects. For instance, 'The GP should always take me seriously' is seen as most important in Israel and as least important in Finland. 'The GP should not keep me in the waiting room for more than 15 minutes' also is seen as most important in Israel but as least important in Italy.
In order to look at the consistency of user views across the different countries, the ten importance aspects were ranked according to their mean value within each country. Table 4 gives the ranks for countries where all ten aspects are available. Some rankings differ between countries. For instance in Denmark 'My GP should inform me, in understandable language, about the medicines that are prescribed for me' is ranked first. In Portugal it is 'My GP should prescribe medicines which are fully covered by the National Health System or social services'. But there is also a general pattern. The service aspect 'My GP should not keep me in the waiting room for more than 15 minutes' is ranked last in all countries, while 'My GP should always take me seriously' is ranked high in all countries.
Importance and performance
Looking at the relationship between importance and performance ratings by means of correlation coefficients, table 5 shows that the correlation coefficients on individual level are all positive and range from small (.080) to moderate (.346). Because of the large number of respondents, even the small correlations are statistically significant. The correlation between the average performance in each country and the individual importance vary also from small (.054) to moderate (.322). Because of the small number of countries these correlation coefficients are not significantly different from zero. Except for one aspect 'My GP should prescribe medicines which are fully covered by the National Health System or social services' all correlations are positive, while we anticipated negative correlations.
If we take into account the variation in performance ratings within countries, the positive relationship between importance and performance ratings is somewhat stronger in the 'high variation' mode compared to the 'low variation' mode. This can be seen by comparing the difference between the first two columns of table 6 and those between the last two columns. We had specifically expected to find a difference between the last two columns of table 6, i.e. within countries with low variation in aggregate performance. However, for only half of the items the difference is statistically significant.
Table 6 Mean scores of ten importance items according to variation in performance items per country and individual scores on corresponding performance items (Low vs High performance)
Country level: High variation in performance Low variation in performance
Individual level: Low performance High performance Low performance High performance
1 should have a good understanding of my problems 2.06 2.92 3.04 3.28
2 should allow me to have an input into the decisions regarding the treatment or help I receive 2.27 3.03 3.11 3.30
3 should always take me seriously 2.99 3.35 3.66 3.55
4 should always keep appointments punctually 3.01 3.32 3.08 3.22
5 should not keep me in the waiting room for more than 15 minutes 2.26 2.82 2.42 2.60
6 should inform me, in understandable language, about the medicines that are prescribed for me 2.80 3.24 3.28 3.43
7 should prescribe medicines which are fully covered by the National Health System or social services 2.74 2.95 2.98 3.09
8 should always be easy to reach by telephone 2.63 3.04 3.29 3.34
9 should make sure that I can see a specialist within 2 weeks after being referred to him/her 2.29 2.67 3.26 3.34
10 should always communicate with other health and social care providers about the services I require 2.42 2.98 3.12 3.30
Italic means in column Low variation at country level/Low performance at individual level differ statistically significant from means in column Low variation at country level/High performance at individual level (Scheffé contrasts).
Overall, importance scores are somewhat lower in countries with high variation in performance ratings as compared to countries with low variation in performance scores.
Discussion
The objective of our study was to gain insight into similarities and differences in the value users of health care in different countries attach to aspects of care. As an indicator of these values, importance scores of the QUOTE-questionnaires were used. These scores reflect what is important in evaluating health care quality according to users. Our results show that health care users in different countries to some extent think differently about the relative importance of various aspects of quality of care. Intra-class correlation coefficients were calculated to measure the difference between countries. They range from low to high. Sex-age adjusted intra-class correlation coefficients were only slightly lower. This means that demographic differences between the groups that filled in the questionnaires in different countries cannot explain the differences in average importance between the countries.
Although there are differences between countries, the importance rankings of the aspects also show consensus. 'My GP should always take me seriously' is nearly always ranked highest, while the item about waiting time is always ranked as least important. Since we only analysed a small sample of countries, it is difficult to generalise this result. However, it might say something about a hierarchy of these instrumental health care values, suggesting that values concerning respectfulness are seen as more important than service aspects, such as waiting time. This is in line with the findings of Grol et al. [9]. There are no accepted explanations for these value differences between countries. General theories about dimensions of culture see culture as the independent variable, explaining differences between countries in institutions and organisations [24,25]. A more specific application in the health care field is Payer's book Medicine and Culture that relates differences in culture to variations in the practice of medicine [26]. In this article, however, differences in values is what we want to explain.
In general, there is a positive relation between what people find important and their experiences, both on an individual level and related to the average experience in a country. The positive relation between importance and experience could probably be explained by a general tendency of cognitive consistency [27] or alternatively by processes of selection where people try to find those health care providers that do what they value most. This alternative hypothesis would mean that the correlation between importance and experience is stronger the more freedom of choice of health care provider people have. One of the assumptions behind the QUOTE-questionnaires is that importance and experience are two different aspects, together constituting quality judgements. The correlation at the individual level is not so high as to invalidate this assumption.
By looking at the variation in the performance scores, we have tried to include social comparison mechanisms into the analysis. It can be argued that if people's individual experience indicates low performance for certain aspects in countries, they will value these aspects as more important. However, such a hypothesis has to be rejected on the basis of the results presented in this article. A contrasting hypothesis, that in countries with low variation in actual experiences, people who experience low performance themselves, will not aspire to something that is apparently (from their own and others' experience) out of reach is only partly supported by our findings. Although people in this low variation condition have significantly lower importance scores for half of the items, still the differences in the high variation condition are higher.
Looking at the individual aspects, it can be argued that people don't find issues important if they are more or less guaranteed by the health care system. An example for this is the issue of prescribing pharmaceuticals that are fully covered by the health care insurance plans of patients. On the basis of the material presented in this article this hypothesis too has to be rejected. People in countries with low levels of cost sharing in this field, found this item more important than people in countries with higher levels of cost sharing. However, an alternative explanation for this finding could be that structural aspects of the health care system, e.g. on the dimension public – private, reflect general values [28,29]. If these general values also relate to instrumental values, than the relationship we found is understandable: the people in countries that took the pain to organise their health care system in a way that financial access is very good, might find this issue more important. In this explanation the mechanism between general and instrumental values is the institutional make-up of health care systems. The importance items in our study reflect instrumental values in the sense that they are low in a hierarchy of values where lower values contribute to the realisation of higher values. Solidarity and equity are examples of general values; prescribing pharmaceuticals that are covered by health care insurance could be seen as contributing to equity, and is thus an instrumental value.
In general, we believe that further theorising about differences between health care systems in what people find important, might start from the positive relation between importance and experience, from the idea that people compare their experiences with those of others, and from the idea that general and instrumental values might be related trough the institutions of the health care system. Hofstede [24] has identified a number of general dimensions of culture that might also be related to instrumental health care values through different types of welfare states [30].
The analysis presented in this article has its shortcomings. The number of countries is small. The analysis of differences between countries is based on only twelve countries, even though large numbers of respondents were involved. The number respondents increase our confidence in the averages per country, but this does not solve the problem of small numbers at the country level. Differences between user groups could not be taken into account because of the small number of groups for all countries except the Netherlands, although one might expect these differences to exist [29].
The multilevel model now contained two levels, respondents and countries. However, as table 1 indicated, the selection of respondents was through health care institutions. Hence, a level between respondents and countries should ideally have been specified. However, the data did not allow this. Differences between user groups are relatively large. However, the estimated intraclass correlations of table 2 seem to refer more to variation between countries than between user groups. Future research with larger numbers of user groups would be helpful to be able to take case mix differences into account.
We used existing data, collected in different studies with different aims and methods. The response rates differed across studies. The user groups in the surveys refer to are very different, ranging from GP-patients in Belarus to disabled people in the UK and IBD patients in Israel. Apart from the apparent differences between user groups, there are also differences between countries in the position and tasks of GPs. The situation in Ukraine and Belarus was transitional, even ten years after the fall of the Iron Curtain [31]. This may have reduced comparability and create variations in respondents and services to be evaluated.
The QUOTE-questionnaires provide a general framework, and researchers have adapted them to their own aims. Against the advantage of flexible adaptation to different research aims and populations stands the disadvantage of reduced (international) comparability. For (international) comparisons more standardisation is very important. Apart from that, the ten QUOTE-aspects we used in our analyses cover different dimensions of the Quality of care concept and can be understood as being comprehensive enough for this explorative type of research.
In conclusion, we believe it is important to continue to do research into health care related values because of the increasing importance of user views, both in the health policies of European countries separately and in the international debate about the performance of health care systems. There is not much ground for strong cultural relativism, saying that what is important in the eyes of health care users is so different that it is not possible to develop performance measures that can be used in a wide range of countries.
Conclusion
There are differences between countries in the importance people attach to aspects of health care. Most of this variation is related to individual differences, but there is also significant variation between countries. The ranking of aspects shows similarities between countries. In nearly all countries, people ranked the item that their GP should take them seriously as most important, while an item about waiting time was always ranked lowest. It is difficult to explain the variation between countries. Further theorising should take into account that importance and performance ratings are positively related, that people compare their experiences with those of others, and that general and instrumental values might be related through the institutions of the health care system.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
PG conceived the study and wrote the first drafts of the article; JK performed the statistical analysis; HS provided data and wrote the final draft of the article; WB provided data; IvdE provided data.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The research presented in this article was funded by the research programme Quality of Care of the Netherlands Organisation for Health Research and Development (ZonMw).
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| 15723701 | PMC554106 | CC BY | 2021-01-04 16:31:52 | no | BMC Health Serv Res. 2005 Feb 21; 5:16 | utf-8 | BMC Health Serv Res | 2,005 | 10.1186/1472-6963-5-16 | oa_comm |
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BMC Dev BiolBMC Developmental Biology1471-213XBioMed Central London 1471-213X-5-51572534810.1186/1471-213X-5-5Research ArticlePossible role of eclosion rhythm in mediating the effects of light-dark environments on pre-adult development in Drosophila melanogaster Paranjpe Dhanashree A [email protected] D [email protected] MK [email protected] Amitabh [email protected] Vijay Kumar [email protected] Chronobiology Laboratory, Evolutionary and Organismal Biology Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, PO. Box. 6436, Jakkur, Bangalore-560064, Karnataka, India2 Evolutionary Biology Laboratory, Evolutionary and Organismal Biology Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, PO. Box. 6436, Jakkur, Bangalore-560064, Karnataka, India2005 22 2 2005 5 5 5 14 10 2004 22 2 2005 Copyright © 2005 Paranjpe et al; licensee BioMed Central Ltd.2005Paranjpe et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
In insects, circadian clocks have been implicated in affecting life history traits such as pre-adult development time and adult lifespan. Studies on the period (per) mutants of Drosophila melanogaster, and laboratory-selected lines of Bactrocera cucurbitae suggested a close link between circadian clocks and development time. There is a possibility of clock genes having pleiotropic effects on clock period and pre-adult development time. In order to avoid such pleiotropic effects we have used wild type flies of same genotype under environments of different periodicities, which phenotypically either speeded up or slowed down the eclosion clock of D. melanogaster.
Results
We assayed pre-adult development time and pre-adult survivorship of four laboratory populations of D. melanogaster, under five different light regimes, continuous light (LL), continuous darkness (DD), and light-dark (LD) cycles of 10:10 h (T20), 12:12 h (T24), and 14:14 h (T28). Although the development time was significantly different in most light regimes, except for females under T24 &T28, pre-adult survivorship remained largely unaffected. The development time was shortest under LL, followed by T20, DD, T24 and T28 regimes, in that order. Interestingly the development time showed a positive correlation with the period of eclosion rhythm, i.e., faster oscillations were associated with faster development, and slower oscillations with slower development.
Conclusion
Based on these results we conclude that periodicity of imposed LD cycles, and/or of eclosion rhythm plays a key role in regulating the duration of pre-adult development in D. melanogaster in a manner that does not involve direct pleiotropic effects of clock genes on both clock period and development time.
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Background
Circadian (Latin: circa = about, dies = day) clocks regulate a number of physiological and metabolic processes in organisms as diverse as unicellular bacteria, fungi, fruit flies and humans [1,2]. The core molecular mechanisms underlying these rhythms are conserved across a range of taxa, and involve the expression of several clock genes, interlocked in transcriptional – translational auto-regulatory feedback loops [2].
Circadian clocks have been implicated in affecting life history traits such as pre-adult development time, and adult lifespan [3-5]. It is generally believed that faster clocks speed up pre-adult development, and shorten adult lifespan, while slower clocks slow down development and lengthen adult lifespan. The role of circadian clocks in the development of Drosophila melanogaster has been quite elegantly addressed in an exhaustive study on the period (per) mutants, which display circadian rhythms with widely different periods [3]. The pre-adult development time of the different per mutants under continuous dim light (LL) and continuous darkness (DD) was positively correlated with the free-running period (τ) of their circadian clocks, i.e. perS mutants (τ = 19 h) developed faster than wild type flies (τ = 24 h), which in turn developed faster than perL mutants (τ = 28 h) [3]. The correlation between development time and clock period remained unchanged even under very bright continuous light (VLL), wherein flies are rendered arrhythmic [6,7]. Moreover, the development time and clock period showed positive correlation even under light-dark (LD) cycles of 12:12 h, and LD 12:12 h superimposed with temperature cycles (LD 12:12 T), wherein flies of different genotypes were entrained to a common 24 h periodicity. A positive correlation between development time and clock period under LD cycles is difficult to explain, unless one considers pleiotropic effects of clock genes that are not mediated by a direct causal relationship between clock period and development time. In a separate study on the melon fly, Bactrocera cucurbitae, which involved selection for faster and slower pre-adult development, the selection regimes yielded faster developing lines with faster circadian clocks (τ ~ 22.6 h), and slower developing lines with slower circadian clocks (τ ~ 30.9 h)[8,9]. A positive correlation between development time and clock period in the above studies suggests pleiotropic effects of clock genes on period of circadian rhythms and pre-adult development time. Therefore, it appears that the genotype, which enables the fly clocks to run faster or slower also aids faster and slower pre-adult development thus leaving the primary question of the role of circadian clocks in regulating pre-adult development in D. melanogaster unresolved. In order to investigate the role of circadian clocks in development time without confounding pleiotropic effects of clock genes one would need to assay development time of flies from similar genetic background under short and long day lengths, wherein their clocks would entrain by speeding up or slowing down oscillations.
In the present study we assayed pre-adult development time and pre-adult survivorship of four large out-bred laboratory populations of D. melanogaster (LL1.. 4) under five different light regimes: LL, DD, LD cycles of 10:10 h (T20), 12:12 h (T24), and 14:14 h (T28). The average periodicity of eclosion rhythm in T20, DD, T24, and T28 was, 20 h, 23.5 h, 24 h and 28 h, respectively, while eclosion was arrhythmic under LL [10]. The results suggest that periodicity of LD cycles and/or of eclosion rhythm play an important role in determining the duration of pre-adult development in D. melanogaster.
Results
ANOVA on pre-adult development time data revealed a significant main effect of light regime (F4,12 = 2411.97, p < 0.001) (Figures 1, 2), while pre-adult survivorship remained largely unaffected (F4,12 = 1.06, p = 0.42). The development time of males and females was shortest under LL, followed by T20, DD, and T24 &T28, in that order (Figures 1, 2; see table 1). Multiple comparisons using 95% Confidence Interval (CI) around mean showed that development time of flies under different light regimes was significantly different from each other, except for T24 and T28 regimes. ANOVA also revealed a significant main effect of sex (F1,3 = 607.85, p < 0.001), and light regime × sex interaction (F4,12 = 6.56, p < 0.05). Multiple comparisons using 95% CI showed that females developed faster than males under all five light regimes and the difference in male-female development time was greatest under T28 regime, followed by DD, T24, LL and T20 regimes. In addition, eclosion appeared to be bimodal under T28, and the pattern of bimodality was more prominent in females than in males (Figure 1). In order to compare the waveform of eclosion under five light regimes, Kruskal-Wallis test was performed, and the test revealed a significant main effect of light regime on the eclosion profile of males [H (4, N = 2566) = 1923.295, p < 0.001] and females [H (4, N = 2709) = 2061.568, p < 0.001]. The Kruskal-Wallis test thus confirmed the results obtained in ANOVA. Although eclosion waveforms under no two light regimes were similar, the dissimilarity was even more striking under T24 and T28. The test revealed that mean development time of males was shortest under LL, followed by T20, DD, T24, and T28, in that order. While the mean development time of females followed a similar trend, it did not differ significantly between T24 and T28.
Figure 1 Eclosion profile of fruit flies D. melanogaster in five different light regimes. Pre-adult development time in hours is plotted along x-axis, and the percentage of eclosing flies is plotted along y-axis. Eclosion profile of males in five different light regimes is shown in the left panels, while that of females is illustrated in the right panels. Graphs were plotted using data pooled over four populations.
Figure 2 Mean pre-adult development time of D. melanogaster populations in five different light regimes. Light regime is plotted along x-axis, and pre-adult developmental time in hours along y-axis. Mean development time of males is shown in the left panels, while those for females in the right panels. The error bars represent 95% CI around the mean. ANOVA revealed significant effect of light regime (F4,12 = 2411.97, p < 0.001), sex (F1,3 = 607.85, p < 0.001), and light regime × sex interaction (F4,12 = 6.56, p < 0.05). The mean pre-adult development time values are provided in table1.
Table 1 Mean pre-adult development time of four laboratory populations of D. melanogaster assayed under five different light regimes.
Light regime Sex Pre-adult development time (hours)
LL M 224.37
LL F 221.91
DD M 258.32
DD F 253.63
T20 M 248.63
T20 F 246.72
T24 M 263.96
T24 F 261.07
T28 M 265.32
T28 F 258.83
The periodicity of eclosion rhythm under DD, T20, T24 and T28, as reported in one of our previous studies on the same populations were 23.5 h, 20 h, 24 h and 28 h, respectively, while eclosion was arrhythmic under LL [10]. In addition, the peak of eclosion rhythm under different light regimes [10] matched closely the peak eclosion in the development time assay (Figure 1). The mean development time of males and females under four light regimes (DD, T20, T24 and T28) showed a significant positive correlation with the mean period of eclosion rhythm under the corresponding environments (r = +0.83 & +0.71, p < 0.001; Figure 3).
Figure 3 Mean development time and the period of the eclosion rhythm show a significant positive correlation. The mean development time in hours under T20, DD, T24 and T28 is plotted along y-axis, and the period of eclosion rhythm under the corresponding light regime is plotted along x-axis. The correlation between mean development time and period of eclosion rhythm for males is shown in left panel while that for females is illustrated in the right panel.
Discussion
In several insect species adult eclosion is gated in a manner that it occurs only during a narrow window of time, generally around dawn when environmental humidity is the highest [11]. In D. melanogaster the clocks that gate adult eclosion are located in the prothoracic gland and ventral lateral neurons [12], and it is believed that these clocks also play a key role in the regulation of pre-adult development [13]. Development time under environments wherein eclosion is arrhythmic, such as bright LL, is solely determined by the developmental state of a fly, and under such a situation pre-adult development time would reflect the minimum time required by flies to complete pre-adult development. On the other hand, environments such as DD and LD cycles, wherein eclosion is rhythmic, the interaction between developmental state and eclosion clock would determine the duration of pre-adult development, and the developmental time would then be expected to be greater than those under LL. In a previous study on four Drosophila populations maintained under LL (JB1..4), the ancestral populations of the flies used in the present study, we had reported shortest development time under LL regime, followed by LD 12:12 h, and DD [14]. In the present study too development time was shortest under LL, followed by T20, DD, and T24 and T28, in that order. As opposed to LL, eclosion under DD is gated in a circadian manner, and as a result flies took longer to develop compared to that in LL. We believe that the slight discrepancy in the results of our two studies could be due to the fact that different set of flies (about 100 generations apart) with different clock periods were used in the two experiments. The periodicity of JB populations under DD was greater than 24 h, whereas those of LL populations was lesser than 24 h. Thus, consistent with our proposal, development time of JB populations was greater under DD compared to development time in LD 12:12 h regime, whereas those of LL populations was shorter in DD compared to those under LD 12:12 h.
The mean development time under four different light regimes (T20, DD, T24, and T28) showed a significant positive correlation with the mean period of eclosion rhythm under the corresponding light regimes; i.e. shorter period of eclosion rhythm under T20 was associated with faster pre-adult development, followed closely by DD, whereas flies took longest to develop under the light regimes wherein their eclosion periodicities were 24 h and 28 h, suggesting that development in D. melanogaster is regulated by eclosion rhythm. The peak of eclosion under three periodic light regimes closely matched phase relationships of eclosion rhythm relative to LD cycles, suggesting that phases of eclosion rhythm also play a key role in the regulation of development time [10]. Finally, flies took different number of environmental cycles to develop under three periodic light regimes. The average number of cycles taken to develop under LD 10:10 h, 12:12 h, and 14: 14 h were approximately 12.5, 11 and 9.5, respectively, which suggests that pre-adult development of D. melanogaster is not entirely regulated by the periodicity of the environment and/or the periodicity of eclosion rhythm. These results are in agreement with the findings of previous studies, where the duration of pre-adult development was positively correlated with clock period [3,9,15]. A subtle but important difference between the outcome of present study and that of most of previous studies is that, the correlation between developmental time and eclosion period in our study is clearly mediated via the periodicity of LD cycles and/or of eclosion rhythm, whereas in previous studies the correlation was independent of external environment and the eclosion rhythm, instead was dependent upon the genotype of the flies [3].
Although development time in the present study was greater under T28 compared to T24, the differences did not reach levels of statistical significance in ANOVA, possibly due to complex interactions between the developmental states, phase of the LD cycles, and eclosion profiles under T24 and T28 regimes. A careful analysis of development time of flies under these two regimes revealed that eclosion under T24 and T28 was bimodal, and bimodality was more prominent under T28 than in T24. To complicate the matter further the eclosion patterns of females had a greater propensity towards bimodality compared to males. The Kruskal-Wallis test revealed that eclosion profiles of flies under T24 and T28 were indeed significantly different, and mean development time of males under T28 was greater than in T24 regime, whereas those of females did not differ between the two light regimes. According to the gating hypothesis ([13]) bimodality could arise when flies are exposed to LD cycles of non-24 periodicity, perhaps due to a mismatch between developmental time and eclosion gate. Indeed, the same phenomenon also occurs under T20, where a small, statistically insignificant eclosion peak appears between 260 h and 270 h in both males and females (fig 1).
Conclusion
Pre-adult development time and circadian rhythm are both multigenic traits, and genes involved in regulating development time as well as circadian rhythms are known to have pleiotropic effects [3]. Our study pre-designed to bypass such pleiotropic effects demonstrates a possible role of the periodicity of light-dark environment and/or of eclosion rhythm in determining the duration of pre-adult development. Taking into account the results of our experiments and those of the per mutant experiments, it appears that the duration of pre-adult development in D. melanogaster is determined by several factors such as the circadian rhythm, developmental state, and the interaction between the phase of eclosion rhythm, and the phase of the LD cycles.
Methods
Fly stock maintenance
The four populations of D. melanogaster used in this study were maintained under constant light (~ 100 lux), at constant temperature of 25°C (± 1°C), and constant humidity of 70 ± 5%, on a 21 day discrete generation cycle (henceforth will be referred as LL1, 2, 3, 4 populations). These populations were maintained at moderate larval densities of ~ 60–80 larvae per 8 dram vial (9.0 mm height × 2.4 mm diameter) containing banana-jaggery food medium (henceforth banana food). The ancestry and maintenance of these populations has been described in detail in an earlier paper [16]. In brief, at every generation, adults of each population are allowed to lay eggs for about 18 hours on petri plates of fresh banana food placed in a plexiglass cage (25 × 20 × 15 cm3). From these petri plates, 60–80 eggs are collected into each of 40 vials in which larvae then develop into adults. Adults eclosing from these vials are transferred to plexiglass cages on 12th day after egg lay. On the 18th day after egg lay, adult flies are supplied with banana food supplemented with live yeast paste for two days, after which eggs are collected to initiate the next generation and the adults discarded. The breeding population typically consists of about 1500 flies.
Pre-adult development time and survivorship assay
From the running culture of each population (LL1..4), eggs laid on banana medium over a 2 h window were collected for the assay. Exactly 30 eggs were dispensed into 8 dram vials containing ~ 6 ml banana food and 50 such vials were set up from each population. Ten vials from each population were introduced into continuous light (LL), continuous dark (DD), light-dark (LD) cycles of 10:10 h (T20), 12:12 h (T24), and 14:14 h (T28). Thus a total of 200 vials were set up for the assay (10 vials × 4 populations × 5 light regimes). These vials were introduced into five different light regimes at 20:00 h, when lights went off simultaneously in all LD regimes. Fluorescent white light of intensity ~ 100 lux was used during light phase, whereas in dark phase red light of λ >650 nm was used. Temperature and relative humidity in the five light environments monitored continuously using a Quartz Precision Thermo-Hygrograph, Isuzu Seisakusho Co, LTD, were found to be comparable. The vials were monitored for eclosion of adult flies after the pupae became dark. Eclosing adults were collected every 2 h, sexed, and counted until all the flies eclosed.
Statistical analyses
Pre-adult development time in hours was calculated as the duration between the midpoint of the egg collection window and the midpoint of the 2-h period during which eclosion occurred. The mean pre-adult development time for a particular sex in a particular light regime was used as data in a mixed model analysis of variance (ANOVA), in which replicate populations were treated as random blocks, and light regime and sex were treated as fixed factors.
In order to detect differences in the eclosion profiles of the flies under different light regime, development time data of individual flies from all four replicate populations were pooled and compared using Kruskal-Wallis test.
Pre-adult survivorship was calculated as the fraction of eggs that successfully developed into adults in each vial. The mean pre-adult survivorship values of each population in each light regime were used as data in a mixed model ANOVA, with replicate populations as random blocks, and light regime as a fixed factor.
Authors' contributions
DAP and AD were involved in collecting eggs, collecting and counting flies for pre-adult development time, estimating pre-adult survivorship, data entry and analyses. VKS conceived of the study and participated in its design and coordination. MKC and AJ gave valuable comments and suggestions throughout the study. All authors read and approved the manuscript.
Acknowledgements
We thank Shailesh Kumar, C. R. Akarsh, Dhanya Kumar, Shahnaz R. Lone, N. Rajanna and M. Manjesh for assistance during assays. We thank the Department of Science and Technology, Government of India, for funding this work. We thank three anonymous reviewers for carefully reading the manuscript and for suggesting improvements.
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| 15725348 | PMC554107 | CC BY | 2021-01-04 16:40:17 | no | BMC Dev Biol. 2005 Feb 22; 5:5 | utf-8 | BMC Dev Biol | 2,005 | 10.1186/1471-213X-5-5 | oa_comm |
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BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-5-201572071510.1186/1471-2407-5-20Research ArticleEvaluation of dendritic cells loaded with apoptotic cancer cells or expressing tumour mRNA as potential cancer vaccines against leukemia Jarnjak-Jankovic Silvija [email protected] Rolf D [email protected]æbøe-Larssen Stein [email protected] Finn [email protected] Gustav [email protected] Department of Pediatric Research, The National Hospital, Oslo, Norway2 Section for Immunotherapy, The Norwegian Radium Hospital, University of Oslo, Norway3 Department of Pediatrics, The National Hospital, Oslo, Norway2005 18 2 2005 5 20 20 21 10 2004 18 2 2005 Copyright © 2005 Jarnjak-Jankovic et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Leukemia is a clonal disorder characterized by uncontrolled proliferation of haematopoietic cells, and represents the most common form of cancer in children. Advances in therapy for childhood leukemia have relied increasingly on the use of high-dose chemotherapy often combined with stem-cell transplantation. Despite a high success rate and intensification of therapy, children still suffer from relapse and progressive disease resistant to further therapy. Thus, novel forms of therapy are required.
Methods
This study focuses on dendritic cell (DC) vaccination of childhood leukemia and evaluates the in vitro efficacy of different strategies for antigen loading of professional antigen-presenting cells. We have compared DCs either loaded with apoptotic leukemia cells or transfected with mRNA from the same leukemia cell line, Jurkat E6, for their capacity to induce specific CD4+ and CD8+ T-cell responses. Monocyte-derived DCs from healthy donors were loaded with tumor antigen, matured and co-cultured with autologous T cells. After one week, T-cell responses against antigen-loaded DCs were measured by enzyme-linked immunosorbent spot (ELISPOT) assay.
Results
DCs loaded with apoptotic Jurkat E6 cells or transfected with Jurkat E6-cell mRNA were both able to elicit specific T-cell responses in vitro. IFNγ-secreting T cells were observed in both the CD4+ and CD8+ subsets.
Conclusion
The results indicate that loading of DCs with apoptotic leukemia cells or transfection with tumour mRNA represent promising strategies for development of cancer vaccines for treatment of childhood leukemia.
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Background
Leukemia represents the most common form of cancer in children. There are two main types of childhood leukemia, acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The success rate in treatment of childhood leukemia has improved continuously over the past decades [1], and today disease-free survival is 70%–80% for ALL and 40%–60% for AML [2-4]. In the Nordic countries the overall event-free survival in ALL has risen from 57% to75% [2]. However, in children with high-risk ALL, the progress has only been modest. The relapse rate has decreased in parallel with the improving results, but the prognosis after relapse has not improved. Only 25%–30% of children who relapse will reach and remain in a second remission. Children with AML have a worse prognosis than those with ALL. Event-free survival for AML is below 55%, whereas the cure rate for children with ALL is near 80%. The complete remission rate differs also, with 5%–10% induction failures due to refractory disease and toxicity in AML, compared to 1%–2% in ALL [2].
Immunotherapy based on vaccination with dendritic cells (DCs) has emerged as an attractive new form of therapy for cancer in general, and DC-based vaccines have already shown promise in follicular non-Hodgkin's lymphoma, and in other hematological malignancies [5-7]. DCs are antigen-presenting cells (APCs) specialized to induce T-cell responses against cells exposing foreign peptides, including tumour-related antigens, in context of MHC molecules [8,9]. DCs reside in tissues in an immature form, where they capture antigens from the environment. After antigen capture, and in response to inflammatory stimuli, DCs mature and migrate to lymph nodes to initiate immunity [9]. Maturation of DCs is associated with up regulation of the co-stimulatory molecules CD80 and CD86, increased expression of HLA molecules, enhancement of their APC function, and expression of CCR7 chemokine receptors that promote migration to the T-cell area in lymph nodes [10]. In several animal studies, it has been shown that immunization with cancer-antigen loaded DCs efficiently primes both CD4+ and CD8+ T-cells, resulting in protective immunity against tumours [11-16].
Vaccination with tumor antigen-loaded DCs has been shown to induce both Th and CTL responses, and tumor regression in some patients [16,17]. An important issue in optimizing DC vaccines is the choice of tumour antigen for loading of DCs. Several clinical trials in patients with melanoma have demonstrated that vaccination against a single antigen can induce tumour specific CTLs [18]. However, for many tumours no specific cancer antigens are known. For such patients, autologous tumor cells or tumor cell lines containing a repertoire of antigens overlapping with the repertoire in the patient's tumor, represents an alternative source of antigens. Effective cross-priming with antigens from tumour cells has been demonstrated with apoptotic cancer cells [19-21]. Transfer of whole tumor mRNA into DCs represents an alternative way of loading DCs. The transfected mRNA can be expressed for a relatively long period of time [22-24] and give rise to specific T-cell responses in vitro and following vaccination of patients [25]. So far, no studies on the relative efficacy of these two antigen loading methods have been performed. The aim of the present study was to compare DCs either loaded with apoptotic Jurkat E6 cells or transfected with mRNA isolated from Jurkat E6 cells, for their ability to generate T-cell responses against antigens derived from the human T-cell line. The Jurkat leukaemic T-cell line is a reference cell line [26], and was chosen as a source of antigen in the model experiments described here. We demonstrate that both strategies can be successfully employed to induce T helper and CTL responses against antigens derived from allogeneic leukemic T-cells.
Methods
Cytokines and chemicals
GM-CSF was purchased from Novartis (Basel, Switzerland), IL-4, TNFα, IL1β and IL-6 from CellGenix (Freiburg, Germany), and Prostaglandin E2 (PgE2), IL-7, IL-2 and IL-12 from R&D Systems (Minneapolis, USA). Staurosporin was obtained from (Sigma Aldrich, Saint Louise, Missouri).
Preparation of DCs and T cells
PBMC from healthy donors (obtained from Buskerud Hospital, Drammen, Norway) were obtained by density gradient centrifugation (Lymphoprep, Nycomed, Norway). Monocyte-derived DCs were generated under serum-free conditions from the adherent fraction of PBMCs cultured in six-well plates at a density of 4 × 106 cells/ml for 1.5 h at 37°C in 3 ml CellGro DC medium (CellGenix, Freiburg, Germany). Non-adherent cells were collected and frozen for later use as responder cells. Adherent cells were cultured in 3 ml CellGro DC medium, supplemented with 800 U/ml GM-CSF and 10 ng/ml IL-4 every second day, until day 5 when maturation of DCs was induced by addition of maturation cocktail (10 ng/ml TNF-, 10 ng/ml IL-11000 U/ml IL-6 and 1 μg/ml PgE2) for 24 h. Characterization of DC phenotype was done by staining 0,5 × 106 cells with fluorochrome-labelled antibodies against the Lin1 panel (CD3, CD14, CD19, CD16, CD20, CD56), HLA-DR, CD1a, CD80, CD83, and CD86 (Becton Dickinson, San Jose, CA), and analyzing by FACSCalibur flow cytometry (Becton Dickinson). The mAb isotypes used were IgG1 FITC, IgG2a PE, IgG1 APC (Becton Dickinson, San Jose, CA).
Assessments of apoptosis and phagocytosis of apoptotic cells
Jurkat E6 cells obtained from American Type Culture Collection (ATCC) were exposed to 1 μM staurosporin for 3 h to induce apoptosis. Apoptotic cell death was assessed using Annexin V-FLUOS as described by the manufacturer (Boehringer Manheim, Manheim, Germany). For assessments of phagocytosis, Jurkat E6 cells were stained with the green fluorescent dye PKH-67 (Sigma Aldrich) as described in the kit manual, exposed to 1 μM staurosporin for 3 h and incubated with immature DCs at a ratio of 3:1. After 6 h, immature DCs were labelled with a red fluorescent antibody (mAb CD1a-PE). Phagocytosis of apoptotic cells was measured quantitatively by flow cytometry. Similarly, phagocytosis was visualized by confocal laser microscopy (Leica TCS SP, equipped with HeNe and Ar lasers) using apoptotic Jurkat E6 cells pre-stained with PKH-26 red fluorescent dye (Sigma Aldrich) and DCs stained with the green fluorescent dye PKH-67.
Preparation of Jurkat E6-cell mRNA and transfection of DCs
Jurkat E6 cells were used as a source of tumor material. Total RNA was isolated from 20–25 × 106 cells using Trizol Reagent as described by the manufacturer (Invitrogen, Basel, Switzerland). Poly (A)+ mRNA was isolated from total RNA using the GenoPrep Direct mRNA kit (GenoVision, Oslo, Norway). Purified mRNA was used fresh or stored at -80°C until use. Transfection of DCs with mRNA was performed as described previously [22], with minor modifications. Briefly, immature DCs were washed once, resuspended in RPMI-1640 (BIO-Whittaker, Walkersville, MD) and placed on ice. 400 μl (approx. 2 × 106 cells) were mixed with mRNA, transferred to a 4-mm-gap cuvette and pulsed with a BTX ECM-830 square-wave electroporator (Genetronics Inc., San Diego, CA) using instrument settings 500 V and 1 ms. Transfected cells were incubated on ice for 30 s followed by addition of 2.0 ml cold CellGro DC medium supplemented with 10 ng/ml IL-4, 800 U/ml GM-CSF and maturation cocktail (see above), and transferred to standard culturing conditions. Transfection with EGFP-pCIpA102 mRNA (10 μg/400 μl) encoding the green fluorescence protein [22] was used to verify transfection efficiency.
Isolation of T-cell subsets CD4 and CD8
The Negative Isolation Kit (Dynal, Biotech) was used for isolation of CD4 and CD8 T cells according to the manufacturer's protocol. Isolation was performed on day 7 after in vitro priming, before setting up the ELISPOT assay.
Induction of primary T-cell responses
Mature DCs (0.3 × 106) expressing Jurkat E6-cell mRNA or loaded with apoptotic Jurkat E6 cells, were co-cultured with 3 × 106 autologous non-adherent PBMC for 7 days in 1.0 ml CellGro DC medium without serum, before setting up the ELISPOT assay. The cultures were tested for INF- production in an ELISPOT assay [27] following restimulation for 24 h with thawed antigen-loaded DC using 0.5 × 105, 1.0 × 105, 2.0 × 105 and 4.0 × 105 responding cells and 0.5 × 104 DCs per well. Mock transfected DCs were used as control. The assay was done in duplicate. Spots were counted manually, and the frequency of reactive T cells was calculated according to the formula: (spots with transfected DC - spots with non transfected DC)/number of T cells added.
Results
Generation of immature DCs and phagocytosis of apoptotic Jurkat E6 cells
Based on previous observations that immature DCs efficiently capture antigens from the environment, we first investigated the ability of immature DCs to similarly phagocytose apoptotic Jurkat E6 cells. Immature DCs were prepared from PBMC in the presence of IL-4 and GM-CSF. To confirm generation of immature DCs, cells were examined by flow cytometry for expression of lineage and differentiation specific markers. The Lin 1 cocktail contains antibodies to CD3, CD14, CD16, CD19, CD20, and CD56 and differentiates DCs from other leukocytes by their lack of staining with Lin1. In contrast, CD1a and HLA-DR are expressed on immature DCs, and maturation is revealed by increased expression of CD83, and the costimulatory molecules CD80 and CD86. As shown in Fig. 1a the generated cells displayed the characteristic phenotype of immature DCs with high expression of CD1a and HLA-DR, and low or no expression of CD80, CD83 and CD86.
For induction of apoptosis, Jurkat E6 cells were treated with staurosporin. In an independent series of experiments, optimal early apoptosis was observed after 3 h (data not shown) and early apoptotic cells were accordingly used in all experiments. To verify apoptosis after 3 h exposure to staurosporin, cells were stained with annexin V-FLUOS and examined by flow cytometry. The assessments showed that more than 90% of the cells were annexin-V positive (Fig. 2).
To study uptake of apoptotic leukemia cells by immature DCs, Jurkat E6 cells were stained with the red fluorescent dye PKH-26 before induction of apoptosis. Immature DCs were stained with the green fluorescent dye PKH-67. Apoptotic Jurkat E6 cells were then co-incubated with immature DCs for various periods of time to determine the optimal conditions for internalization of apoptotic Jurkat E6 cells. We observed that immature DCs ingested apoptotic Jurkat E6 cells within 6 h of co-incubation. Phagocytosed apoptotic Jurkat E6 cells (red stained) were observed inside or in the process of being phagocytozed by immature DCs (green stained) by confocal microscopy (Fig. 3b).
Flow cytometry was used to further determine the efficiency of DC loading with apoptotic leukemic cells. In these experiments, apoptotic Jurkat E6 cells had been pre-stained with the green fluorescent dye PKH-67 and DCs were identified by staining with PE-conjugated anti-CD1a. Highly efficient uptake of apoptotic Jurkat E6 cells was confirmed, since virtually all CD1a positive cells showed green PKH-67 staining (Fig. 3a).
Following antigen loading, DCs were matured in the presence of pro-inflammatory cytokines for 24 h. Assessments by flow cytometry confirmed that this treatment led to up-regulation of CD83, and the co-stimulatory molecules CD80 and CD86, in compliance with a mature DC phenotype (Fig. 1b).
Transfection of immature DC with mRNA from Jurkat E6 cells
Transfection of cells with tumor-derived mRNA is an alternative method for loading of DCs with tumor antigens. mRNA from the Jurkat E6 cells was isolated and electroporated into DCs according to previously optimized methods [22]. Following this protocol, optimal conditions for electroporation were 500 volt and 1 ms when using a 4-mm-gap cuvette. These conditions produced both efficient transfections (142 × background fluorescence; Fig. 4) and a survival rate indistinguishable from untransfected cells (data not shown). The transfected DCs were matured as described above and induction of the characteristic phenotype was confirmed by flow cytometry (Fig. 1c).
Analysis of T-cell responses
ELISPOT assay of INF-γ producing cells is the method of choice for assessments of T-cell responses against cancer vaccines representing a heterogeneous mixture of antigens. This assay measures in a quantitative way the number of reactive T cells in pre and post-vaccination samples and thus directly relates the effect of vaccination to in-vivo expansion of reactive T cells. Autologous T cells were stimulated with tumour-mRNA transfected DCs and with DCs loaded with apoptotic Jurkat E6 cells. Fig. 5 shows the results from experiments with cells derived from three different donors. In all experiments a specific T-cell response against antigen-loaded DCs as compared to control DCs (mock transfected/non-loaded) could be demonstrated. No clear-cut difference between the two modes of antigen-loading was observed. In all experiments with un-fractionated T cells, we also observed T-cell reactivity against control DCs. This background completely obscured the specific response if in vitro priming was performed in the presence of exogenously added recombinant human IL-2 (results not shown). Antigen loading of DC by mRNA transfection and phagocytosis will introduce the Jurkat antigens into two different antigen processing pathways, cytosolic expression and processing for mRNA-encoded antigens and endosomal processing for phagocytosed apoptotic cells. As a result, one might expect that mRNA loading would preferentially result in activation of specific CD8+ CTLs, while loading with apoptotic cells would mainly result in activation of specific CD4+ Th1 cells. To investigate if this was the case, we separated the responding T-cell population into a CD4+ containing fraction and a CD8+ containing fraction using negative selection with Dynabeads coated with CD8 and CD4 antibodies respectively. The results shown in Fig. 5 demonstrate that a specific Th1 response was obtained in all donors and that both methods of loading resulted in a Th1 response. The frequency of specific Th1 cells varied between donors, with a trend indicating that mRNA loading in general is more efficient than apoptotic cells in priming of a Th1 response. The results depicted in Fig. 5 clearly show that relatively high frequencies of specific CTLs can be generated in all donors and that both methods of antigen-loading result in CTL priming. In donor 2 and 3, mRNA loading was clearly superior to apoptotic cells, indicating that expression of mRNA-encoded antigens more efficiently entered the proteasomal pathway for processing of HLA class I restricted antigens.
Discussion
Immunotherapy for childhood leukemia has the potential to contribute to long-term control or cure of the disease. Until now immunotherapeutic approaches for leukemia have been limited to trials of cytokine therapy [3]. Further development of biologically based treatments may prove to be effective in therapy of patients suffering from this disease. Several forms of DC-mediated immunotherapy are currently being investigated using a wide variety of vaccination protocols summarized in [28]. Two very important issues are the choice of antigen and the method of antigen loading. In the present study we have chosen to use the complex antigen mixture represented by whole tumor cells. Reports comparing the ability of apoptotic and necrotic cells to induce DC maturation [29] found that incubation of DCs with necrotic, but not apoptotic, tumor cell lines induce maturation. However, other reports concluded that incubation with apoptotic cells is sufficient to induce DC maturation [30-34]. In our study we have used apoptotic cells, and the requirement for DC maturation signals was provided by a standardized maturation cocktail. We accordingly analyzed human monocyte-derived DCs for their ability to: (a) take up apoptotic leukemia cells and express transfected mRNA, (b) express a mature phenotype following tumour-antigen capture and culture in maturation cocktail and (c) prime un-fractionated T cells as well as the CD4+ and CD8+ T-cell subsets. Due to the complexity of the antigens represented by the allogeneic tumor cells, the aim of these model experiments was not to use this allogeneic system to prove that we could elicit tumour specific T-cell responses in this way, but to provide data to demonstrate efficient antigen transfer and compare the relative efficacy of DCs loaded by the two different procedures, in eliciting complex T-cell responses. Our results demonstrate that immature DCs can efficiently take up apoptotic Jurkat E6 cells, and that phagocytosis was mainly confined to the CD1a+ subset of immature DC. Furthermore, support for expression of transfected mRNA derived from the allogeneic leukemia cell line is indirectly provided by its ability to prime T-cell responses specific for transfected cells. We also show that the two different methods of antigen-loading did not result in any apparent differences in the phenotype of the mature DCs. In terms of immune responses both methods of antigen loading produced DCs capable of inducing INF- secreting T cells. However, it appeared that DCs loaded with tumour-mRNA in general were most potent in inducing T-cell responses.
We observed that the frequencies of induced INF-γ producing T cells depended on the individual donor, the method of antigen-loading and the subset of T cells studied. Such variations are not surprising, since the model system used employs allogeneic cells and no effort was done to HLA match the blood donors with the Jurkat cell line in these experiments. Since the experimental system is based on the use of an allogeneic cell line, we expect multiple antigens, encoded by a broad array of polymorphisms, including other HLA alleles to be involved. We have therefore taken advantage of the genetic differences between responding cells and the leukaemia cell line by using the combined repertoires of membrane expressed and cross presented allo-antigens as a sensitive readout for immunological response in our experiments.
We expected that the two different procedures would provide some differences in loading of HLA molecules with tumour-derived antigens and subsequently in the responding T-cell subsets. According to the current dogma, processed peptides from phagocytosed apoptotic cells would be directed to HLA class I molecules by a process known as cross-presentation and to HLA class II molecules by the classical pathway. Cross-priming of CTL with antitumor activity has been demonstrated with DCs loaded with apoptotic tumour cells [19,21,35]. Specifically, Schnurr et al. demonstrated the antigens from apoptotic pancreatic carcinoma cell lines, either in form of whole cells or as released particles, were potent in inducing CTL-cell priming and activation by DC. In addition, Hoffman et al. reported stronger CTL responses with apoptotic tumour cells in a squamous cell carcinoma model. The enhanced CTL activation by antigens from apoptotic cells may be attributed to several mechanisms. After ingestion, most particulated antigens requiring phagocytosis are digested into peptides associating with HLA class-II molecules in the endocytic compartments and are presented to T-helper cells [36]. Conversely, scavenger receptor-mediated phagocytosis of apoptotic tumour cells allows antigens to gain access to HLA class-I compartments, resulting in cross-presentation of the antigens to CTL [20]. In addition, enhanced CTL responses to tumours might be mediated by heat shock proteins expressed by stress induced apoptotic tumour cells [37]. On the basis of this theoretical background and reported observations [21,31] we believe that antigen preparations from apoptotic tumour cells can also represent an alternative in DC-based tumour vaccines. On the other hand, tumour mRNA expressed in DCs would be processed for presentation by HLA class I molecules. In accordance with this, DCs loaded with apoptotic leukaemia cells stimulated both CD4+ and CD8 positive T cells and mRNA loaded DCs were superior in inducing CD8+ T-cell responses [38,39]. Interestingly, mRNA-loaded DCs were also able to induce specific CD4+ T-cell responses in all donors tested, suggesting some leakage of endogenously produced proteins into the lysosomal antigen-processing compartments. Similar results have recently been published by Su et al., who demonstrated a significant Th response against the defined tumour antigen hTERT following in vitro stimulation of un-fractionated T cells with hTERT mRNA transfected DC. The Th response could be further augmented by targeting the antigen to the lysosomal compartment using mRNA encoding a chimeric hTERT/lysosome-associated membrane protein (LAMP-1) protein.
The aim of the present study was to determine if loading of DCs with antigens derived from a tumour cell line, either as apoptotic cells or as mRNA would provide a basis for an efficient vaccine, using ELISPOT as a read-out of immune responses. It has been shown that DCs transfected with antigens encoded in tumor mRNA is capable of inducing potent T-cell responses against tumour-specific epitopes [40]. While protein antigens from tumour lysate are rapidly proteolysed following endocytosis by antigen-presenting cells, model experiments using mRNA encoding a fluorescent protein, EGFP, has shown that protein is still being produced 24 hrs after transfection of DCs, with peak expression after 48 hrs [22]. Thus, tumor mRNA transfected DCs may not only represent a potent strategy for CTL priming but may also represent a general method for DC-based vaccines. In vaccine preparations using DCs, mRNA is thus preferable to a protein lysate. Similarly, immunization with DCs loaded with mRNA from leukaemia cells could represent a feasible approach in treatment of these cancers. It is now widely accepted that not only CTLs but also CD4 (+) T-helper cells are critical to the generation and maintenance of potent antitumor responses in vivo. In this context, our observation and that of others demonstrating that DCs loaded with mRNA also are equally capable of inducing Th responses strongly argue in favour of this type of vaccination. Our preclinical results further support that vaccination of leukemia patients with tumour-mRNA transfected autologous DCs should be clinically evaluated as therapeutic strategy.
Conclusion
In our study we demonstrate that both DCs loaded with apoptotic Jurkat E6 cells or transfected with mRNA isolated from Jurkat E6 cells, can induce T-helper and CTL responses against antigens derived from allogeneic leukemic T-cells. We also show that the two different methods of antigen-loading did not result in any apparent differences in the phenotype of the mature DCs. In terms of immune responses both methods of antigen loading produced DCs capable of inducing INF- secreting T cells. However, it appeared that DCs loaded with tumour mRNA in general were most potent in inducing T-cell responses.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
SJJ performed preparation of DCs and T cells, assessments of apoptosis and phagocytosis of apoptotic cells, preparation of Jurkat E6-cell mRNA and transfection of DCs, isolation of T-cell subsets CD4 and CD8 and induction of primary T cell responses. SSL did the transfection of DCs with EGFP mRNA and fluorescence microscopy of DCs. RP, FW and GG planned the project.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Figures and Tables
Figure 1 Phenotype of generated DCs. Expression of antigens were determined by flow cytometry (a) Before loading with tumor antigens (b) After loading with apoptotic Jurkat E6-cells and following maturation with TNFα, IL1β, IL6 and PgE2 for 24 h and (c) After transfection with Jurkat E6-cell mRNA and maturation for 24 h. The histograms show staining with the appropriate mAb.
Figure 2 Staurosporin-induced apoptosis of Jurkat E6-cells. T cells were cultured for 3 h with 1 μM staurosporin and examined by Annexin V-FLUOS staining and flow cytometry.
Figure 3 Phagocytosis of apoptotic Jurkat E6 cells by immature DCs. At day 5 of culture, DCs were co-cultivated with apoptotic Jurkat E6 cells, previously labelled with green fluorescent dye. (a) Flow cytometry of immature DCs stained with anti CD1a-PE and apoptotic Jurkat E6 cells stained with PKH-67 green. (b) Confocal microscopy analysis shows the presence of intracellular apoptotic cells labelled with PKH-26 red within DCs labelled with PKH-67 green (yellow cell, arrow) or in the process of being phagocytized (yellow and red cell, arrow).
Figure 4 mRNA transfectation of DCs. (a) Flow cytometric analysis of DCs after transfection with EGFP/pCIpA102 mRNA (10 μg/400 μl) by square-wave electroporation and maturation for 24 h in medium with maturation cocktail. Control cells were mock electroporated without mRNA. (b) Fluorescence microscopy of DCs 24 h after transfection with EGFP/pCIpA102 mRNA.
Figure 5 ELISPOT analysis of T-cell activation. T cells were stimulated with DCs loaded with tumour antigen (mRNA or apoptotic cells) or control DCs as indicated. (a) Mean number of IFN-γ positive spots obtained with the indicated numbers of un-fractionated T cells, and CD4+ and CD8+ T-cell subsets from individual donors. (b) Frequency of reactive T cells. The data represent mean values of all donors calculated by the formula: [(spots loaded DC - spots control DC)/105 T cells].
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| 15720715 | PMC554108 | CC BY | 2021-01-04 16:03:07 | no | BMC Cancer. 2005 Feb 18; 5:20 | utf-8 | BMC Cancer | 2,005 | 10.1186/1471-2407-5-20 | oa_comm |
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BMC Int Health Hum RightsBMC International Health and Human Rights1472-698XBioMed Central London 1472-698X-5-11572369610.1186/1472-698X-5-1Research ArticleRural Indian tribal communities: an emerging high-risk group for HIV/AIDS Naik Eknath [email protected] Arun [email protected] Richard [email protected] Balasubramaniam [email protected] Seetharam [email protected] Bindu [email protected] Sagar [email protected] John [email protected] Sarah [email protected] Hamisu M [email protected] Department of Epidemiology and Biostatistics, College of Public Health, University of South Florida, Tampa, Florida, USA2 Swami Vivekananda Youth Movement, Mysore, Karnataka, India3 Division of Infectious Diseases, Department of Internal Medicine, University of South Florida, Tampa, Florida, USA4 Department of Maternal & Child Health, School of Public Health, University of Alabama at Birmingham, USA2005 21 2 2005 5 1 1 28 9 2004 21 2 2005 Copyright © 2005 Naik et al; licensee BioMed Central Ltd.2005Naik et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Rural Indian tribes are anthropologically distinct with unique cultures, traditions and practices. Over the years, displacement and rapid acculturation of this population has led to dramatic changes in their socio-cultural and value systems. Due to a poor health infrastructure, high levels of poverty and ignorance, these communities are highly vulnerable to various health problems, especially, communicable diseases including HIV/AIDS. Our study sought to assess knowledge, attitudes and practices regarding sexuality, and the risk factors associated with the spread of HIV/AIDS and STDs among these communities.
Methods
A nested cross sectional study was undertaken as part of the on going Reproductive and Child Health Survey. A total of 5,690 participants age 18–44 were recruited for this study. Data were obtained through home interviews, and focused on socio-demographics, knowledge, attitudes and behaviors regarding sexuality, HIV/AIDS and other STDs.
Results
The study revealed that only 22% of adults had even heard of AIDS, and 18 % knew how it is transmitted. In addition, only 5% knew that STDs and AIDS were related to each other. AIDS awareness among women was lower compared to men (14% vs.30 %). Regarding sexual practices, 35% of the respondents reported having had extramarital sexual encounters, with more males than females reporting extramarital affairs.
Conclusion
Lack of awareness, permissiveness of tribal societies for premarital or extra-marital sexual relationships, and sexual mixing patterns predispose these communities to HIV/AIDS and STD infections. There is a dire need for targeted interventions in order to curtail the increasing threat of HIV and other STDs among these vulnerable populations.
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Background
India is the second most populous nation in the world and has changing sociopolitical and demographic characteristics as well as varied morbidity and mortality patterns [1]. These changes, in conjunction with the country's high population growth rate, have exacerbated the prevailing and emerging public health challenges the country is facing. Since 1986 when the first case of human immunodeficiency virus (HIV) was reported in India [2], it has become imperative to include acquired immunodeficiency syndrome (AIDS) on its long list of public health issues that need to be addressed.
As a direct result of these challenges, India has begun to assess and monitor the impact of HIV/AIDS throughout the country's various states and regions with the assistance of several international health organizations. According to 2003 estimates from UNAIDS, approximately 5.1 million individuals in India are infected with the HIV virus [3]. Furthermore, recent studies indicate that transmission of HIV is no longer confined to high-risk urban populations, but is spreading across rural settings as well [4]. This trend is a cause for concern as AIDS is increasingly hampering social and economic development throughout the country.
For effective control of the spread of HIV/AIDS, it is crucial to have data on knowledge, attitudes and behavioral practices for specific population as research has shown that socio-cultural influences, traditional lifestyles, societal norms, and traditions influence HIV/AIDS transmission rates [5,6]. Because India's HIV/AIDS transmission pattern is predominantly heterosexual (85% of all newly reported cases) [7], subcultures that have relaxed marital structures or are tolerant of high-risk sexual practices (e.g., sex with a commercial sex worker) are particularly vulnerable to the spread of HIV/AIDS and STDs within their communities [7,8]. With more Indian men reporting premarital and extramarital sexual activity, women who marry as teenagers are vulnerable to HIV/AIDS infection and STDs [9,10].
The rapid spread of HIV/AIDS in rural Indian communities has been attributed to the country's poor health infrastructure, poverty and lack of awareness [4,10]. Despite these indicators, little is known about the risk factors, transmission rates, or the impact AIDS will have in these areas in the future. Traditionally, there has been little research and only a paucity of health-related research conducted among this potentially high-risk vulnerable population.
Throughout India, approximately 8% of the population lives within rural tribal communities, which are collectively referred to as 'Tribes'. These communities are geographically distinct; with each tribe having its own unique customs, traditions, beliefs and practices. Even within a particular tribal entity, differences in dialect, health practices, unique customs, values, and traditions are apparent. In rural Indian communities indices of reproductive health are typically very poor: maternal mortality rate is about 230 per 100,000 live births and 61.2% of the women suffer from at least one gynecologic pathology [11]. Because tribal groups have existed on the fringe of Indian society, they may still be unaware or indifferent to the potential health threats from HIV/AIDS. Ascertaining whether or not tribal communities are potentially a high risk group warranting intervention is a necessary step in India's war on AIDS. Accordingly, we undertook this study to explore the risks for this special group of people.
Methods
We conducted a cross-sectional study nested within an existing enumerative study referred to as the Reproductive and Child Health Survey (RCHS). The RCHS was initially designed to enumerate and ascertain basic demographic and health profiles for all tribal members. Data collection for this particular study was done in two phases. Phase one involved adding additional questions to the original RCHS study to assess risk factors (knowledge, attitudes and behavioral practices) associated with the transmission of HIV/AIDS and other communicable diseases.
Study population
The study population comprised of tribal communities living in the southern region of Karnataka (Figures 1,2). Members of these tribes have traditionally derived much of their livelihood from the country's vast reserve of natural forest resources. However over the years, these tribes have been forced to migrate from their ancestral land and are currently living within poor rural communities throughout the state. The initial displacement was as result of the submergence of their traditional homelands through the construction of the Kabini dam, and the second displacement was as a result of 'Project Tiger,' a wildlife conservation project that displaced them to their current location in the southern region of Karnataka, where this study was conducted.
Figure 1 Map of India showing the region of Karnataka where the study was conducted. Used with permission from India-tourism.com
Figure 2 The reproductive and child health program area in the southern part of Karnataka. Used with permission from the Reproductive Child Health Survey (RCHS) project
Sampling
Because of the enumerative nature of the RCHS, all persons within the age group 18–44 years who participated in the RCHS study were included for phase one of this study. This age group was selected as the focus of the initial study was on reproductive health issues.
Survey instruments
A semi-structured questionnaire with both open- and close-ended questions was developed to collect information on knowledge, attitudes and behaviors regarding HIV/AIDS, as well as other relevant demographic information not included in the RCHS. The instrument was developed in English, translated to the native language, and subsequently back translated to English for content and language verification. The survey instrument was field-tested for validity purposes and modified accordingly.
Data collection
A team of ten interviewers from the local tribal communities with a minimum of high school education were selected and trained for two weeks to ensure uniform and high-quality data collection. All adults were interviewed separately to ensure confidentiality. Each interviewer read out each of the questions and response choices (where appropriate) to the interviewees and recorded all answers directly on the questionnaire. Verbal informed consent was obtained from each respondent prior to starting the interview.
Data analysis
Data were entered into an electronic database using Sybase Central Software (Sybase, Inc. Dublin, CA). To ensure confidentiality, all respondent identifiers were expunged to create a secondary data set that was used for the final analysis. Frequency tables were generated for selected demographics and health related categorical variables. In addition, univariate analysis was performed on relevant continuous variables. The findings are presented below.
Ethical consideration
Ethical clearance was obtained from ethical boards within each of the tribal communities, and appropriate government agencies were informed about the study objectives. Permission was also obtained from the Institutional Review Board (IRB) at the University of South Florida in Tampa, Florida. Verbal informed consent was obtained from the local leaders, and the individual participants. Because of the high levels of illiteracy, it was not feasible for us to obtain written consent.
Results
Demographic profile
A total of 11,379 individuals from the 5 tribal communities had been enumerated as part of the RCHS. Of these, 5,690 were within the study age range (18–44 years) and formed the basis for this analysis.
Table 1 shows the demographic profile of the study participants. The mean age of the study group was 31 years. There were more males than females; 53% vs.47%. Eighty four percent were married (91% females & 78% males). The average age at marriage was 13 years for females, and 22 years for males. Only 28% (27% female, & 30% male) of the population was literate i.e. able to read and write in any of the Indian languages. The majority of respondents (67 %) reported living in tiled roof houses with mud flooring, while only 40 % indicated easy access to potable water. Agriculture was the major source of income in these communities. The reported average daily income ranged from US $1.50 to $2.00. Approximately 35% of the respondents migrated on average three to four months each year to nearby areas for work.
Table 1 Demographic profile of the study participants
Characteristics Number (%) (N = 5,690)
Mean age 31 years
Sex
Men 3,016 (53)
Women 2,674 (47)
Currently living in tiled-roof housing 3,812 (67)
Access to potable water 2,276 (40)
Migrated to find work 1,992 (35)
Currently married
Men 2,353 (78)*
Women 2,433 (91)**
All 4,786 (84)
Literacy***
Men 814 (27)*
Women 802 (30)**
All 1,616 (28)
Mean age of first marriage Mean years (95% CI)
Men 22.0 (19.5–24.5)
Women 13.0 (11.2–14.7)
All 15.0 (11.2–18.8)
*Percentages are based on the total number of men in the study.
**Percentages are based on the total number of women in the study.
***Literacy as defined by census operations of India is the ability to read and write in any of the Indian languages.
CI = Confidence interval
Unique sexual practices among tribal members
The findings revealed that these tribal communities did not have a structured marital system; instead members practiced a form of serial monogamy in which they change partners and remarry every four to five years. Regarding sexual practices, 35% of the respondents reported either premarital affairs or extramarital affairs (Table 2). However such practices were more common in men compared to women. Furthermore, 20% of the male participants reported having had sex with a commercial sex worker (CSW) during the period the wife had had a child.
Table 2 Self reported sexual practices of respondents
Characteristics N = 5,690
Age at first sexual activity Mean years (95% CI)
Men 17.0 (13.4–20.6)
Women 13.0 (11.5–14.5)
Premarital or extramarital sexual encounters Number (n) (%)
Males 1,434 72.0
Females 558 28.0
Total 1,992 35*
Sex with commercial sex worker within a year after spouse giving birth (men only n = 3,016) 470 20
* Percent based on the total sample of 5,690 respondents
Knowledge and beliefs about HIV/AIDS and STDs
Among these communities, there was a low level of knowledge on HIV/AIDS; only 22 % of all study participants (n = 1,252) had heard of AIDS (Table 3). Among those who have heard of AIDS, less than 20 % (n = 250) knew how HIV/AIDS was transmitted (16.8 % male vs. 8% females). About 98 % were not aware of the methods to prevent HIV/AIDS transmission. As many as 30 % (n = 376) of those who had heard of AIDS believed that "sinners" will get AIDS, while 10 % (n = 125) believed that AIDS and STDs could be prevented by the sterilization of women. Fifteen percent (n = 188) thought "AIDS is acquired by looking at a person who has AIDS," and 18 % (n = 225) believed that "AIDS is acquired by talking to a person who has AIDS." Only 5 % knew that a relationship exists between HIV/AIDS and STDs. Interestingly, 4 percent (n = 51) believed that there was a cure for AIDS. Most had not heard of STDs, and of those who had heard of them only 1 percent (n = 16) were aware of associated symptoms.
Table 3 Knowledge and beliefs about HIV/AIDS and STDs
Knowledge and beliefs about HIV/AIDS No. of study subjects
N = 5,690
No. of study subjects who have ever heard of HIV/AIDS
N = 1,252
N Percent of 5,690 Percent of 1,252
Have ever heard of HIV/AIDS 1,252 22.0 100.0
Know how HIV/AIDS is transmitted 250 4.4 20.0
Know methods to prevent the transmission of HIV/AIDS 114 2.0 9.1
Believe "sinners" will get AIDS 376 6.6 30.0
Believe AIDS and STDs can be prevented by sterilization of women 125 2.2 10.0
Believe AIDS is acquired by looking at an infected person with AIDS 188 3.3 15.0
Believe AIDS is acquired by talking to a person who has AIDS 225 4.0 18.0
Believe there is a cure for HIV/AIDS 51 0.9 4.1
Discussion
In today's modern world, it is difficult to imagine societies that are still socially and culturally isolated from the rest of civilization; however, they do exist. The tribal societies throughout India have remained socially and culturally alienated from mainstream Indian society until developmental and conservation activities in tribal areas forced interactions between them. Displacement of the tribal people of southern Karnataka has led to a complex process of rapid acculturation and loss of cultural identity; as they struggle to maintain their traditional social structure, they must adopt new skills, beliefs, and practices necessary for success in their new environment. During this acculturation process, they have been faced with a myriad of public health challenges complicated by poverty, ignorance, and reluctance to abandon traditional beliefs and practices that would allow them to assimilate successfully.
To date this is the first health related study among the displaced tribal communities of southern Karnataka that has attempted to assess risk threshold for the transmission and spread of HIV/AIDS and other STDs. It is not surprising that knowledge and awareness about HIV/AIDS and STDs was very low among tribal communities compared to the national figures given the degree of isolation, low literacy rates, and minimal access to information.
The high level of poverty, inadequate health resources, ignorance and high-risk beliefs and practices among the tribal communities has contributed to the vulnerability of this population. As such it has created a highly susceptible population for the rapid spread of HIV/AIDS and other STDs' as well.
Because tribal members are forced to migrate outside of their communities in search for work and increased wages, this may contribute to the spread of HIV/AIDS as many engage in extramarital affairs, seek commercial sex partners, or are under the threat of sexual harassment (females). While there has been limited scientific research exploring the cultural context of extramarital sexual behaviors, it is generally noted that in these communities, extramarital affairs are condoned and widely practiced especially during periods when women are pregnant or nursing or during period of travel for work [8]. This kind of behavior creates a fertile ground for HIV transmission and spread. Our data indicate that tribal women are particularly vulnerable for HIV/AIDS in this population since many of them commence sexual activity at an early age, and get married early as well. Also, they are in a culture that condones extramarital sex, and this exposes the women to a particularly precarious situation, increasing their risk for acquiring HIV.
Conclusion
It is evident from this study that the Indian tribal community is experiencing a latent phase that is potentially a precursor for an HIV/AIDS epidemic. There is a high prevalence of behavioral risk factors, coupled with ignorance, and inadequate health infrastructure thus creating a potential risk for rapid spread of HIV/AIDS, as well as other related diseases. In a country that is struggling to contain the spread of HIV, it is particularly important for concerned parties to pay attention to this population. Currently, virtually no resources are allocated toward the treatment of those infected with HIV/AIDS; the main stay of management is through education and preventive measures to control the spread of the scourge as they represent the most practical and cost effective strategies in this developing nation. It therefore becomes imperative and urgent to address the health concerns revealed in our study in order to formulate effective, culture-sensitive and appropriate intervention programs so that an imminent disaster (i.e. HIV/AIDS epidemic) in this remote and isolated communities can be averted.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
All the authors were involved in the design of the study, analysis, interpretation and development of the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We would like to acknowledge and thank staff of Swami Vivekananda Youth Movement for their valuable logistic support in the data collection. We would also like to acknowledge India-tourism.com and the RCHS for allowing us access to their maps.
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| 15723696 | PMC554109 | CC BY | 2021-01-04 16:29:56 | no | BMC Int Health Hum Rights. 2005 Feb 21; 5:1 | utf-8 | BMC Int Health Hum Rights | 2,005 | 10.1186/1472-698X-5-1 | oa_comm |
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BMC NeurosciBMC Neuroscience1471-2202BioMed Central London 1471-2202-6-141573332410.1186/1471-2202-6-14Research ArticleEffects of prostaglandin E2 on the electrical properties of thermally classified neurons in the ventromedial preoptic area of the rat hypothalamus Ranels Heather J [email protected] John D [email protected] Department of Biology, College of William and Mary. Williamsburg, Virginia 23187, USA2005 27 2 2005 6 14 14 26 6 2004 27 2 2005 Copyright © 2005 Ranels and Griffin; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Physiological and morphological evidence suggests that activation of the ventromedial preoptic area of the hypothalamus (VMPO) is an essential component of an intravenous LPS-dependent fever. In response to the endogenous pyrogen prostaglandin E2 (PGE2), the majority of temperature insensitive neurons in the VMPO show an increase in firing rate, while warm sensitive neurons are inhibited. We have hypothesized that these PGE2 dependent effects on firing rate are due to changes in the inherent electrical properties of VMPO neurons, which are regulated by the activity of specific ionic currents.
Results
To characterize the electrical properties of VMPO neurons, whole-cell recordings were made in tissue slices from male Sprague-Dawley rats. Our results indicate that PGE2 dependent firing rate responses were not the result of changes in resting membrane potential, action potential amplitude and duration, or local synaptic input. However, PGE2 reduced the input resistance of all VMPO neurons, while increasing the excitability of temperature insensitive neurons and decreasing the excitability of warm sensitive neurons. In addition, the majority of temperature insensitive neurons responded to PGE2 with an increase in the rate of rise of the depolarizing prepotential that precedes each action potential. This response to PGE2 was reversed for warm sensitive neurons, in which the prepotential rate of rise decreased.
Conclusion
We would therefore suggest that PGE2 is having an effect on the ionic currents that regulate firing rate by controlling how fast membrane potential rises to threshold during the prepotential phase of the action potential.
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Background
Fever, an elevation in body temperature, is thought to play an adaptive role in the immune system's ability to fight infection [1]. A suggested mechanism for its production and maintenance is a shifting of the thermostatic set-point into the hyperthermic range [1,2]. Through the integration of both central and afferent thermal information, this set-point is established by the activity of neurons in the preoptic and anterior regions of the hypothalamus (PO/AH) that can be thermally classified on the basis of their inherent ability to respond to changes in temperature [3]. The majority of PO/AH neurons are considered temperature insensitive, showing little or no temperature dependent changes in firing rate. Approximately 30% of PO/AH neurons can be classified as warm sensitive, responding to local warming with an increase in firing rate [4]. While there has been considerable debate as to the criteria that should be used to classify a neuron as warm sensitive, we have used a regression coefficient of at least 0.8 impulses·s-1·°C-1. This criterion is based on previous studies that indicate a functional difference for neurons which show this degree of inherent thermosensitivity [3,4]. In addition to responding to local changes in temperature, some of these warm sensitive neurons are also responsive to changes in skin or spinal temperature, while others show thermally dependent changes in their firing rates that may directly correlate with the activation of specific thermoregulatory responses. Although this integrative ability seems to be restricted to warm sensitive neurons in the PO/AH, temperature insensitive neurons may also play an important role in determining the set-point temperature through their synaptic interactions with thermoregulatory effector neurons [3]. Regardless of thermosensitivity, many PO/AH neurons may respond to adjustments in other homeostatic conditions or the presence of endogenous pyrogens such as prostaglandin E2 (PGE2), which could shift the thermostatic set-point and alter the activation of thermoregulatory mechanisms [2].
In response to stimulation of the immune system, changes in the activity of neurons in specific regions of the PO/AH may be responsible for the adjustment of the thermostatic set-point that results in an elevation in body temperature. Physiologic evidence suggests that in response to endotoxins such as lipopolysaccharide (LPS), this shift in set-point is mediated by the activation of afferent neural pathways or the production of systemic pyrogens, which ultimately leads to the local production of PGE2 within the hypothalamus [5,6]. Early microinjection studies clearly established a role for prostaglandins in the production of a fever and later identified the importance of the region surrounding the OVLT in this response [7-10]. More recently, it has been shown that fever in response to intravenous LPS is dependent on the presence of the PGE2 producing enzyme cyclooxygenase-2 in the ventromedial preoptic area of the hypothalamus (VMPO) [11]. In addition, it has now been demonstrated that unlike other regions of the PO/AH, PGE2 has a selective effect on the firing rates of VMPO neurons, based on thermosensitivity, with PGE2 increasing the firing rates of temperature insensitive neurons and inhibiting the firing rates of warm sensitive neurons [12]. Anatomical studies also support the importance of the VMPO in the production of a fever, demonstrating that either the intravenous injection of LPS or microinjection of PGE2 directly into the VMPO will produce a fever that can be correlated with an increase in the cellular activation of neurons within the VMPO [13,14].
Using a functional criterion for determining the thermosensitivity of hypothalamic neurons, a clear difference in the effects of PGE2 on the firing rates of VMPO neurons has been demonstrated [12]. Based on current models of set-point temperature regulation, this PGE2 dependent increase in the firing rates of temperature insensitive neurons or decrease in the firing rates of warm sensitive neurons could lead to a hyperthermic shift in the thermostatic set-point and production of a fever [3]. Yet, little is known about the electrical responses by which PGE2 regulates the firing rates of VMPO neurons. We have hypothesized that these PGE2 dependent changes in firing rate are not the result of a change in the frequency of synaptic input to these neurons, but a selective effect on specific electrical properties of VMPO neurons. To characterize these responses, whole-cell recordings were made from VMPO neurons in tissue slices from male Sprague-Dawley rats, in response to changes in temperature and PGE2.
Results
For forty two VMPO neurons, temperature sensitivity and PGE2 dependent changes in firing rate, electrical activity, and the frequency of synaptic input were determined. The majority of these neurons were classified as temperature insensitive (n = 32). The remaining ten neurons were classified as warm sensitive. With respect to thermosensitivity or responses to PGE2, there was no specific pattern to the distribution of these neurons throughout the VMPO (Fig. 1).
Using the cellularly invasive procedure of whole cell recording, the PGE2 dependent changes in firing rate that were recorded from VMPO neurons were similar to those reported in an earlier extracellular single-unit recording study [12]. In response to PGE2, fourteen temperature insensitive neurons showed significant increases in firing rate. Thirteen of these neurons had thermosensitivities ≤ 0.4 impulses·s·°C-1. The firing rates of temperature insensitive neurons, having thermosensitivities ≤ 0.4 impulses·s-1.°C-1, significantly increased in response to PGE2, from 5.75 ± 1.31 impulses·s-1 to 6.5 ± 1.31 impulses·s-1 (paired T test, P = 0.04; firing rates at least 10 minutes into the following washout period = 6.37 ± 1.32 impulses·s-1). Of the thirteen temperature insensitive neurons with thermosensitivities of 0.41 – 0.79 impulses·s-1.°C-1, the majority (n = 10) showed little or no change in firing rate in response to PGE2. The firing rates of these neurons did not significantly change from a baseline of 8.4 ±1.21 impulses·s-1 (PGE2 = 8.55 ± 1.24 impulses·s-1 (paired T test, P = 0.38); washout = 8.34 ± 1.24 impulses·s-1). In contrast to the responses of temperature insensitive neurons, the majority (n = 8) of VMPO warm sensitive neurons showed a significant decrease in firing rate during perfusion with PGE2. The firing rates of warm sensitive neurons significantly decreased from 15.28 ± 4.93 impulses·s-1 to 12.35 ± 4.85 impulses·s-1 in response to PGE2 (paired T test, P = 0.003; firing rates at least 10 minutes into the following washout period = 12.87 ± 4.12 impulses·s-1).
Electrical properties
All VMPO neurons recorded in this study had resting membrane potentials of -45.0 ± 1.1 mV (n = 42). There was no significant difference between the resting membrane potentials of temperature insensitive neurons (-45.6 ± 1.2 mV; n = 32) and warm sensitive neurons (-43.1 ± 2.4 mV; n = 10). In addition, resting membrane potential did not change in response to PGE2 and was not responsible for PGE2 dependent changes in firing rate.
The top panels of Figure 2 show the action potential activity of a temperature insensitive neuron during baseline conditions, perfusion with PGE2, and the washout period. While the resting membrane potential did not change from a baseline mean of -43.94 mV, firing rate increased 43.4% in response to PGE2, from a mean of 5.11 impulses·s-1 to 7.33 impulses·s-1. The onset of this response occurred several minutes after perfusion with PGE2 had begun and lasted approximately 15 minutes beyond the point when perfusion with PGE2 ended. This was typical of all temperature insensitive neurons which had a significant change in firing rate in response to PGE2. These neurons showed response latencies of 3.5 ± 0.69 minutes and durations that ranged from 7 to 40 minutes before firing rate returned towards the baseline level.
The lower panels of Figure 2 show the action potential activity of a warm sensitive neuron during baseline conditions, perfusion with PGE2, and the washout period. While the resting membrane potential did not change from a baseline mean of -51.62 mV, the firing rate of this neuron decreased in response to PGE2, from a mean of 10.07 impulses·s-1 to 8.40 impulses·s-1. The onset of this response occurred several minutes after perfusion with PGE2 had begun and lasted approximately 25 minutes beyond the point when perfusion with PGE2 was stopped. Similar changes were recorded for the other warm sensitive neurons that were inhibited by PGE2, which showed response latencies of 3.90 ± 0.95 minutes and durations that ranged from 10 to 25 minutes.
Throughout the entire length of a recording (46.7 ± 1.8 minutes), the amplitude and duration of action potentials recorded from VMPO neurons slowly decreased by an average of 4.6 mV and 0.16 milliseconds, respectively (n = 42). It is presumed that this was due to minor changes in ionic gradients, resulting from the technique of whole-cell recording. Based on the PGE2 dependent changes in firing rate reported in this study and previous extracellular recordings [12], these small changes in action potential amplitude and duration did not affect the ability of VMPO neurons to respond to PGE2. There were also no significant changes in the amplitudes or durations of action potentials recorded from temperature insensitive and warm sensitive neurons in response to PGE2.
The input resistance of VMPO neurons recorded in this study significantly decreased in response to PGE2 from 367.1 ± 24.5 MΩ. to 339.7 ± 23.7 MΩ (n = 42; paired T test, P = 0.0001). This PGE2 dependent decrease in resistance was independent of thermosensitivity. Figure 3 shows current-voltage plots from a temperature insensitive neuron (Fig. 3A) and a warm sensitive neuron (Fig. 3B). For both of these neurons, input resistance decreased similarly in response to PGE2, with a reversal potential at or near the resting membrane potential.
During the application of a depolarizing current, VMPO neurons did not show PGE2 dependent changes in the frequency of action potentials (frequency response). However, the majority of VMPO neurons did show PGE2 dependent changes in another characteristic of neuronal excitability, the first spike latency. Temperature insensitive neurons with thermosensitivities ≤ 0.4 impulses·s-1.°C-1showed a significant PGE2 dependent decrease in the first spike latency, from 7.4 ± 1.2 ms during baseline conditions to 5.2 ± 0.6 ms in response to PGE2 (n = 18; paired T test, P = 0.008; Figure 4). The first spike latency for these temperature insensitive neurons returned to baseline levels during the washout period (7.6 ± 1.4 ms). In contrast, warm sensitive neurons showed a significant increase in this latency response during perfusion with PGE2(n = 7), similar to the response of the warm sensitive neuron in Figure 5. (baseline = 4.9 ± 0.5 ms; PGE2 = 7.9 ± 1.1 ms (paired T test, P = 0.046); washout = 4.2 ± 0.5 ms). Temperature insensitive neurons with thermal coefficients of 0.41 to 0.79 impulses·s-1.°C-1 showed little change in this measurement of neuronal excitability (n = 8), in response to PGE2 (baseline = 7.0 ± 1.3 ms; PGE2 = 6.8 ± 1.1 ms (paired T test, P = 0.69); washout = 6.8 ± 1.1 ms).
In Figure 6, the averaged pre- and post-spike activity of a temperature insensitive neuron (A) and a warm sensitive neuron (B) are shown during baseline conditions and in response to PGE2. In a similar manner to the neuron shown in Figure 6A, the majority of temperature insensitive neurons with thermal coefficients ≤ 0.4 impulses·s-1.°C-1 showed a significant increase in the rate of rise of the depolarizing prepotential in response to PGE2 (n = 12; baseline = 0.46 ± 0.04 mV·ms-1, PGE2 = 0.55 ± 0.05 mV·ms-1; paired T test, P = 0.01). In contrast, the majority of warm sensitive neurons showed a significant decrease in the rate of rise of the depolarizing prepotential in response to PGE2, similar to the response of the neuron in Figure 6B (n = 8; baseline= 0.61 ± 0.08 mV·ms-1, PGE2 = 0.50 ± 0.08 mV·ms-1; paired T test, P = 0.012). Although small changes in the voltage deflections that occurred at the end of an action potential showed some degree of change in response to PGE2, these changes were inconsistent in both temperature insensitive or warm sensitive neurons. Temperature insensitive neurons with thermosensitivities of 0.41 – 0.79 impulses·s-1.°C-1 did not show a significant change in the rate of rise of the depolarizing prepotential in response to PGE2.
Synaptic input
The frequency of post synaptic potentials (PSPs) recorded from VMPO neurons in the localized environment of coronal tissue slices was predominately inhibitory and insensitive to changes in temperature (Table 1). In 92% of the recordings, temperature had little or no effect on the frequencies of either inhibitory post synaptic potentials (IPSPs; m = 0.25 ± 0.06 PSPs·s-1.°C-1) or excitatory post synaptic potentials (EPSPs; m = 0.23 ± 0.06 PSPs·s-1.°C-1). In response to PGE2, the frequency of synaptic input recorded from VMPO neurons did not change (Table 1).
Discussion
PGE2 dependent changes in the firing rates of VMPO neurons
As current models for temperature regulation suggest, the integrated responses of hypothalamic warm sensitive neurons and temperature insensitive neurons play an important role achieving and maintaining a discrete set-point for temperature control [2]. While their importance in thermoregulatory pathways has yet to be determined, in vitro recordings from tissue slices have also identified neurons in the PO/AH that lack spontaneously generated activity (silent neurons), are predominantly driven to produce action potentials by synaptic input (EPSP-driven neurons), or produce action potentials in a bursting pattern [2,15]. However, only prepotential driven warm sensitive and temperature insensitive neurons were identified in the VMPO.
In the generation of a fever, current thermoregulatory models of neural networking suggest that either an increase in the activity of temperature insensitive neurons or the inhibition of warm sensitive neurons would shift the set-point to a more hyperthermic temperature [2]. Through competing synaptic inputs, either of these responses would alter the temperature at which thermoeffector neurons begin to activate thermoregulatory responses. However, previous studies have not been able to show a correlation between the thermosensitivity of hypothalamic neurons and firing rate responses to PGE2 or other endogenous pyrogens [16-19]. This may be the result of the various criteria that were used to define thermosensitivity in many of these studies, as well as recordings from more general areas of the PO/AH. In a recent study of the extracellular single-unit activity of neurons in the VMPO, a clear thermal distinction in firing rate responses to PGE2 was reported, with temperature insensitive neurons excited by PGE2 and warm sensitive neurons inhibited [12]. In this previous study, a functionally significant criterion was used to define warm sensitivity (m ≥ 0.8 impulses·s-1.°C-1) [3,15]. As this criterion provides a method for identifying integrative warm sensitive neurons in the tissue slice preparation, it allows the experimental findings to have a functional significance in modeling thermoregulation in vivo. In the present study, which uses whole-cell recording techniques to record the intracellular properties of VMPO neurons and the same functional criterion for defining warm sensitivity, similar firing rate responses were recorded. As suggested above, either an increase in the firing rates of temperature insensitive neurons or a decrease in the firing rates of warm sensitive neurons could lead to a hyperthermic shift in the thermostatic set-point and a fever.
It is also important to note that while the majority of PGE2 responsive neurons showed a significant recovery in firing rate during the washout period, some did not. This lack of complete recovery may have resulted from responses to PGE2 that were considerably long in duration, lasting up to 40 minutes, and minor changes in ionic gradients that can occur over time during whole-cell recordings. As mentioned in the methods, all recordings were closely monitored for any changes that would make them unacceptable.
Cellular properties of VMPO neurons and responses to PGE2
In addition to the direct effects of PGE2 on the electrical properties of VMPO neurons, the frequency of localized synaptic input was characterized. In the present study, we were specifically interested in monitoring firing rate responses to PGE2 and characterizing the resulting voltage potential changes (i.e., action potentials and membrane potentials). In order to ensure that we did not interrupt the firing rate responses, we did not perform any voltage clamp measurements and did all recordings in current clamp, with current = 0 pA. This prevented any detailed characterization of synaptic potentials, other than frequency. As has been shown in other regions of the PO/AH [15], the majority of this synaptic input to VMPO neurons was inhibitory and predominantly insensitive to temperature. In response to PGE2, the frequency of PSPs recorded from most VMPO neurons either did not change or showed an increase. However, PGE2 dependent increases in the frequency of PSPs were not recorded from neurons that showed PGE2 dependent changes in firing rate. This may have resulted from a smaller degree of synaptic influence on firing rate, due to a change in the input resistance [20]. Additionally, the lack of local synaptic input from warm sensitive neurons would suggest that the axonal projections of these neurons may not terminate within the VMPO, but form efferent projections to other hypothalamic nuclei such as the paraventricular nucleus.
As shown in Figure 3, the input resistance recorded from most of the VMPO neurons decreased in response to PGE2, regardless of temperature sensitivity or PGE2 dependent changes in firing rate. Since this response had little or no influence on firing rate, we would suggest that decreases in resistance resulted from changes in the activity of multiple currents, which continued to maintain equilibrium at or near the resting membrane potential. While we have also suggested that PGE2 may cause cAMP concentrations to either increase or decrease within these neurons [12,21], the net effect on hyperpolarizing K+ currents and depolarizing Na+ or Ca++ currents may still lead to an increase in conductance that does not result in a change in resting membrane potential. Therefore, although these changes in the input resistance may be an important response to PGE2, they are not directly responsible for PGE2 dependent changes in firing rate.
In response to a depolarizing current pulse, PGE2 dependent changes in the excitability of VMPO neurons were recorded. Using two primary measurements of excitability, the frequency response and first spike latency (see: Results & Methods), our recordings indicate that PGE2 selectively increased the excitability of temperature insensitive neurons, while decreasing the excitability of warm sensitive neurons. Although there was no significant PGE2 dependent changes in the frequency responses of VMPO neurons, temperature insensitive neurons showed a significant decrease in first spike latency in response to PGE2 (Fig. 4), while warm sensitive neurons showed a significant increase (Fig. 5). This would suggest that PGE2 is having a direct effect of the activity of transient voltage dependent currents, which are responsible for regulating the excitability and rhythmic firing rates of these neurons. While these current pulses were not matched so that they would result in the same level of depolarization, making it difficult to discern which voltage-gated conductances may be responding to PGE2, this data does provide support for our finding of PGE2 dependent changes in the prepotential [22].
Several transient voltage dependent currents have been identified in hypothalamic neurons which regulate excitability and rhythmic firing rate activity. These include a slow component sodium current and a calcium dependent potassium current (IK, Ca) in suprachiasmatic neurons [23], a low voltage activated calcium current and a IK, Ca current in posterior hypothalamic neurons [24], a non-inactivating potassium current in neurhypophyseal nerve terminals [25], and a IA current in PO/AH neurons [22]. Although any or all of these currents may be present in VMPO neurons, only the IA has been implicated in temperature dependent changes in firing rate, through a mechanism in which the thermally dependent inactivation rate of this current influences the rate of rise of the prepotential that precedes each action potential [22]. In a similar manner, our data suggests that PGE2 has a direct effect on the firing rates of VMPO neurons through changes in the prepotential, increasing the rate of rise in temperature insensitive neurons, while decreasing the rate of rise of the prepotential in warm sensitive neurons (Fig. 6). Therefore, PGE2 dependent changes in the firing rates of VMPO neurons may also depend on an ability to influence the inactivation rate of the IA type current.
Within the VMPO, overlapping expression of EP3 and EP4 receptors may provide PGE2 with the ability to selectively affect the activity of neurons in this region [26]. Activation of either receptor subtype is known to influence cellular activity through the regulation of intracellular cAMP concentrations, with EP4 activation leading to an increase in cAMP and EP3 activation leading to a decrease [26-28]. Support for this mechanism is provided by evidence showing that cAMP plays a role in thermoregulation and more specifically, in the generation of a fever [29]. With respect to excitability and rhythmic firing rate activity, several studies have demonstrated that cAMP modulates PGE2 dependent changes the activity of certain potassium currents, including the IA [25,30,31]. Therefore, selective activation of either EP3 or EP4 receptors may be responsible for the changes in firing rate and electrical activity we have recorded from VMPO neurons.
Conclusion
In response to intravenous LPS, the local production of PGE2 within the PO/AH region results in a thermally dependent change in the firing rates of VMPO neurons. Through a direct effect on the rate of rise of the depolarizing prepotential, which is also a determinant of thermosensitivity, PGE2 increases the firing rates of temperature insensitive neurons, while decreasing the firing rates of warm sensitive neurons in the VMPO. While the results of this study provide a clear focus for future studies into the conductance mechanisms of these responses, it also supports a functional and important role for the VMPO region and both groups of thermally classified neurons in the production of this type of fever.
Methods
Anterior hypothalamic tissue slices containing the VMPO were prepared from male Sprague-Dawley rats (100 – 150 grams), which were housed under standard conditions and given food and water ad lib. Prior to each recording session, an animal was anesthetized (isoflurane) and sacrificed by quick decapitation, according to procedures approved by the National Science Foundation and the Animal Care and Use Committee of the College of William and Mary. Following removal of the brain, a tissue block of the hypothalamus was cut using a vibratome into 400 μm thick coronal slices. Two or three slices containing the VMPO were then placed in a recording chamber and allowed to equilibrate for 1–2 hours. Tissue slices were continually perfused with pyrogen free artificial cerebral spinal fluid (aCSF), which consisted of (in mM), 124 NaCl, 26 NaHCO3, 10 glucose, 5 KCl, 2.4 CaCl2, 1.3 MgSO4, and 1.24 KH2PO4. This nutrient medium was oxygenated (95% O2 – 5% CO2), warmed to a stable temperature of ~36°C by a thermoelectric assembly, and allowed to gravity flow into the chamber at 1 – 1.5 ml· min-1 (chamber volume = 1.2 ml) [32]. A small thermocouple, positioned in the recording chamber just below the tissue slices, was used to continuously monitor tissue temperature.
Tight-seal whole-cell recordings were made using glass microelectrodes with tip inner diameters of ~2 μm (3–5 MΩ), filled with a solution that consisted of (in mM) 130 K-gluconate, 10 EGTA, 2 ATP, 1 MgCl2, 1 CaCl, having a pH of 7.2 and an osmolarity of approximately 295 mOsmols/liter. As described previously [33], a liquid junction potential of 12.0 mV was subtracted from all recorded potentials. All recordings were made using an integrated patch-clamp amplifier (Axopatch 200B, Axon Instruments) and along with temperature, were stored on digital tape for later analysis. Firing rate was continuously recorded as a voltage measurement from a rate/interval monitor and was determined by action potential triggered input from a window discriminator (FHC Inc.). Specific stimulation protocols to measure input resistance and excitability were generated by a computer that was interfaced with the amplifier (pClamp software, Axon Instruments). Acceptable recordings consisted of action potential amplitudes through 0 mV and stable recordings of at least 20 minutes.
Using a stereomicroscope, the recording electrode was positioned in the VMPO, which encompasses 700 μm of the hypothalamus just rostral to the suprachiasmatic nucleus. It extends laterally 900 μm from the third ventricle and dorsally 500 μm from the ventral brain surface [34]. Once a tight seal (> 2 GΩ) was achieved between the electrode and the surface of a neuron, the cell membrane was ruptured by suction, establishing an intracellular recording. When the activity of a neuron was stable for several minutes, temperature in the recording chamber was varied 2–3°C above and below 36°C, by changing the input voltage to the thermoelectric assembly. Neuronal thermosensitivity (impulses· s-1·°C-1) was characterized by plotting firing rate as a function of temperature to determine the regression coefficient (m) of this plot. As in previous studies [12,15,35], warm sensitivity was defined as a regression coefficient of at least 0.8 impulses· s-1·°C-1. All other neurons in this study were defined as temperature insensitive.
After thermosensitivity had been characterized, each neuron was tested for its response to PGE2. At a stable temperature (~36°C), the perfusion medium was switched to one containing PGE2 (200 nM or 1 μM, Sigma Chemical Co.). The duration of exposure to PGE2 ranged from 5 – 15 minutes, with durations of less than 10 minutes occurring only when there was a clear indication of a response. Exposure to PGE2 was followed by perfusion with aCSF for a washout period of at least 10 minutes.
To determine if PGE2 had a significant effect on firing rate, one-minute segments of stable activity were digitized for comparison (60 Hz; pClamp Software, Axon Instruments). These segments were collected during baseline conditions (just prior to perfusion with PGE2) at the end of perfusion with PGE2, and at the end of a 10-minute washout period (or when firing rate returned to baseline levels). For each segment, a mean and standard error were calculated (Sigmaplot software, SPSS Inc.). A significant response to PGE2 was determined by comparison to the baseline level using a standard T-test (P ≤ 0.05).
Changes in resting membrane potential and the action potential waveform in response to PGE2 were also characterized. As in a previous study [32], the resting membrane potential was continuously recorded. After rapid changes in membrane potential (including action potentials) were filtered out (half amplitude response setting at 0.5 Hz), one minute segments of voltage activity were digitized (60 Hz) during baseline conditions, perfusion with PGE2, and the washout period. To characterize changes in the action potential, an averaged action potential from ten inherently activated action potentials was produced for each of the experimental conditions (sampling rate = 66.7 kHz). Measurements were made of action potential amplitude (resting membrane potential to peak) and duration (at one half peak). Measurements were also made of the rate of rise of the depolarizing prepotential that precedes each action potential [22]. For resting membrane potential and action potential measurements, significant responses to PGE2 were determined by comparison to baseline using a standard T-test (P ≤ 0.05).
Throughout each recording, input resistance and excitability were measured every 5 – 10 minutes to determine changes in response to PGE2 and to insure that recordings remained stable. Large changes that did not return towards baseline levels were a characteristic of a deteriorating recording and marked the end of legitimate data. Input resistance was determined by the slope of a current-voltage plot obtained from a computer generated protocol in which a series of ten current pulses (-10 to -100 pA) were administered [32]. In addition, two primary characteristics of neuronal excitability were measured in response to a depolarizing current [36]. The frequency response was determined by the number of action potentials produced during the depolarizing current. The first spike latency was also measured and was defined as the duration from the start of the depolarizing current to the peak of the first action potential. All significant responses to PGE2 were again determined by comparison to baseline using a standard T-test (P ≤ 0.05).
The frequencies of synaptic potentials (EPSPs and IPSPs) recorded from each VMPO neuron were determined during baseline conditions, perfusion with PGE2, and the washout period. As in previous whole-cell recordings, individual potentials were identified as rapid changes in membrane potential of at least 1 mV greater than background noise [15,20]. For each experimental condition, blind counts of PSPs were made over a 20 second duration to produce frequency averages (PSPs·s-1). As with the other measurements, all significant responses to PGE2 were determined by comparison to baseline using a standard T-test (P ≤ 0.05). In addition, the thermosensitivities of EPSPs and IPSPs were characterized by calculating frequency averages (using the same methods as detailed above) at different temperatures (≥ 3°C range) and plotting the results as a function of temperature [15].
Once a recording had been completed, a stereomicroscope was used to visually confirm the location of the recording electrode. The ventral edge of the third ventricle was used as a reference to determine the lateral-medial and dorsal-ventral coordinates. The coronal position was also specified by the depth of the electrode from the surface of the tissue slice. In addition, the side of the brain from which the recording was made was identified by preparing the tissue slices with more lateral tissue on the left. Tissue slices were then removed from the recording chamber, fixed in a 10% formalin solution, and sectioned again to a thickness of 50 μm. Sections were then stained with giemsa to identify specific hypothalamic areas so that the location of each recording within VMPO could be reconfirmed [12] [38].
Authors' contributions
HJR, carried out the majority of cellular recordings and data analysis. JDG conceived of the study and participated in its design, coordination and completion. Both authors contributed equally to the drafting of this manuscript.
Acknowledgements
We want to thank Erin Waller, Sarah Norcross and Elizabeth Wallis for their assistance in the histological processing of neural tissue. This work was supported by NSF grant IBN-9983624, and in part by a Howard Hughes Medical Institute grant through the Undergraduate Biological Sciences Program to the College of William and Mary.
Figures and Tables
Figure 1 Intracellular whole-cell recordings from VMPO Neurons. The positions of all recorded neurons in this study are indicated on the above coronal diagrams (temperature insensitive = ●, warm sensitive = ▲ modified from Figures 18–20, Paxinos & Watson, 1998). A corresponds to the anterior regions of VMPO, approximately 400 μm rostral to the areas shown in B, which correspond to the midpoint of the VMPO. C corresponds to the posterior regions of VMPO, approximately 400 μm caudal to the areas shown in B. 3 V, third ventricle; AVPe, anteroventral periventricular nucleus; MPA, medial preoptic area; Pe, periventricular hypothalamic nucleus; ox, optic chiasm; VMPO, ventromedial preoptic nucleus.
Figure 2 The effects of temperature and PGE2 on the activity of a VMPO temperature insensitive neuron and a VMPO warm sensitive neuron. The top panels show one second records of the action potential activity of a VMPO temperature insensitive neuron during all three experimental conditions (resting membrane potential = -43.94 mV). The firing rate significantly increased from a baseline of 5.11 ± 0.12 impulses·s-1 to 7.33 ± 0.13 impulses·s-1 during perfusion with 1 μM PGE2 (washout = 4.90 ± 0.14 impulses·s-1). The lower panels show one second records of the action potential activity of a VMPO warm sensitive neuron during all three experimental conditions (resting membrane potential = -51.62 mV). The firing rate significantly decreased from a baseline of 10.07 ± 0.13 impulses·s-1 to 8.40 ± 0.12 impulses·s-1 during perfusion with 1 μM PGE2 (washout = 10.20 ± 0.16 impulses·s-1).
Figure 3 The effects of PGE2 on the input resistance of a VMPO temperature insensitive neuron (A) and a VMPO warm sensitive neuron (B). For each, changes in membrane potential are plotted in response to a series of hyperpolarizing current pulses (-10 to -100 pA, 200 ms), during baseline conditions (●), perfusion with PGE2 (▲), and after PGE2 has been washed out of the chamber (■). For the temperature insensitive neuron in A (m = 0.05), input resistance decreased from a baseline of 406.33 MΩ, to 332.40 MΩ during perfusion with PGE2. After PGE2 had been washed out of the chamber, input resistance increased to 372.32 MΩ. For the warm sensitive neuron in B (m = 1.30), input resistance decreased from a baseline of 388.00 MΩ, to 244.86 MΩ during perfusion with PGE2. After PGE2 had been washed out of the chamber, input resistance increased to 327.63 MΩ.
Figure 4 The effect of depolarizing current and PGE2 on the activity of a VMPO temperature insensitive neuron. A shows the initial action potential that was produced during baseline conditions, in response to a depolarizing current injection (50 pA, 500 msec). The onset of the current is indicated on the line at the bottom of the figure and only the initial 34 mSec are shown. The first spike latency was 8.0 mSec. In B, the initial action potential that was produced in response to a depolarizing current injection is shown during perfusion with PGE2 (1 μM). The first spike latency was 3.0 mSec.
Figure 5 The effect of depolarizing current and PGE2 on the activity of a VMPO warm sensitive neuron. A shows the initial action potential that was produced during baseline conditions, in response to a depolarizing current injection (50 pA, 500 msec). The onset of the current is indicated on the line at the bottom of the figure and only the initial 22.0 mSec are shown. The first spike latency was 4.0 mSec. In B, the initial action potential that was produced in response to a depolarizing current injection is shown during perfusion with PGE2 (200 nM). The first spike latency was 6.6 mSec.
Figure 6 Averaged pre- and post-spike activity of a temperature insensitive VMPO neuron (A) and warm sensitive VMPO neuron (B). For each, averages of 10 action potentials (truncated) during baseline conditions and during perfusion with PGE2 (1 μM), are superimposed on the spike threshold (post-pike activity during PGE2 is indicated by an asterisk). These averages do not include pre-spike activity that contained putative postsynaptic potentials. For the temperature insensitive neuron in A, the rate of rise of the depolarizing prepotential increased from 0.17 mV·mSec-1 to 0.27 mV·mSec-1. For the warm sensitive neurons in B, the rate of rise of the depolarizing prepotential decreased from 0.38 mV·mSec-1 to 0.18 mV·mSec-1.
Table 1 Effect of PGE2on the Frequency of IPSPs and EPSPs recorded from VMPO Neurons.
IPSPs·s-1 EPSPs·s-1
Thermosensitivity (impulses·s-1.°C-1) N Baseline PGE2 Washout Baseline PGE2 Washout
≤0.4 19 5.1 ± 1.2 5.6 ± 1.0 5.3 ± 1.0 3.0 ± 0.0 3.1 ± 0.7 3.2 ± 0.1
0.41 – 0.79 13 6.4 ± 1.1 7.3 ± 1.3 6.2 ± 1.1 3.3 ± 0.7 4.2 ± 3.3 3.7 ± 0.8
≥ 0.8 10 4.2 ± 1.1 3.8 ± 0.9 3.8 ± 0.9 2.4 ± 0.7 3.2 ± 0.4 1.4 ± 0.3
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| 15733324 | PMC554110 | CC BY | 2021-01-04 16:03:48 | no | BMC Neurosci. 2005 Feb 27; 6:14 | utf-8 | BMC Neurosci | 2,005 | 10.1186/1471-2202-6-14 | oa_comm |
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BMC Musculoskelet DisordBMC Musculoskeletal Disorders1471-2474BioMed Central London 1471-2474-6-111572768610.1186/1471-2474-6-11Research ArticleAbdominal fat and hip fracture risk in the elderly: The Dubbo Osteoporosis Epidemiology Study Nguyen Nguyen D [email protected] Chatlert [email protected] Jacqueline R [email protected] John A [email protected] Tuan V [email protected] Bone and Mineral Research Program, Garvan Institute of Medical Research, St Vincent's Hospital, Sydney, UNSW, Australia2 Division of Endocrinology and Metabolism, Department of Medicine, Faculty of Medicine Khon Kaen University, 40002 Thailand2005 23 2 2005 6 11 11 10 1 2005 23 2 2005 Copyright © 2005 Nguyen et al; licensee BioMed Central Ltd.2005Nguyen et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Fat mass, which is a major component of body weight, is directly related to bone mineral density and reduced fracture risk. It is not known whether abdominal fat is associated with hip fracture. The present study was designed to examine the association between abdominal fat and hip fracture in women and men aged 60+ years.
Methods
This was a nested case-control study with one fracture case being matched with two controls of the same age. In women 63 cases were matched with 126 controls, and in men 26 cases were matched with 52 controls. Hip fracture was confirmed by X-ray and personal interview. Other measurements included weight, height, body mass index (BMI), abdominal fat, and femoral neck bone density (FNBMD). Conditional logistic regression model was used to analyse data.
Results
The odds ratio of hip fracture risk associated with each 10% lower abdominal fat was 1.5 (95% CI, 1.1 to 2.1) in women and 1.2 (95% CI, 0.7 to 2.0) in men. However after adjusting for FNBMD or body weight, the abdominal fat-fracture association was no longer statistically significant. Similarly, body weight and BMI was each significantly associated with hip fracture risk (in women), but after taking with account the effect of FNBMD, the association become statistically non-significant.
Conclusion
Lower abdominal fat was associated with an increased risk of hip fracture in elderly women, but the association was not independent of FNBMD or weight. The contribution of abdominal fat to hip fracture risk is likely to be modest.
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Background
Hip fracture is a public health concern, because it is associated with increased mortality, morbidity, reduced quality of life, and incurs significant economic and social costs [1]. Bone mineral density (BMD), a measure of bone strength, is a strong predictor of hip fracture risk [2], and is used as a surrogate measure of the severity of osteoporosis [3], the mechanism of BMD-hip fracture relationship is not well understood. Body weight is strongly related to bone mineral density, such that higher weight is associated with both higher BMD [4-7], and reduced fracture risk [8,9]. Body weight is the sum of lean and fat mass, and the relative importance of each component to hip fracture risk is contentious [10-14]. Lower fat mass was associated with an increase in the risk of hip fracture after adjusting for body weight and age [15], but it is not clear whether the significant relationship is independent of BMD.
Central abdominal fat, which can be derived from dual-energy X-ray absorptiometry (DXA) scans, is highly correlated with, and has been suggested to be a surrogate measure of body fat [16]. Therefore, it is hypothesized that the BMD-fracture relationship may be partly mediated by fat mass, represented by central abdominal fat. The aim of this study was to test this hypothesis in a sample of elderly men and women of Caucasian background.
Methods
Setting and subjects
The present study was designed as a nested case-control study within the larger Dubbo Osteoporosis Epidemiology Study (DOES), which has been on going since 1989 [17,18]. Briefly, in 1989, all men and women aged 60 or above living in Dubbo, a city of approximately 32,000 people 400 km north west of Sydney (Australia), were invited to participate in the DOES. At that time, the population comprised 1,581 men and 2095 women aged ≥ 60 years, of whom, 98.6 % were Caucasian and 1.4 % were indigenous Aboriginal. Dubbo was selected for the study site because the age and gender distribution of the population closely resembles the Australian population and it is relatively isolated in terms of medical care, so that virtually complete ascertainment of all fractures occurring in the target population is possible. This study has been approved by the St Vincent's Hospital Ethics Committee, and informed written consent was obtained from each participant.
By mid 2003, 2560 subjects aged 60+ have participated in the study. Within this population, 89 (63 women and 26 men) hip fracture cases, which had had abdominal fat measured were identified from radiologists' reports from the two centres providing X-ray services as previously described [17]. Fractures were only included if the report of fracture was definite and, on interview, had occurred with minimum or no trauma, including a fall from standing height or less. Fractures clearly due to major trauma (such as motor vehicle accidents) and due to underlying diseases (such as cancer or bone-related diseases) were excluded from the analysis.
For every fracture case, two non-fracture controls of the same age were randomly selected from the database. Age matching tolerance of ± 5 years was applied for women 85+ years and men 81+ years. In total, data from 267 subjects were included in the analysis.
Measurements
Subjects were interviewed by a nurse co-ordinator who administered a structured questionnaire to collect data including age, life-style factors such as past and present tobacco intake (assessed as pack-years) and alcohol consumption, physical activity. Anthropometric variables (height, weight) were measured and a dietary assessment was performed based on a frequency questionnaire for calcium intake as described elsewhere [19].
Femoral neck bone mineral density (FNBMD, g/cm2) was measured by DXA using a LUNAR DPX-L densitometer (GE-LUNAR, Madison). The radiation dose with this method is <0.1 μGy. The coefficient of reliability of BMD in our institution in normal subjects is 0.96 and 0.98 at the proximal femur and lumbar spine, respectively [20].
Abdominal fat of the subjects was directly measured from the spinal DXA scan. Abdominal fat was derived from a standard window extending for 4 cm on either side of the first to fifth lumbar vertebrae. The DXA software expresses the fat mass in this abdominal window as a percentage of the total soft tissue. The coefficient of variation of this measurement as determined for dual scans performed on the same day in 60 people was 1.8% [21].
Statistical analysis
The magnitude of correlation of associations between abdominal fat, body weight and FNBMD were estimated the product moment correlation coefficients and simple linear regression analysis. Differences in these measures between fracture cases and controls expressed as standardized difference (95% confidence interval- CI) were tested by paired t-test or Wilcoxon signed ranks test with significance level of 5%, depending on the distribution of data. The association between abdominal fat and hip fracture risk was assessed by the conditional logistic regression via the PROC PHREG [22] of the Statistical Analysis System (SAS) [23].
Results
Abdominal fat in both women and men was normally distributed with no significant skewness. In the entire sample, there was no significant difference in percent of abdominal fat between women and men (23.9 ± 9.5 % vs. 24.0 ± 10.1 %, P = 0.993). Abdominal fat significantly decreased with age (r = -0.21, P = 0.003) with 1.4% (SE = 0.47) per 5 year in women. In men the rate of decrease was 0.5% (SE = 0.87) with each 5 year of age; however the decrease was not statistically significant (r = -0.07, P = 0.561). The correlation between abdominal fat and weight (r = 0.7, p < 0.001 for both genders) was higher than that between abdominal fat and FNBMD (r = 0.4, p < 0.001 in women and r = 0.2, p = 0.041 in men), (Figure).
Figure 1 Correlations between abdominal fat and weight and femoral neck bone mineral density. (Abdominal fat was expressed as percentage of the total soft tissue).
After matching for age and gender, compared to the controls, women with hip fracture had significantly lower weight (-0.7SD, p < 0.001), BMI (-0.6SD, P = 0.001), abdominal fat (-0.5SD, P = 0.014) and FNBMD (-0.9SD, P < 0.001). In contrast, in men there was no significant difference between those with hip fracture and those without fracture with respect to weight, BMI and abdominal fat; however FNBMD in men with a hip fracture was 1.1SD lower than those without a fracture (Table 1).
Table 1 Baseline characteristics of participants as at 1989
Hip fracture Non fracture P value Standardized difference (95% CI)
Women (n = 63) (n = 126)
Age (y) 76.3 ± 7.1 76.4 ± 7.2 0.067b 0.0 (-0.3 to 0.3)
Height (cm) 155.8 ± 6.9 158.2 ± 6.1 0.008b -0.4 (-0.7 to -0.1)
Weight (kg) 55.6 ± 11.3 63.7 ± 10.9 <0.001b -0.7 (-1.0 to -0.4)
BMI (kg/m2) 22.8 ± 4.0 25.3 ± 4.7 0.001b -0.6 (-0.9 to -0.3)
Abdominal fata (%) 21.3 ± 9.2 25.5 ± 8.6 0.014b -0.5 (-0.8 to -0.2)
FNBMD (g/cm2) 0.64 ± 0.10 0.75 ± 0.14 <0.001b -0.9 (-1.2 to -0.5)
Home physical activity (METs) 85.5 ± 34.9 76.6 ± 30.6 0.075b 0.2 (-0.0 to 0.6)
Calcium intake (mg/d) 608 ± 401 580 ± 370 0.455c 0.1 (-0.2 to 0.5)
Duration of smoking (y) 40.8 ± 16.6 33.2 ± 14.4 0.204c 0.5 (-0.1 to 10.8)
Smoking intake (c/d) 13.8 ± 8.0 11.8 ± 7.5 0.384c 0.3 (-0.3 to 0.8)
Men (n = 26) (n = 52)
Age (y) 75.2 ± 6.0 75.1 ± 5.9 0.329b 0.0 (-0.5 to 0.5)
Height (cm) 169.8 ± 8.0 172.2 ± 5.5 0.219b -0.4 (-0.9 to -0.2)
Weight (kg) 71.7 ± 15.5 75.2 ± 9.0 0.277b -0.3 (-0.8 to 0.2)
BMI (kg/m2) 24.7 ± 3.9 25.3 ± 2.2 0.442b -0.2 (-0.7 to 0.3)
Abdominal fata (%) 26.2 ± 10.2 24.8 ± 6.2 0.581b 0.2 (-0.4 to 0.7)
FNBMD (g/cm2) 0.64 ± 0.10 0.75 ± 0.11 0.002b -1.1 (-1.6 to -0.5)
Home physical activity (METs) 78.4 ± 37.4 78.5 ± 25.1 0.990b 0.0 (-0.5 to 0.5)
Calcium intake (mg/d) 546 ± 322 572 ± 242 0.450c -0.1 (-0.6 to 0.5)
Duration of smoking (y) 46.4 ± 13.4 36.2 ± 15.9 0.036c 0.7 (-0.01 to 1.3)
Smoking intake (c/d) 16.3 ± 5.5 16.4 ± 4.8 0.168c -0.01 (-0.7 to 0.7)
Results are expressed as mean ± SD; BMI, Body mass index; FNBMD, femoral neck bone mineral density; METs, metabolic equivalents
aAbdominal fat was expressed as percentage of the total soft tissue;
bPaired t-test;
cWilcoxon Signed Ranks test
The risk of hip fracture was estimated to increase by 1.5-fold (95%CI: 1.1 to 2.1) in women and 1.2-fold (95% CI: 0.7 to 2.0) in men for each 10% lower abdominal fat. However after adjusting for BMD or body weight, the abdominal fat-fracture association was no longer statistically significant. (Table 2)
Table 2 Odds-ratio (OR) of the risk factors for hip fracture in elderly women and men by conditional logistic regression analysis
Unadjusted OR (95% CI) OR (95% CI) adjusted for FNBMD OR (95% CI) adjusted for weight
Women
Abdominal fata (-10%) 1.5 (1.1 to 2.1) 1.1 (0.7 to 1.5) 1.1 (0.7 to 1.7)
Weight (-10 kg) 2.0 (1.4 to 2.8) 1.3(0.9 to 1.7) -
BMI (-4 kg/m2) 1.7 (1.2 to 2.4) 1.2 (0.8 to 1.7) 1.5 (0.7 to 3.0)
FNBMD (-0.12 g/cm2) 2.4 (1.7 to 3.9) - 2.1 (1.3 to 3.5)
Men
Abdominal fata (-10%) 1.2 (0.7 to 2.0) 1.5 (0.7 to 2.9) 1.8 (0.8 to 4.0)
Weight (-10 kg) 1.4 (0.8 to 2.3) 1.5 (0.7 to 3.2) -
BMI (-4 kg/m2) 1.3 (0.7 to 2.5) 1.7 (0.6 to 4.5) 1.3 (0.9 to 1.9)
FNBMD (-0.12 g/cm2) 2.3 (1.3 to 4.0) - 3.0 (1.3 to 6.5)
BMI, Body mass index; FNBMD, femoral neck bone mineral density.
aAbdominal fat was expressed as percentage of the total soft tissue.
Bold-faced values are statistically significant.
Similarly, body weight and BMI was each significantly associated with hip fracture risk (in women), but after taking with account the effect of FNBMD, the association become statistically non-significant. In both women and men, the association between BMD and fracture risk was consistently significant either in unadjusted or in adjusted analysis (Table 2).
Discussion
It has been known for some time that body weight and whole body fat mass are significant predictors of hip fracture risk in women [9,15], however, it is not clear whether this association is independent of BMD. Abdominal fat has been shown to be well correlated with whole body fat mass [21]. The present study's finding of lower abdominal fat among hip fracture cases compared with the controls is consistent with previous observations [9,15]. However, it further suggests that the association between fat and hip fracture risk is not independent of BMD. Women with lower weight and fat mass may have lower FNBMD because of lower gravitational loading on the bone [24,25], or may have lower level of endogenous estrogens produced in adipose tissue and muscle [26,27]. On the basis of these findings, it may be proposed that BMD is a direct predictor of hip fracture risk, and that central abdominal fat (or fat mass) is a determinant of BMD. Thus, the previously observed relationship between fat and fracture risk is an indirect, rather than a causal association.
Interestingly, in men abdominal fat or body weight was not significantly associated with hip fracture risk either before or after adjusting for BMD. Moreover, the magnitude of difference between fracture versus non-fracture cases in body weight or abdominal fat in men was generally more modest compared to that in women. For example, men with hip fracture had 0.2SD lower abdominal fat and 0.4SD lower in weight than non-fracture men. These differences were not significant. In contrast, in women the corresponding differences were significant and were 0.5SD lower for abdominal fat and 0.7SD for weight. This may suggest that the BMD-hip fracture association in men is not mediated via fat mass, despite a similar correlation between fat mass and BMD. However the lack of significance of the association between abdominal fat and hip fracture in men in the present study may be due to the small sample size.
In recent years, there has been considerable interest in the relationships between osteoporosis, diabetes and cardiovascular disease [28-30]. A common characteristic of individuals with diabetes and cardiovascular diseases is that the majority have higher body weight and fat mass [31-38], and on this basis together with the well-known relationship between weight and BMD, it is expected these individuals would have higher BMD and lower risk of fracture. However, epidemiological data point out that individuals with cardiovascular diseases have lower BMD, and a higher risk of fracture [29,39]. The present study also found that, without BMD adjustment, men and women with lower body weight had a higher risk of hip fracture. Hypertension has been suggested as a potential contribution to the risk of hip fracture [39]. A previous study showed that abdominal fat is positively correlated with blood pressure [40]. However, in this study lower, not higher, abdominal fat was a risk for hip fracture in women. These data suggest that the association between diabetes, cardiovascular diseases and hypertension and fracture risk is also not mediated via fat mass.
From a public health point of view, the present study's finding suggests that abdominal fat does not add to the discriminatory value of hip fracture risk that is already provided by BMD. Indeed, in this study, none of the body size measurements (weight, height, BMI and abdominal fat) was a significant predictor of hip fracture risk after adjusting for BMD. This suggests that these measures have limited value in the prediction of hip fracture in a population or an individual.
A number of issues should be kept in mind before extrapolating the present finding. First, the participants in this study were Caucasian aged 60 years and above, so it may not be generalizable to younger populations and to different races. Second, neither total body fat, nor waist and hip circumferences (WHC) were measured and these may have had stronger predictive value. However, these measurements may underestimate abdominal adiposity in those with both large waist and hip circumferences, while DXA determination of regional body fat distribution may indeed be more valid than WHC [16].
Conclusion
These data have demostrated that in the elderly, abdominal fat was significantly associated with hip fracture risk in women but the association was not independent of BMD, whereas in men abdominal fat was not a significant predictor of hip fracture risk. Measurement of DXA abdominal fat does not contribute to hip fracture prediction over and above that provided by BMD.
List of abbreviations
All abbreviations are defined in the text.
Competing interests
The author(s) declare that they have no competing interest
Authours' contributions
NDN obtained and analysed the data, and drafted the manuscript. CP had an active role in data analysis and interpretation of results. JRC had an active role in the conduct of the Dubbo Osteoporosis Epidemiology Study and helped with the interpretation of results. JAE established the Dubbo Osteoporosis Epidemiology Study. TVN had an active role in the conception if this project, involved in study design, analysis data and interpretation of results. All authors contributed to the last version of the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We gratefully acknowledge the assistance of Sr Janet Watters and Donna Reeves for the interview, data collection and measurement bone mineral density. We also appreciate the invaluable help of the staff of Dubbo base hospital. We thank Natasa Ivankovic for management of the database. This works has been partly supported by the National Health and Medical Research Council of Australia. and untied educational grants from GE-Lunar, Merck Australia, Eli Lilly International and Aventis Australia. The first author is supported by an untied educational grant from Merck Australia Pty Ltd.
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| 15727686 | PMC554111 | CC BY | 2021-01-04 16:32:04 | no | BMC Musculoskelet Disord. 2005 Feb 23; 6:11 | utf-8 | BMC Musculoskelet Disord | 2,005 | 10.1186/1471-2474-6-11 | oa_comm |
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BMC Evol BiolBMC Evolutionary Biology1471-2148BioMed Central London 1471-2148-5-181572535210.1186/1471-2148-5-18Research ArticleAnalysis of the human Alu Ye lineage Salem Abdel-Halim [email protected] David A [email protected] Dale J [email protected] Jerzy [email protected] Mark A [email protected] Department of Biological Sciences, Biological Computation and Visualization Center, Center for Bio-Modular Multi-scale Systems, Louisiana State University, 202 Life Sciences Building, Baton Rouge, Louisiana 70803 USA2 Department of Anatomy, Suez Canal University, Ismailia, Egypt3 Genetic Information Research Institute, 2081Landings Drive, Mountain View, CA 94043 USA2005 22 2 2005 5 18 18 7 12 2004 22 2 2005 Copyright © 2005 Salem et al; licensee BioMed Central Ltd.2005Salem et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Alu elements are short (~300 bp) interspersed elements that amplify in primate genomes through a process termed retroposition. The expansion of these elements has had a significant impact on the structure and function of primate genomes. Approximately 10 % of the mass of the human genome is comprised of Alu elements, making them the most abundant short interspersed element (SINE) in our genome. The majority of Alu amplification occurred early in primate evolution, and the current rate of Alu retroposition is at least 100 fold slower than the peak of amplification that occurred 30–50 million years ago. Alu elements are therefore a rich source of inter- and intra-species primate genomic variation.
Results
A total of 153 Alu elements from the Ye subfamily were extracted from the draft sequence of the human genome. Analysis of these elements resulted in the discovery of two new Alu subfamilies, Ye4 and Ye6, complementing the previously described Ye5 subfamily. DNA sequence analysis of each of the Alu Ye subfamilies yielded average age estimates of ~14, ~13 and ~9.5 million years old for the Alu Ye4, Ye5 and Ye6 subfamilies, respectively. In addition, 120 Alu Ye4, Ye5 and Ye6 loci were screened using polymerase chain reaction (PCR) assays to determine their phylogenetic origin and levels of human genomic diversity.
Conclusion
The Alu Ye lineage appears to have started amplifying relatively early in primate evolution and continued propagating at a low level as many of its members are found in a variety of hominoid (humans, greater and lesser ape) genomes. Detailed sequence analysis of several Alu pre-integration sites indicated that multiple types of events had occurred, including gene conversions, near-parallel independent insertions of different Alu elements and Alu-mediated genomic deletions. A potential hotspot for Alu insertion in the Fer1L3 gene on chromosome 10 was also identified.
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Background
The proliferation of Alu elements has had a significant impact on the architecture of primate genomes [1]. They comprise over 10% of the human genome by mass and are the most abundant short interspersed element (SINE) in primate genomes [2]. Alu elements have achieved this copy number by duplicating via an RNA intermediate in a process termed retroposition [3]. During retroposition the RNA copy is reverse transcribed by target primed reverse transcription (TPRT) and subsequently integrated into the genome [4-6]. While unable to retropose autonomously, Alu elements are thought to borrow the factors that are required for their amplification from the LINE (long interspersed element) elements [6-9], which encode a protein with endonuclease and reverse transcriptase activity [10,11]. Because of their high copy number, Alu repeats have been a significant source of new mutations as a result of insertion and post-integration recombination between elements [12,13].
The majority of Alu amplification occurred early in primate evolution, and the current rate of Alu retroposition is at least 100 fold slower than the peak of amplification that appears to have occurred 30–50 million years ago [2,14-16]. Even though there are over one million Alu elements within the human genome, only a small number of these elements are capable of movement [17]. As a result of the limited amplification capacity of Alu elements, a series of discrete subfamilies of Alu elements that share common diagnostic mutations have been identified in the human genome [18-21]. A small subset of "young" Alu repeats are so recent in origin that they are present in the human genome and absent from the genomes of non-human primates, with some of the elements being polymorphic with respect to insertion presence/absence in diverse human genomes [16,22-25]. Individual SINE elements have proven to be essentially homoplasy-free characters which are therefore quite useful for resolving phylogenetic and population genetic questions [2,26-34]. For example, young Alu subfamilies which arose around the radiation of Subtribe Hominina (gorillas, chimpanzees, and humans) four to six million years ago [35] were used as homoplasy free phylogenetic markers to resolve the branching order in hominids [36]. Relationships among other primates have also been resolved using relatively large numbers of Alu elements as phylogenetic markers [28,37-40]
We have previously characterized a large number of recently integrated Alu elements found in the human genome that fall in six distinct lineages, termed Ya, Yb and Yc, Yd, Yg and Yi based upon their diagnostic mutations [41-52]. Here, we describe the distribution in the human genome of three Alu subfamilies that are members of the Alu Ye lineage [53] and are characterized by four (Ye4), five (Ye5) and six (Ye6) diagnostic mutations, respectively.
Results
Subfamily size and age
Alu Ye elements were identified in the draft sequence of the human genome using BLAST [54] queries of the draft sequence to identify exact complements to an Alu Ye specific oligonucleotide (Fig. 1). See the Materials and Methods section for details on the search. Using this approach we identified 25 Ye4 subfamily members that shared four diagnostic base positions and thus comprised the Alu Ye4 subfamily. We also identified 103 elements that shared five diagnostic base positions and comprise the Alu Ye5 subfamily and 25 Ye6 subfamily members that shared six diagnostic base positions and comprised the Alu Ye6 subfamily. Each of the subfamilies was named in accordance with standard nomenclature for new Alu subfamilies [55].
Figure 1 Sequence alignment of Alu Ye subfamilies. The consensus sequence for the Alu Y subfamily is shown at the top. The sequences of Alu Ye4, Ye5 and Ye6 subfamilies are shown below. The dots below represent the same nucleotides as the consensus sequence. Deletions are shown as dashes and mutations are shown as the correct base for each of the subfamilies.
To estimate the copy number of the Ye4, Ye5 and Ye6 Alu subfamilies, we preformed BLAST searches of the draft sequence of the human genome using an Alu Ye lineage-specific oligonucleotide to query the database (as outlined in the methods). Seventeen of the 25 Alu Ye4 elements were unique (non-paralogous). There were also 76 unique Ye5 Alu elements and 23 unique Ye6 Alu subfamily members. Multiple alignments of the Alu elements from each subfamily were constructed and the number of mutations from the consensus sequence for each Alu subfamily was determined. In each case the mutations were divided into those that occur at CpG dinucleotides and those that occur at non-CpG positions without including small insertions or deletions as described previously [47-49]. The mutations are divided into these two different classes to estimate the average age of each subfamily because the CpG base positions in repeated sequences mutate at a rate that is about six times higher than non-CpG positions [56] as a result of the spontaneous deamination of 5-methylcytosine residues [57].
Mutation densities were calculated for each Alu Ye subfamily. For 17 elements from the Alu Ye4 subfamily, the non-CpG and CpG mutation densities were 2.1% (83/3944) and 12.5 % (106/850). Using a neutral rate of evolution of 0.15% per million years for non-CpG positions [58] and 0.9% per million years for the CpG base positions [56] along with the average mutation density yields age estimates of 14.03 and 13.86 million years old for the Ye4 subfamily. For the Alu Ye5 subfamily 76 elements were analyzed that contained a total of 17632 non-CpG nucleotides and 3800 CpG nucleotides that contained 351 non-CpG and 431 CpG mutations. The mutation densities of the Ye5 subfamily were 1.99% and 11.34% for the non-CpG and CpG nucleotides yielding age estimates based on the average mutation density of 13.27 and 12.60 million years old. For the Alu Ye6 subfamily 23 elements were analyzed that contained a total of 5336 non-CpG nucleotides and 1150 CpG nucleotides that contained 86 non-CpG and 92 CpG mutations. The mutation densities of the Ye6 subfamily were 1.61% and 8% for the non-CpG and CpG nucleotides yielding age estimates based on the average mutation density of 10.75 and 8.89 million years old.
Evolutionary analysis
In order to determine the approximate time of insertion for each Alu Ye4, Ye5 and Ye6 subfamily member, we performed a series of PCR reactions using human and non-human primate DNA samples as templates. Unfortunately, not all of the loci identified in the draft sequence were amenable to PCR analysis, as some of them had inserted into other repetitive regions of the genome making the design of flanking unique sequence PCR primers difficult.
For the Ye subfamilies, 120 of the 153 elements identified in the draft human genomic sequence were amplified by PCR. Examination of the orthologous regions of the various species genomes displayed a series of different PCR patterns indicative of the time of retroposition of each of the elements into the primate genomes. Results from a series of these experiments showed a gradient of Ye Alu repeats beginning with some elements that are recent in origin and unique to the human genome (e.g. Ye5AH110) and ending with elements that are found within all ape genomes (e.g. Ye5AH148). The distribution of all the Ye elements in various primate genomes is summarized in Additional File 2.
Gene conversion
Gene conversion between Alu elements and in other regions of the human genome exerts a significant influence on the accumulation of single nucleotide diversity within the human genome [2,50]. To estimate the frequency of gene conversion in the Alu Ye subfamily members, we compared the sequences of the elements found in the human genome to the consensus sequences of other Alu subfamilies. Using this approach, we identified two Alu Ye5 subfamily members that appeared to have been subjected to partial gene conversion at their 3' ends. Alu Ye5AH70 contains three mutations that are diagnostic for the Yb8/9 subfamily. Similarly, Alu Ye5AH173 contains three Alu Sc mutations. Each of the sequence exchanges occurred in a short contiguous sequence suggesting that they were products of gene conversion rather than homoplasic point mutations.
We identified one Alu-containing locus that was involved in full gene conversion/ replacement event, (Ye5AH181). In this case, the orthologous Alu elements have similar flanking sequences and direct repeats, although they are not precisely identical due to the random mutations that accumulated over time. DNA sequence analysis of this locus showed that the Alu element of selected new world monkey genomes (spider monkey, woolly monkey and tamarin) belonged to the Alu Sg subfamily. This suggests that a gene conversion of an older, pre-existing Alu Sg may have introduced the Ye5 sequence in the common ancestor of humans, chimpanzees, gorillas and orangutans. Amplification of this locus was unsuccessful in the old world monkey taxa tested.
Alu-mediated genomic deletions
Two deletions of part of the human genome appeared to be associated with newly inserted Alu Ye elements. These deletions were identified at loci Ye5AH24 and Ye5AH27. In the case of Ye5AH24, the deletion was associated with a gene conversion of an Alu Y in both orangutan and siamang to AluYe5 in human, bonobo, common chimpanzee and gorilla and involved the removal of about 500 bp from the 3' flanking region. For Alu Ye5AH27, the deletion was associated with a gene conversion of an Alu Sx element (orangutan and siamang) to AluYe5 (human, bonobo, common chimpanzee and gorilla) and involved the removal of 142 bp from the 3' flanking region. Based on this data, we estimate the frequency of Alu retroposition mediated deletions of approximately 1.67% (2/120).
The pre-integration sites for three elements (Ye5AH11, Ye5AH40 and Ye5AH173) did not amplify in any non-human primate species. Previously, the insertion of L1 elements has been shown to be associated with large genomic deletions [59]. Thus, one possible explanation for the absence of pre-integration PCR products would be that a large deletion (>1 kb) occurred at each of these loci during Alu integration. If a deletion occurred during the integration of an Alu element in the human genome, then the pre-integration product size calculated computationally would be an underestimate of the true size of the locus. To investigate this possibility, we utilized long template PCR reactions of these loci that would facilitate the amplification of larger (up to 25 kb) products. Unfortunately, PCR amplicons were not generated by any of these loci, suggesting that the retrotransposition of these Alu elements in humans may have generated deletions greater than 25 kb in size. Alternately, the orthologous loci in non-human primate genomes may have undergone additional mutations at the oligonucleotide primer sites, preventing PCR amplification.
Independent Alu insertions
We have also identified one locus (Ye5AH161) that contained multiple paralogous Alu insertions in human, chimpanzee, gorilla lineage, old world monkey and new world monkey lineages (Fig. 2). In the human, chimpanzee and gorilla lineage (subtribe Hominina) there was an independent insertion of an Alu Ye5 in the 5' flank of an Alu Sx that is common to all taxa. In all the old world monkey genomes tested (Green monkey, Macaque and Rhesus monkey), an Alu Sp has inserted in the 5' flank of the shared Sx element about 58 bp away of the Alu Ye5 present in Hominina. Also, in the woolly and spider monkeys (new world monkeys), there was an independent insertion of an Alu Sx in the 5' flank of the shared Alu Sx. In gibbon, siamang and orangutan, there were no independent Alu insertions at this locus, only the common Alu Sx is present. In orangutan, however, there was an extra 145 bp of genomic sequences inserted inside the old Alu Sx. The pattern discussed suggests that these three independent parallel insertion events occurred sometime after the divergence of these primates from one another. This locus on chromosome 10q23.33 lies in intron 39–40 of the Human Fer1L3 gene, about 50 bp from exon 39. This locus may be considered a hot spot for Alu insertion. An alignment of locus Ye5AH161 is available as Additional file 1 and at .
Figure 2 Parallel insertions at the Ye5AH161 locus. A) The figure shows an agarose gel chromatograph of the PCR products resulting from amplification at the Ye5AH161 locus in 13 primate species. The ~795 bp PCR product is found in the human, common chimpanzee, pygmy chimpanzee, gorilla, green monkey, Rhesus monkey, macaque, woolly monkey and spider monkey genomes. Smaller bands were found in orangutan, gibbon and siamang. Sequence analysis of the PCR products shows three independent insertions; a Ye5 in subtribe Hominina (human, chimpanzee and gorilla), a second insertion of an Alu Sp in old world monkeys, and an Alu Sx insertion in new world monkeys. Suspected non-homologous recombination has inserted 145 bp in the orangutan genome at this locus. B) A schematic representation of the multiple Alu independent insertions and the distance between the shared Alu Sx and the independently inserted Alu elements. The sequence of Fer1L3-Exon 39 is shown. Silent mutations are highlighted and the distance from the inserted Alus are indicated. Abbreviations used in the figure are: Human (H), Chimpanzee (C), Gorilla (G), Orangutan (O), Gibbon (Gn), Siamang (S), Green monkey (Gm), Rhesus monkey (R), Macaque (M), Woolly monkey (W) and Spider monkey (Sm).
We also identified another near-parallel independent Alu insertion event at human Ye5AH16 locus in all the old world monkey genomes tested (Green monkey, Macaque and Rhesus), within the same locus where an Alu Ye5 element was located in the human, chimpanzee, gorilla and orangutan genomes. Thus, the near-parallel insertion most likely occurred after the divergence of humans and apes from old world monkeys, but before the radiation of the old world monkeys. The element present in the old world monkey genomes is an Alu Y and is 80 bp from the human insertion site.
Human genomic diversity
To determine the human genomic diversity associated with each of the Alu Ye4, Ye5 and Ye6 subfamily members, we performed a series of PCR reactions on a collection of 80 geographically-diverse human genomes. Using this approach, we identified one new Alu insertion polymorphism (Ye5AH167) from the loci analyzed in this report. The allele frequencies, genotypes and heterozygosities for the Alu insertion polymorphism are shown in Table 1.
Table 1 Human genetic diversity of Ye5AD167.
Genotypes
Ye5AD167 +/+ +/- -/- fYe5 Het1
African American 6 8 6 0.50 0.51
Asian 2 16 2 0.50 0.51
European/German Caucasian 3 9 7 0.39 0.49
South American 5 13 1 0.61 0.49
Average Heterozygosity2 0.50
1. Unbiased heterozygosity.
2. The average heterozygosity for all populations.
Discussion
Our detailed analysis of the Alu Ye5 subfamily resulted in the recovery of two new Alu subfamilies, Ye4 and Ye6. Each of these Alu subfamilies has a relatively small copy number in the human genome. The proportion of polymorphic elements within each of the subfamilies is quite low with only 0.83% of the Alu Ye elements being polymorphic, only one member of Ye subfamilies (Ye5AD167) is polymorphic with respect to insertion presence/absence in the human genome. In contrast, many other young Alu subfamilies have levels of insertion polymorphism in excess of 20% [2]. Therefore, the amplification of these Alu subfamilies within the human genome has occurred at a very low rate, and may have recently ceased entirely. The estimated average ages of ~14, ~13 and ~9.5 million years old for the Alu Ye4, Ye5 and Ye6 subfamilies, respectively are consistent with their relatively recent origin in primate genomes. It is also consistent with the master gene model of SINE retroposition which suggests that as a master element accumulates mutations over time, the resulting elements will share those mutations [60].
Members of the Alu Ye lineages are dispersed throughout the genomes of all hominoids (humans, greater and lesser apes) suggesting that this subfamily of Alu elements began to amplify about 15–20 million years ago. Therefore, the Ye subfamily appears to have been retroposition competent during hominoid evolution, but must have been relatively inefficient at producing copies. Although the rate of Ye amplification has not been dramatic within the human lineage, it may be quite interesting to recover Alu Ye subfamily members from other ape genomes and to determine the rate of Ye subfamily amplification in these genomes to see if there has been any differential amplification of these elements in non-human primate genomes. The differential amplification of ID SINEs within various members of the rodent lineage has been reported previously suggesting that the amplification of SINEs within various genomes is subject to changes [61,62].
Gene conversion between Alu repeats has been reported previously [26,63,64]. The gene conversion events involve in three Alu Ye subfamily members were quite interesting. In one case (Ye5AH181), the Alu-containing locus was involved in full gene conversion event where Alu Sg in new world monkeys is replaced by an Alu Ye5 in Humans, chimpanzees, gorillas and orangutan. In the other two cases (Ye5AH70 and Ye5AH173), only a small portion of the 3' end of the Ye elements were involved in the gene conversion. This is in good agreement with the molecular nature of gene conversion events recently reported for the Ya5 and Yb8/9 Alu subfamilies [47,48,64,65]. The detection of three gene conversion events from about 153 Alu Ye elements suggests that gene conversion of these events has been relatively rare, with a rate of 1.96%. However, this rate is comparable to that reported previously for the Alu Ya5 and Yb8 subfamilies within the human genome, as well as that for the Ta subfamily of human LINE elements [64-66].
In all cases, the Ye Alu family members that were involved in the gene conversion were monomorphic for insertion presence within the human genome. In the partial gene conversion events, the Ye Alu repeats were gene converted by Yb8/9 and Sx Alu elements. The Yb8/9 Alu subfamily was one of the first groups of Alu repeats that was ever reported to be involved in gene conversion, and may be more prone to these types of events as a result of a retroposition rate that is slightly higher than other recently integrated Alu subfamilies in the human genome [48,64,65]. The gene conversion between Alu elements may in part be a function of the length of time that the individual Alu elements have resided in the human genome [26,50]. Based on an examination of low copy number transgenes in the mouse, it has been suggested that the germline recombination machinery in mammals has been evolved to prevent high levels of ectopic recombination between repetitive sequences [67]. It is quite possible that the high copy number of Alu elements allows for pairing between regions of sequence identity of different Alu elements initiating the start of gene conversion before cellular control systems can terminate the process resulting in the production of small gene conversion tracts.
The identification of multiple paralogous Alu insertions involving an Alu Ye element (Ye5AH161) in humans, bonobo, common chimpanzee and gorilla lineage, Alu Sp in old world monkeys lineage and Alu Sx in new world monkeys lineage is also interesting. The paralogous insertion of an Alu repeat into the orthologous regions of human and non-human primate genomes is an independent evolutionary event [26]. To date there are no known cases of the independent insertion of paralogous Alu elements into identical sites within different genomes. The detection of parallel insertions is a function of the rate of retroposition of Alu elements within various primate lineages and the time since the most recent common ancestor [26]. However, this locus (Ye5AH161) supports the idea of hotspots for the integration of Alu repeats within primate genomes. Future studies on the integration of different SINE elements in syntenic regions of human and rodent genomes may yield new insight into the molecular nature of hotspots for SINE element integration.
Genomic deletions created upon LINE-1 retrotransposition using cell culture assays have been recently identified [59]. The rate of LINE element deletion was estimated indirectly in the human genome to be about 3% [68] or 8–13% through sequencing variable sizes of the preintegration sites of L1HS in primates [69]. The precise molecular mechanism of the LINE mediated genomic deletions is still unclear. Recently, an Alu-mediated deletion that resulted in the inactivation of the human CMP-N-acetylneuraminic acid hydroxylase gene [70] and Alu mediated deletions of noncoding genomic sequences have been identified [71]. Here we report two new examples of Alu retroposition-mediated deletions that may have happened by a mechanism similar to that of the LINE element mediated genomic deletions since Alu and L1 elements utilize a common mobilization pathway [6,8,72]. In both cases, Alu Ye5AH24 and Alu Ye5AH27, the deletion appears to have occurred, after the separation of human, chimpanzee and gorillas from orangutan and Siamang, during the process of gene conversion similar to the lineage specific Alu deletion reported previously [70,71].
Here, we have estimated the frequency of Alu retroposition associated genomic deletions as approximately 1.67%. The size of the deleted sequences was over 300 bp on average. New Alu integrations have been estimated to occur in vivo at a frequency of one new event in every 10 to 200 births [12]. If sizable deletions accompany one in every 100 new Alu retroposition events in vivo, the genomic impact of these events could be substantial. This is not a trivial number of deletions when extrapolated to the copy number of Alu elements in the human genome which is over one million [2]. Approximately about 16,700 Alu elements may have been involved in retroposition mediated deletion events within primate genomes. If each of these deletion events removes an average of 300 bp of genomic sequence, this would mean that Alu retroposition mediates the deletion of about 5 Mb of the primate genomic sequences. However, if the Alu associated deletions have involved larger sequences similar to those recently reported for LINE elements [59], then the impact of these events may be 50–500 Mb of lineage specific deletions. In either case, these types of events represent a novel mechanism of lineage-specific deletion within the primate order. Detailed studies of the orthologous regions of primate genomes deleted in this manner may prove instructive for understanding the genetic basis of the difference between humans and non-human primates.
Conlcusion
The Alu Ye lineage has had an extended history of expansion in the human lineage. Its expansion appears to have begun soon after the divergence of the hominoids from the remainder of the catarrhine primates and proceeded at a relatively low level since then. Extended periods of relatively low levels of retrotransposition may allow some mobile elements to retain duplication capability for long periods of time. Despite a relatively low level of retrotransposition, the Alu Ye lineage has contributed to the architecture of the human genome through insertion mutations, retrotransposition associated genomic deletions, and gene conversion.
Methods
Computational analysis
To identify Alu Ye elements in the draft sequence of the human genome (August 6, 2001, UCSC GoldenPath assembly), we used Basic Local Alignment Search Tool (BLAST) [54] queries of the draft sequence to identify exact complements to the oligonucleotide 5'- GAACCCCGGGGGGCGGAGCCTGCAG-3' that is diagnostic for the Ye lineage as shown in Fig. 1. All of the exact complements to the oligonucleotide queries along with 1000 bp of adjacent flanking unique DNA sequence were excised and stored as unique files and subjected to additional analysis as outlined previously [47-49]. A complete list of all the Alu elements identified in the searches is located in Additional file 2 and is available at .
DNA samples and PCR amplification
Oligonucleotide primers and PCR amplification reactions for each of the Alu Ye lineage loci analyzed were performed as previously described [47-49] using the primers and annealing temperatures shown in Additional file 2 for Alu Ye lineage members. Diverse human DNA samples were available from previous studies [47-49]. The cell lines used to isolate DNA samples were as follows: chimpanzee (Pan troglodytes), WES (ATCC CRL1609); gorilla (Gorilla gorilla) lowland gorilla Coriell AG05251B, Ggo-1 (primary gorilla fibroblasts) provided by Dr. Stephen J. O'Brien, National Cancer Institute, Frederick, MD, USA; bonobo (Pan paniscus) Coriell AG05253A; orangutan (Pongo pygmaeus) ATCC CRL6301; green monkey (Chlorocebus aethiops) ATCC CCL70 (old world monkey); and owl monkey (Aotus trivirgatus) OMK (OMKidney) ATCC CRL 1556 (new world monkey). Cell lines were maintained as directed by the source and DNA isolations were performed using Wizard genomic DNA purification (Promega). DNA samples from peripheral lymphocytes or tissue were prepared from the gibbon (Hylobates lar) and siamang (Hylobates syndactylus). Additional non-human primate DNA samples (Pan troglodytes, Pan paniscus, Gorilla gorilla, Pongo pygmaeus, Macaca mulatta (old world monkey), Macaca nemestrina (old world monkey), Saquinus labiatus (new world monkey), Lagothrix lagotricha (new world monkey), Ateles geoffroyi (new world monkey) and Lemur catta (prosimian) available as a primate phylogenetic panel (PRP00001) were purchased from the Coriell Institute for Medical Research.
Sequence analysis
DNA sequencing was performed on a gel purified PCR products that had been cloned using the TOPO TA cloning vector (Invitrogen) using chain termination sequencing [73] on an Applied Biosystems 3100 automated DNA sequencer. The sequence of the orthologous loci (that contained a paralogous Alu element) has been assigned accession numbers AY849282-AY849301. Sequence alignments of the Ye lineage subfamily members were performed using MegAlign software (DNAStar version 3.1.7 for Windows 3.2). The ages for each of the Alu Ye subfamilies were calculated using mutation densities as previously described [43,47-49,65] with rates suggested by Xing et al. [56].
Authors' contributions
AS performed all experimental work for the project, shared in the analysis and interpretation of the results and wrote the first draft of the manuscript. DAR provided assistance with analysis and interpretation of the data and in preparing the manuscript for submission. DJH wrote the software used to extract Ye elements and the associated flanking sequences from the human genome draft sequence. JJ provided assistance with the analysis and interpretation of the data and input on late drafts of the manuscript. MAB provided the initial input for the project as well as valuable input on each draft of the manuscript.
Supplementary Material
Additional File 1
This supplemental file represents a sequence alignment for for locus Ye5AH161 in fasta format.
Click here for file
Additional File 2
This supplemental table lists all Alu Ye elements recovered with information on PCR conditions, chromosomal location and phylogenetic origin. It is in Microsoft Word format.
Click here for file
Acknowledgements
This research was supported by Louisiana Board of Regents Millennium Trust Health Excellence Fund HEF (2000-05)-05, (2000-05)-01, and (2001-06)-02 (MAB), National Science Foundation BCS-0218338 (MAB) and EPS-0346411 (MAB) and the State of Louisiana Board of Regents Support Fund (MAB).
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| 15725352 | PMC554112 | CC BY | 2021-01-04 16:37:16 | no | BMC Evol Biol. 2005 Feb 22; 5:18 | utf-8 | BMC Evol Biol | 2,005 | 10.1186/1471-2148-5-18 | oa_comm |
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BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-6-171571590510.1186/1471-2164-6-17Research ArticleProtein domains and architectural innovation in plant-associated Proteobacteria Studholme David J [email protected] J Allan [email protected] Gail M [email protected] The Sainsbury Laboratory, Norwich, NR4 7UH, UK2 Department of Molecular Microbiology, John Innes Centre, Norwich, NR4 7UH, UK3 Department of Plant Sciences, University of Oxford, Oxford, OX1 3RB, UK2005 16 2 2005 6 17 17 18 8 2004 16 2 2005 Copyright © 2005 Studholme et al; licensee BioMed Central Ltd.2005Studholme et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Evolution of new complex biological behaviour tends to arise by novel combinations of existing building blocks. The functional and evolutionary building blocks of the proteome are protein domains, the function of a protein being dependent on its constituent domains. We clustered completely-sequenced proteomes of prokaryotes on the basis of their protein domain content, as defined by Pfam (release 16.0). This revealed that, although there was a correlation between phylogeny and domain content, other factors also have an influence. This observation motivated an investigation of the relationship between an organism's lifestyle and the complement of domains and domain architectures found within its proteome.
Results
We took a census of all protein domains and domain combinations (architectures) encoded in the completely-sequenced proteobacterial genomes. Nine protein domain families were identified that are found in phylogenetically disparate plant-associated bacteria but are absent from non-plant-associated bacteria. Most of these are known to play a role in the plant-associated lifestyle, but they also included domain of unknown function DUF1427, which is found in plant symbionts and pathogens of the alpha-, beta- and gamma-Proteobacteria, but not known in any other organism. Further, several domains were identified as being restricted to phytobacteria and Eukaryotes. One example is the RolB/RolC glucosidase family, which is found only in Agrobacterium species and in plants. We identified the 0.5% of Pfam protein domain families that were most significantly over-represented in the plant-associated Proteobacteria with respect to the background frequencies in the whole set of available proteobacterial proteomes. These included guanylate cyclase, domains implicated in aromatic catabolism, cellulase and several domains of unknown function.
We identified 459 unique domain architectures found in phylogenetically diverse plant pathogens and symbionts that were absent from non-pathogenic and non-symbiotic relatives. The vast majority of these were restricted to a single species or several closely related species and so their distributions could be better explained by phylogeny than by lifestyle. However, several architectures were found in two or more very distantly related phytobacteria but absent from non-plant-associated bacteria. Many of the proteins with these unique architectures are predicted to be secreted.
In Pseudomonas syringae pathovar tomato, those genes encoding genes with novel domain architectures tended to have atypical GC contents and were adjacent to insertion sequence elements and phage-like sequences, suggesting acquisition by horizontal transfer.
Conclusions
By identifying domains and architectures unique to plant pathogens and symbionts, we highlighted candidate proteins for involvement in plant-associated bacterial lifestyles. Given that characterisation of novel gene products in vivo and in vitro is time-consuming and expensive, this computational approach may be useful for reducing experimental search space. Furthermore we discuss the biological significance of novel proteins highlighted by this study in the context of plant-associated lifestyles.
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Background
The Proteobacteria comprise a phylum of Gram-negative bacteria that includes an extraordinary diversity of lifestyles, ecology and metabolism. At one end of a spectrum are free-living organisms such as Pseudomonas aeruginosa, which has a relatively large genome that encodes enormous regulatory and metabolic flexibility, allowing it to colonise diverse niches. At the other extreme are highly specialised intracellular symbionts (Buchnera species, Rickettsia species), whose small genomes have undergone reductive evolution and which lack many common metabolic and regulatory features. With the availability of complete genome sequences for many model plant-associated bacteria, we are particularly interested in how genome analyses can be used to gain insights into the mechanisms and evolution of associations between bacteria and plants.
There are complete annotated genome sequences available for several phylogenetically diverse proteobacterial plant pathogens and symbionts, along with many of their non-pathogenic and non-symbiotic relatives. For example, among the alpha-Proteobacteria, complete genome sequences are available for the phytopathogen Agrobacterium tumefaciens [1-3], the nitrogen-fixing symbionts Bradyrhizobium japonicum [4], Mesorhizobium loti [5] and Sinorhizobium meliloti [6,7], the non-pathogenic free-living Caulobacter crescentus [8], and the animal pathogenic Rickettsia species [9-11]. Ralstonia solanacearum [12] is the sole completely sequenced plant pathogen amongst the beta-Proteobacteria, a division that also includes animal pathogens in the genera Neisseria [13,14] and Bordetella [15] and the free-living chemolithoautotroph Nitrosomonas europaea [16] whose genomes have been sequenced. Among the available complete genome sequences for the gamma-Proteobacteria are those of the plant pathogens Xylella fastidiosa [17,18], Xanthomonas campestris [19], Xanthomonas axonopodis [19] and Pseudomonas syringae pathovar tomato [20] as well as P. aeruginosa [21], which is an occasional pathogen of plants as well as animals.
Each of these three divisions of the Proteobacteria contains a wide variety of different lifestyles, so it is logical to assume that bacteria-plant interactions have evolved independently in multiple separate Proteobacterial lineages. Ultimately the differences between these lifestyles are determined by the organisms' genes acting through their expressed proteins and RNAs. Given the abundance of complete genome sequence data now available, a high priority is to understand which features of an organism's proteome determine its lifestyle, and the evolutionary processes underlying environmental adaptation and evolution of novel traits. Two main sources have been proposed for the evolution and acquisition of novel traits by bacteria: (i) duplication, mutation and recombination of existing genes within a single lineage, and (ii) lateral gene transfer between lineages. A combination of both bioinformatic and experimental studies are needed to determine the relative importance of these two processes in the evolution of plant-associated lifestyles in bacteria.
Evolution of new complex biological behaviours tends to arise (but not exclusively) by novel combinations of existing building blocks. The functional and evolutionary building blocks or units of the proteome are protein domains. Protein domains can be classified into families; examples of widely used classification schemes are those of Pfam [23] and SMART [24]. We hypothesised that systematic identification of proteins having domain architectures that are exclusive to plant-associated bacteria would identify good candidates for proteins with specific involvement in plant-microbial interactions, or in a plant-associated lifestyle, and would also generate insight into the distribution and evolution of novel traits in plant-associated bacteria.
Results and discussion
Hierarchical clustering of completely-sequenced prokaryotic proteomes
To gain an overview of the similarities and differences between their protein domain content, we classified representative prokaryotes into hierarchical clusters based on their complement of protein domain families described. For each proteome we generated a 7,677 binary state element vector where each element represented the presence or absence of one of the 7,677 Pfam protein domain families. Pairwise distances were calculated for each pair of proteomes based on the level of similarity between the pair of vectors, and tree was built by neighbour-joining (see Methods for more details). One hundred trees were built, each time leaving out 10 % of the vector elements, selected at random. The tree shown in Figure 1 represents the consensus of these 100 jacknife trials.
Figure 1 Clustering of complete prokaryotic proteomes based on their protein domain content. 100 jacknife trials were performed, each leaving out a random 10% of the data.
The tree in Figure 1 illustrates the similarities and differences between prokaryotes with respect to their repertoire of recognisable protein domain families. There is clearly a correlation between domain complement and phylogeny; for example, the Archaea form a distinct cluster that is clearly separated from the Bacteria. Furthermore, within the Bacteria, the Cyanobacteria, Gram-positive Bacteria, chlamydias and mycoplasmas each fall into distinct clusters. However, there are some striking discrepancies between the protein domain-based clustering and phylogenetic classification. For example, the oral pathogen Treponema denticola (marked with an asterisk in Figure 1) clusters with the dental bacterium Fusobacterium nucleatum rather than with its fellow spirochetes T. pallidum and Borrelia burgdorferi.
It is notable that the Proteobacteria do not form a single distinct cluster in the protein-domain based classification in Figure 1. The cluster that contains the gamma-proteobacterial Pseudomonas and Xanthomonas species also contains the beta-Proteobacteria R. solanacearum and Chromobacterium violaceum. This probably reflects that these organisms have relatively large genomes and therefore share in common some common protein domains that are not encoded in smaller more streamlined genomes. Conversely X. fastidiosa, which has a relatively small genome, falls into a cluster with Neisseria meningitidis.
Interestingly, the plant pathogen E. caratovora fell into a cluster with Yersinia pestis, Salmonella species and E. coli, which are animal pathogens and commensals. This indicates that despite differing lifestyles, these species have diverged relatively little with respect to loss and gain of protein domain families.
Overall, the results of clustering bacterial proteomes on the basis of their domain content suggested that in addition to phylogeny, an organism's domain repertoire may reflect other factors, possibly including genome size and lifestyle. These preliminary observations led us to investigate whether it is possible to identify any particular domains or domain architectures that may be characteristic of a plant-associated lifestyle.
Protein domain families restricted to plant-associated bacteria
We queried the Pfam 16.0 database to determine the species distribution of each of the 7,677 domain families. Of these, 85 were found in at least one of the completely sequenced plant associated bacteria but absent from all other completely sequenced bacteria. Most of these domain families are restricted to a single species or group of very closely related organisms. For example, domain of unknown function DUF1484 (Pfam:PF07363) appears to be restricted to Ralstonia solanacearum, whilst DUF1520 (Pfam:PF07480) is restricted to Bradyrhizobium japonicum and Sinorhizobium meliloti. Although it is possible that these species-specific domain families are involved in pathogenesis or symbiosis it is equally likely that they have some unrelated function. However, several domains are potentially interesting from the point of view of plant-microbe interactions either because they are found in phylogenetically disparate species of phytobacteria or because they are also found in eukaryotes. Table 1 lists the domain families that are found in plant-associated members of more than one subdivision of the Proteobacteria, but are not found in any non-plant-associated bacteria. Several of these are already implicated in host-plant interactions. For example, proteins belonging to the NolX family (Pfam:PF05819) include HrpF from the gamma-proteobacterium X. campestris and NolX from the alpha-proteobacterium Rhizobium fredii and Rhizobium species NGR234. In these rhizobia, NolX (also referred to as NopX) has been shown to play a role in nodulation specificity and is exclusively expressed during the early stages of interactions with plants [25,26]. NolX is thought to facilitate protein secretion into the plant host via a type III secretion system [27], and a similar role has been postulated for X. campestris HrpF [28]. The importance of members of the NolX family in microbe-plant interactions is reinforced by our observation that they are also found in several other plant-associated alpha- and gamma-Proteobacteria as well as in the phytopathogenic beta-proteobacterium R. solanacearum (see Table 1), but are not found in any other completely sequenced genomes. Similarly, the Avirulence domain (Pfam:PF03377) is restricted to the phytopathogens R. solanacearum and Xanthomonas species [29].
Table 1 Pfam protein domain families found in phylogentically disparate plant-associated bacteria and not found in non-plant associated bacteria.
Pfam domain family Species distribution
Avirulence PF03377 X. avirulence protein, Avr/PthA R. solanacearum; X. axonopodis (pv. citri); X. campestris (pv. citri); X. campestris (pv. vesicatoria); X. campestris; X. manihotis; X. oryzae (pv. oryzae); X. oryzae;
DspF PF06704 DspF/AvrF protein Erwinia amylovora; E. carotovora subsp. atroseptica SCRI1043; Erwinia pyrifoliae; Erwinia stewartii; Pantoea agglomerans (pv. gypsophilae) (Erwinia herbicola); Pectobacterium atrosepticum; P. syringae (pv. tomato); P. syringae;
DUF1427 PF07235 Domain of unknown function A. tumefaciens (strain C58 / ATCC 33970); B. japonicum; P. aeruginosa; R. solanacearum; Rhizobium leguminosarum (biovar trifolii); Rhizobium meliloti (Sinorhizobium meliloti); X. campestris (pv. campestris);
DUF811 PF05665 Domain of unknown function P. aeruginosa; R. solanacearum;
HrpE PF06188 HrpE protein Erwinia amylovora; E. carotovora subsp. atroseptica SCRI1043; Erwinia chrysanthemi; Erwinia pyrifoliae; Erwinia stewartii; Pectobacterium atrosepticum; Pectobacterium carotovorum (subsp. carotovorum) (E. carotovora (subsp. carotovora)); P. fluorescens; P. syringae (pv. glycinea); P. syringae (pv. phaseolicola); P. syringae (pv. savastanoi); P. syringae (pv. syringae); P. syringae (pv. tabaci); P. syringae (pv. tomato); P. syringae;
HrpF PF06266 HrpF protein Erwinia amylovora; E. carotovora subsp. atroseptica SCRI1043; Erwinia chrysanthemi; Erwinia pyrifoliae; Erwinia stewartii; Pectobacterium atrosepticum;Pectobacterium carotovorum (subsp. carotovorum) (E. carotovora (subsp. carotovora)); P. syringae (pv. glycinea); P. syringae (pv. phaseolicola); P. syringae (pv. savastanoi); P. syringae (pv. syringae); P. syringae (pv. tabaci); P. syringae (pv. tomato);
Ice_nucleation PF00818 Ice nucleation protein repeat Bordetella phage BPP-1; Erwinia herbicola; Pantoea ananas (Erwinia uredovora); P. fluorescens; P. syringae (pv. syringae); P. syringae; X. campestris (pv. campestris); X. campestris (pv. translucens);
NolX PF05819 NolX protein R. solanacearum; Rhizobium fredii (Sinorhizobium fredii); Mesorhizobium loti; Rhizobium sp. (strain NGR234); X. axonopodis (pv. citri); X. axonopodis pv. glycines; X. campestris (pv. campestris); X. campestris (pv. vesicatoria); X. oryzae (pv. oryzae);
VirK PF06903 VirK protein A. tumefaciens (strain C58 / ATCC 33970); A. tumefaciens; B. japonicum; P. syringae (pv. tomato); R. solanacearum; Rhizobium sp. (strain NGR234); X. axonopodis (pv.citri); X. campestris (pv. campestris); X. fastidiosa (strain Temecula1 / ATCC 700964); X. fastidiosa;
A further protein family limited to plant-associated bacteria is characterised by the ice nucleation repeat (Pfam:PF00818)and is found in proteins that may have a role in frost damage to host plants. It remains to be seen whether the remaining two domain families (DUF811 and DUF1427) are involved in the plant-associated lifestyle. DUF1427 (Pfam:PF07235) is restricted to several plant-associated alpha-Proteobacteria, the beta-proteobacterium R. solanacearum and the gamma-Proteobacteria P. aeruginosa and X. campestris (Table 1). Although their functions are unknown, proteins containing DUF1427 are thus candidates for involvement in interactions with plants or may at least have a role in plant-associated lifestyles. Several of these proteins have predicted signal peptide sequences and / or predicted transmembrane regions, suggesting an extracytoplasmic location. This may be indicative of a role in extracellular interactions with plants or with other components of the environment. Table 2 lists the 13 protein domain families that appear to be restricted to plant-associated bacteria and to eukaryotes and/or Archaea. Interestingly, this highlights at least one example of a protein domain that has probably been recruited into plant-associated bacteria from a plant host. Proteins containing a RolB/RolC-like domain (Pfam:PF02027) are found to be restricted to plant-associated alpha-Proteobacteria and to plants of the genus Nicotiana (see Table 2 and Figure 2). The activity of these proteins in plants may lead to an increase in intracellular auxin activity caused by the release of active auxins from inactive beta-glucosides [30,31]. The presence of many Agrobacterium-like proteins in Rhizobium (Agrobacterium) vitis reflects another key feature of the biology of these plant-associated bacteria, the fact that many of the genes involved directly in Agrobacterium and Rhizobium- plant interactions are encoded on large plasmids that facilitate lateral gene transfer of complex and novel traits between bacteria. Rhizobium (Agrobacterium) vitis is not a symbiont, but rather causes a tumorigenic disease of grapevine through the action of a number of A. tumefaciens-like genes [32].
Table 2 Pfam protein domain families restricted to plant-associated bacteria and eukaryotes.
Pfam domain family Species distribution (not exhaustive)
CBM_14 PF01607 Chitin binding Peritrophin-A domain Ralstonia solanacearum; Metazoa; Fungi; Viruses
CD225 PF04505 Interferon- induced transmembrane protein Xanthomonas campestris (pv campestris); Metazoa;
DUF726 PF05277 Protein of unknown function (DUF726) Pseudomonas syringae (pv tomato); Metazoa; Plants;
DUF763 PF05559 Protein of unknown function (DUF763) Mesorhizobium loti; Sinorhizobium meliloti; Xanthomonas axonopodis (pv. citri); Xanthomonas campestris (pv. campestris); Archaea;
GDA1_CD39 PF01150 GDA1/CD39 (nucleoside phosphatase) family Pseudomonas syringae (pv. Tomato); Plants; Fungi; Metazoa;
Het-C PF07217 Heterokaryon incompatibility protein Het-C Pseudomonas syringae (pv. tomato); Fungi;
PAX PF00292 'Paired box' domain Rhizobium etli; Mesorhizobium loti; Metazoa;
PPR PF01535 PPR repeat Ralstonia solanacearum; Plants; Metazoa; Fungi;
Rhamnogal_lyase PF06045 Rhamnogalacturonate lyase family Erwinia carotovora subsp. atroseptica SCRI1043; Erwinia chrysanthemi; Plants;
Ribosomal_60s PF00428 60s Acidic ribosomal protein Ralstonia solanacearum (Pseudomonas solanacearum); Plants; Metazoa; Archaea;
RolB_RolC PF02027 RolB/RolC glucosidase family Agrobacterium rhizogenes; Agrobacterium tumefaciens (strain Ach5), and Agrobacterium tumefaciens (strain 15955); Agrobacterium tumefaciens (strain Ach5), and Agrobacterium tumefaciens; Agrobacterium tumefaciens (strain Ach5); Agrobacterium tumefaciens (strain C58 / ATCC 33970); Agrobacterium tumefaciens; Agrobacterium vitis (Rhizobium vitis); Plants;
SBP56 PF05694 56 kDa selenium binding protein (SBP56) Bradyrhizobium japonicum; ; Plants; Metazoa; Archaea;
ST7 PF04184 ST7 protein Rhizobium loti (Mesorhizobium loti); Metazoa;
Figure 2 Examples of proteins containing a RolB/RolC domain.
Protein domain families that are over-represented in plant-associated bacteria
Bacterial physiology and behaviour is determined not only by the presence or absence of particular proteins but also by numbers of representatives of protein families. For example, gene duplication events may lead to a lineage-specific expansion that results in novel orthologues that can take on novel functions different from that of the parent gene. Therefore we investigated whether any protein domain families were over-represented in the plant-associated proteobacteria with respect to the background distribution of domains in all Proteobacteria for which complete sequences were available. For each of the 7,677 Pfam domain families, we counted the numbers of proteins in which that domain family occurs in the complete proteomes of Erwinia carotovora, Pseudomonas syringae pathovar tomato, Ralstonia solanacearum, Sinorhizobium meliloti, Bradyrhizobium japonicum, Mesorhizobium loti, Agrobacterium tumefaciens (Washington strain and Dupont strain), Xanthomonas campestris pathovar campestris, Xanthomonas axonopodis pathovar citri, Xylella fastidiosa and Xylella fastidiosa (strain Temecula1). We then calculated a P value for the probability of observing at least this number of occurrences given the background frequency in the Proteobacteria and assuming a binomial distribution. The smaller the P value, the less likely that the observed frequency occurred by chance. In other words, the smaller the P value, the more over-represented is the domain family. The most over-represented domains are listed in Table 3.
Table 3 Protein domain families over-represented in plant-associated proteobacteria.
Domain family Expected number of proteins Observed number of proteins P
Pfam accesion Pfam ID
PF00211 Guanylate_cyc 33.39 70 2.17E-008
PF00296 Bac_luciferase 46.36 81 2.56E-006
PF04828 DUF636 36.58 65 1.40E-005
PF04679 DNA_ligase_A_C 17.65 38 1.76E-005
PF01068 DNA_ligase_A_M 24.03 47 2.18E-005
PF02738 Ald_Xan_dh_C2 35.72 63 2.33E-005
PF03758 SMP-30 19.35 40 2.63E-005
PF01638 DUF24 37 64 3.51E-005
PF01757 Acyl_transf_3 54.86 87 3.75E-005
PF00067 p450 24.24 46 5.31E-005
PF02746 MR_MLE_N 50.18 80 6.30E-005
PF02894 GFO_IDH_MocA_C 66.35 100 6.97E-005
PF01799 Fer2_2 31.26 55 7.69E-005
PF06169 DUF982 11.06 26 8.88E-005
PF07536 HWE_HK 23.82 44 1.35E-004
PF01022 HTH_5 68.47 101 1.38E-004
PF03573 OprD 14.03 30 1.41E-004
PF00656 Peptidase_C14 14.89 31 1.73E-004
PF03459 TOBE 83.78 139 2.48E-004
PF02627 CMD 51.89 79 2.75E-004
PF01188 MR_MLE 56.78 85 2.79E-004
PF07506 RepB 10.84 24 3.81E-004
PF01261 AP_endonuc_2 85.48 122 4.91E-004
PF00150 Cellulase 11.06 24 4.97E-004
PF01408 GFO_IDH_MocA 85.7 130 5.36E-004
PF00941 FAD_binding_5 21.05 38 5.58E-004
PF01315 Ald_Xan_dh_C 29.35 49 5.60E-004
PF00353 HemolysinCabind 36.15 57 8.12E-004
PF06823 DUF1236 8.93 20 9.64E-004
The domain with the statistically most significant over-representation in the plant-associated bacteria was the guanylate cyclase domain (Pfam:PF00211). This domain was particularly abundant in B. japonicum (32 proteins) and S. meliloti (24 proteins). No other fully-sequenced proteobacterium encodes more than three, although the spirochaete Leptospira interrogans encodes 17 proteins matching PF00211). Cyclic-diGMP, the product of guanylate cyclase, is a secondary messenger that plays a role in cell-cell and cell-surface contact in several bacteria by regulating cellular adhesion genes [33]. Such interactions are very important in initiating bacterial infection of eukaryotic organisms and this may account in part for the high numbers of such domains in these plant-associated bacteria. Of particular interest is the observation that one response regulator from C. crescentus has been shown to become sequestered to the cell pole following phosphorylation [35]. This is coupled to the activation of the guanylate cyclase domain, suggesting that localised synthesis of this secondary message could induce local effects within specific regions of the bacterial cell.
Another domain with statistically significant over-representation in the plant-associated bacteria was the bacterial luciferase-like monooxygenase domain (Pfam:PF00296). This domain was particularly abundant in the plant-associated alpha-Proteobacteria with 15 proteins in Agrobacterium tumefaciens, 11 proteins in B. japonicum and 9 proteins in M. loti containing this domain. The related alpha-Proteobacteria C. crescentus, B. melitensis, B. suis and Rhodopseudomonas palustris have 3, 2, 2 and 0 luciferase (PF00296) proteins respectively. Other species containing large numbers of luciferase-like proteins include Mycobacterium bovis (13 proteins) and M. tuberculosis (14 proteins).
Several domains of unknown function are amongst those most over-represented in the phytobacteria. For example, DUF636 is unusually abundant in the rhizobia with 16 representative proteins in B. japonicum and 14 and 13 in M. loti and S. meliloti respectively. Other prokaryotes encode between 0 and 5 DUF636 proteins, whilst Arabidopsis thaliana and Homo sapiens each encode one.
Domain architectures
The functionality of the proteome depends not only on the repertoire of protein domains but also on the interactions and cellular context of those domains. One important aspect of this context is the range of combinations of domains within a protein; that is the domain architecture of proteins.
We used the Pfam database to ascertain the domain architecture of every protein sequence from each bacterial species for which a complete annotated genome sequence was available. 3,774 distinct protein domain architectures were found in R. solanacearum, P. aeruginosa, E. carotovora (subspecies atroseptica), P. syringae (pathovar tomato), B. japonicum, S. meliloti, M. loti, A. tumefaciens, X. fastidiosa, X. campestris, X. axonopodis. 459 of the 3,774 domain architectures encoded in genomes of plant-associated bacteria were absent in all other bacteria for which complete genome sequences were available. These 459 architectures are listed in the supplementary data. However, many of these architectures were restricted to a single species or several closely related species and so were of limited interest for this study.
We were particularly interested to discover whether any domain architectures are related to plant-associated lifestyle rather than simply resulting from phylogeny. The 15 protein architectures illustrated in Table 4 were each found in plant-associated bacteria from at least two different divisions of the Proteobacteria and were not found in any other non-plant-associated organisms. For example, polypeptide sequences consisting of an N-terminal domain of unknown function DUF442 fused to a metallo-beta-lactamase domain are restricted to A. tumefaciens, M. loti, S. meliloti, X. fastidiosa and X. fastidiosa.The metallo-beta-lactamase domain (Pfam:PF00753) is common and widespread, being found in over 2000 different proteins from a wide range of organisms. However, only in these proteins from plant-associated bacteria is the metallo-beta-lactamase domain fused to DUF442. This suggests that the catalytic domain may have been recruited to some new function connected to a plant-associated lifestyle in these bacteria.
Table 4 Domain architectures found in phytobacteria of two or more subdivisions of the Proteobacteria and not found in non-plant-associated bacteria.
Domain architecture Species distribution Proteins
DUF763 Aeropyrum pernix; Archaeoglobus fulgidus; Bradyrhizobium japonicum; Methanobacterium thermoautotrophicum; Methanopyrus kandleri; Picrophilus torridus; Pyrobaculum aerophilum; Pyrococcus abyssi; Pyrococcus furiosus; Pyrococcus horikoshii; M. loti; S. meliloti; Sulfolobus solfataricus; Sulfolobus tokodaii; Thermoplasma acidophilum; Thermoplasma volcanium; X. axonopodis (pv. citri); X. campestris (pv. campestris); Hypothetical protein XCC1094. (Q8PBM5); Hypothetical protein XAC1190. (Q8PN83); Hypothetical protein APE1824. (Q9YAX1); Hypothetical protein ST0586. (Q974S6); Hypothetical protein PF0611. (Q8U361); Hypothetical protein. (Q97VZ2); Hypothetical protein PH0745. (O58515); Hypothetical protein SMb21455. (Q92U57); Hypothetical protein. (Q9UZ46); Mlr6856 protein. (Q987Y3); Bll3834 protein. (Q89NK4); Uncharacterized conserved protein. (Q8TYA4); Hypothetical protein PAE0766. (Q8ZYH9); Hypothetical protein TVG0468151. (Q97BH6); Hypothetical protein Ta1095. (Q9HJ77); Hypothetical protein AF1496. (O28776); Hypothetical protein. (Q6L1J8); Hypothetical protein MTH448. (O26548); Hypothetical protein MTH449. (O26549);
VirK A. tumefaciens (strain C58 / ATCC 33970); A. tumefaciens; Bradyrhizobium japonicum; P. syringae (pv. tomato); R. solanacearum; Rhizobium sp. (strain NGR234); X. axonopodis (pv. citri); X. campestris (pv. campestris); X. fastidiosa (strain Temecula1 / ATCC 700964); X. fastidiosa; VirK (Tiorf135 protein). (O50246*); VirA/G regulated gene. (Q7CNV8); Hypothetical 15.8 kDa protein in pinF2 3'region (ORF2). (Q44433*); Hypothetical 15.6 kDa protein y4WH. (P55686*); PUTATIVE SIGNAL PEPTIDE PROTEIN. (Q8XX33*); VirK protein. (Q8PDC2*); VirK protein. (Q8PQ93); ID299. (Q9ANE2*); Blr1847 protein. (Q79UP9); VirK protein. (Q87D31); VirK protein. (Q9PC40*); Hypothetical protein. (Q880Z8);
DUF1427 A. tumefaciens (strain C58 / ATCC 33970); Bradyrhizobium japonicum; P. aeruginosa; R. solanacearum; Rhizobium leguminosarum (biovar trifolii); S. meliloti; X. campestris (pv. campestris); Hypothetical protein XCC2052. (Q8P914); Bsl6958 protein. (Q89EW2); Hypothetical protein. (Q93EB2); HYPOTHETICAL TRANSMEMBRANE PROTEIN. (Q8Y2U1*); AGR_L_1747p. (Q8U4X9*); Hypothetical protein. (Q92Y85); Bsr4258 protein. (Q89MD5); Hypothetical protein. (Q9I0E5*);
DUF1486 A. tumefaciens (strain C58 / ATCC 33970); Neurospora crassa; P. aeruginosa; P. syringae (pv. tomato); R. solanacearum; M. loti; S. meliloti; Hypothetical protein. (Q7SFH5); Hypothetical protein Atu3018. (Q8UBJ8); Hypothetical protein. (Q92YL1); Mlr2224 protein. (Q98IW1); Hypothetical protein. (Q9I3U3); Hypothetical protein. (Q9JP27); AGR_L_3571p. (Q7CRD4); Hypothetical protein RSc0819. (Q8Y171);
RepB A. tumefaciens (strain C58 / ATCC 33970); P. syringae (pv. tomato); M. loti; S. meliloti; Msr9757 protein. (Q98P91); Mll8115 protein. (Q983Y2); Hypothetical protein. (Q88BH6); Hypothetical protein Atu5040. (Q8UKR0); AGR_pAT_52p. (Q7D423); Hypothetical protein. (Q92XS2); Hypothetical protein. (Q930E6); Hypothetical protein. (Q930E5);
DUF442~Lactamase_B A. tumefaciens (strain C58 / ATCC 33970); M. loti; S. meliloti; X.fastidiosa (strain Temecula1 / ATCC 700964); X. fastidiosa; Metallo-beta-lactamase superfamily protein. (Q8UAA9); Hypothetical protein. (Q92ZB8); AGR_L_2726p. (Q7CSJ2); Hypothetical protein. (Q87AD6); Mlr2158 protein. (Q98J12); Hypothetical protein. (Q9PFB0);
GAF~Phytochrome Bradyrhizobium sp. ORS278; X. axonopodis (pv. citri); Phytochrome-like protein. (Q8PEQ2); Bacteriophytochrome. (Q8VUB6);
Glyco_hydro_6~CBM_2 Microbispora bispora; Micromonospora cellulolyticum; R. solanacearum; Thermomonospora fusca; X. fastidiosa (strain Temecula1 / ATCC 700964); X. fastidiosa; Cellulose 1,4-beta-cellobiosidase. (Q87E00); 1,4-beta-cellobiosidase. (Q9PDW2); PROBABLE EXOGLUCANASE A (1,4-BETA-CELLOBIOSIDASE) PROTEIN (EC3.2.1.91). (Q8XS97); Endoglucanase A precursor (EC 3.2.1.4) (Endo-1,4-beta-glucanase) (Cellulase). (P26414*); Endoglucanase E-2 precursor (EC 3.2.1.4) (Endo-1,4-beta-glucanase E-2)(Cellulase E-2) (Cellulase E2). (P26222*); Endo-beta-1,4-glucanase. (Q53488);
DUF811 P. aeruginosa; R. solanacearum; Hypothetical protein. (Q9I6E4*); Hypothetical protein. (Q9I6E5*); Hypothetical protein RSc3082. (Q8XUV1);
Condensation~Condensation~AMP-binding~PP-binding~Condensation~AMP-binding~PP- binding~Condensation~AMP-binding~PP- binding~Condensation~AMP-binding~PP- binding~Condensation~AMP-binding~PP- binding~Thioesterase~Thioesterase P. syringae (pv. tomato); R. solanacearum; Probable peptide synthesis protein. (Q8XS39); Non-ribosomal peptide synthetase, terminal component. (Q881Q3);
NolX R. solanacearum; Rhizobium fredii (Sinorhizobium fredii); M. loti; Rhizobium sp. (strain NGR234); X. axonopodis (pv. citri); X. axonopodis pv. glycines; X. campestris (pv. campestris); X. campestris (pv. vesicatoria); X. oryzae (pv. oryzae); HrpF protein. (Q8PBA6); HrpF protein. (Q8PQD2); HrpF. (Q83XD5); HrpF. (O33967); HrpF. (Q6F5A9); HrpF. (Q9KW22); Type III secretion system component. (Q6QJ83); SECRETED PROTEIN POPF2. (Q8XRF4); SECRETED PROTEIN POPF1. (Q8XPT2); Nodulation protein; NolX. (Q989P8); Nodulation protein nolX. (P55711); Nodulation protein NolX. (Q93LZ2); Nodulation protein NolX. (Q9EUG7); Nodulation protein nolX. (P33213);
DUF802~DUF802 R. solanacearum; X. axonopodis (pv. citri); Hypothetical protein XAC3753. (Q8PG64*); Probable transmembrane protein (Q8XQ05*);
Avirulence~Avirulence R. solanacearum; X. axonopodis (pv. citri); X. campestris (pv. citri); X. campestris (pv. vesicatoria); X. campestris; X. oryzae (pv. oryzae); X. oryzae; Avirulence protein AvrXa7-3M. (Q6GWX1); Avirulence protein AvrXa7-1M. (Q6GWX7); Avirulence protein. (Q9EZV3); Avirulence protein AvrXa7-4M. (Q6GWX4); Avirulence protein. (Q9F0D0); Hypothetical 122 kDa avirulence protein in avrBs3 region. (P14727); AvrBs3-2 protein. (Q07061); PROBABLE AVRBS3-LIKE PROTEIN. (Q8XYE3); Apl3 protein. (Q9Z3F5); Avirulence protein. (Q8PRG7); PthA protein. (Q56780); Apl1 protein. (Q9R7J3); Avirulence protein AvrXa7-2M. (Q6GWX3); Avirulence protein. (Q8PRN6); Avirulence protein AvrXa10. (Q56830); PthB. (Q7X130); Apl2 protein. (Q9Z3F6); Avirulence protein. (Q8PRM3); Avirulence protein. (Q8PRK7);
RgpF-RgpF M. loti; Rhizobium sp. (strain NGR234); X. axonopodis (pv. citri); X. campestris (pv. campestris); Mll4799 protein. (Q98D97); Hypothetical protein XAC3576. (Q8PGP0); Hypothetical protein wxcX. (O34262); Hypothetical 45.0 kDa protein y4gN. (P55470);
TPR_2~TPR_1~Sulfotransfer_1 M. loti; X. axonopodis (pv. citri); uncultured bacterium 560; TPR domain/sulfotransferase domain protein. (Q6SGF7); Mlr4028 protein. (Q98EY4); Hypothetical protein XAC3051. (Q8PI47);
One regulatory domain found in large numbers in Pseudomonas genome is the PAS domain (Pfam PF00989) [36], which is present in 25 ORFs in P. aeruginosa PAO1 and 30 ORFs in P. syringae pathovar tomato. The average number of PAS-containing ORFs in complete proteobacterial genomes is about 10. Although PAS domains are only found in a limited subset of bacterial regulators, they are at the forefront of molecular innovation with 9 of the novel architectures identified in P. aeruginosa, and 5 of those in P. syringae pathovar tomato containing PAS domains (see supplementary data for more details). Xanthomonas genomes also encode a large number of PAS-containing polypeptides, (18 and 21 in X. axonopodis and X. campestris respectively). However, each X. fastidiosa encodes only one: PhoR, a regulator generally associated with responses to phosphate limitation. Ten novel PAS architectures are present in each Xanthomonas genome, of which 7 are common and 3 are unique to each strain (some of which are illustrated in Figure 3). PAS domains, which are involved in sensing light, oxygen and other environmental factors, have particular importance in helping bacteria to adapt to a changing environment, an ability of little value to X. fastidiosa in its restricted and relatively constant niches.
Figure 3 Examples of proteins containing phytochrome domains.
One intriguing signal transduction domain identified in unique domain architectures from both P. syringae and Xanthomonas was a phytochrome domain (Pfam:PF00360) (Figure 4). This domain enables light-mediated signal transduction in plants and bacteria, through binding a light-sensitive chromophore [37,38]. Phytochrome-containing proteins are used to detect light, and to discriminate between different wavelengths of light. Phytochromes are used for shade avoidance by plants, and to detect depth in soil or water or other conditions where light is attenuated. The short list of bacteria that contain phytochromes includes photosynthetic species (e.g. Rhodospirillum centenum, Anabaena species strain PCC7120 and Synechocystis species strain PCC6803) as well as plant associated bacteria (e.g. R. leguminosarum, A. tumefaciens) and soil bacteria (e.g. P. putida) [38,39]. An unusual photosynthetic strain, Bradyrhizobium species ORS278 uses phytochrome to regulate the photosynthesis gene cluster and a similar induction was seen with Rhodopseudomonas pallustris but not with several other photosynthetic bacteria [40]. It is not known why phytochrome proteins are retained in non-photosynthetic bacteria but it has been suggested that the phytochrome-like sensor kinases in Agrobacterium may play a role in detecting depth in soil strata as a means of optimising interactions with roots [39]. Most of the bacterial phytochrome proteins have a PAS domain and a GAF domain at the N-terminus and a histidine kinase domain at the C-terminus (see Figure 4), though a phytochrome from Rhodobacter sphaeroides (UniProt:Q8VRN4; see Figure 4) has a more complex domain architecture [40]. The presence of two phytochromes in P. syringae, one of them with a unique architecture, may reflect the recruitment of phytochrome to a novel regulatory function unique to P. syringae. Protein PSPTO2652 from P. syringae is unique in that it has an additional C-terminal histidine kinase. Another unusual domain architecture is the PAS-GAF-Phytochrome-PAS organisation found in Xanthomonas proteins XAC4293 and XCC4154 (Figure 4), which, if shown to be functional, may represent a new phytochrome protein family.
Figure 4 Examples of proteins containing phytochrome domains.
Further analysis of novel Pseudomonas protein domain architectures
The availability of multiple finished and unfinished Pseudomonas genomes allowed us to study in more detail the distribution, genomic context and properties of Pseudomonas gene products highlighted by this analysis. Closer examination of the genomic context of the P. syringae genes encoding proteins with unusual domain architectures showed that most were flanked on either or both sides by genes that have few or no orthologues in other Pseudomonas strains, suggesting that these novel genes have been recruited simultaneously with other genes, possibly of related function, or that they have recombined into the genome at hotspots for recombination and insertion of alien DNA.
To further address the hypothesis that at least some of these architectures have been acquired by horizontal gene transfer we examined the GC content and third position GC content of each of these genes, in comparison to the total genome (0.593 GC, 0.716 GC3). Sixteen of the genes deviated from the average GC3 content by more than 0.05. High GC3 content genes include pvsA, PSPTO4084, PSPTO2413 and cfa6. Low GC3 content genes include hrpZ, PSPTO3210, glf, PSPTO4696, hopPtoS(1,2 & 3), PSPTO2259, PSPTO0400, avrF and PSPTO1070. The GC content of flanking genes frequently reflected that of the novel gene, most strikingly for glf, PSPTO2441, PSPTO4696, hopPtoS(1,2 &3), PSPTO4699, PSPTO1070 & PSPTO2632, which were each associated with low GC regions containing few ORFs with orthologues in other Pseudomonas genomes.
One other feature frequently associated with horizontally transferred genes is the presence of IS elements, tRNAs, plasmid and phage genes in flanking regions. PSPTO3229, PSPTO4569, PSPTO2312, PSPTO2829, PSPTO2310, Glf, PSPTO2441, PSPTO4696 and PSPTO2326 are all located in close proximity to IS elements and phage-like sequences, or in defined regions of the genome flanked by IS elements and phage-like sequences (see Figure 5).
Figure 5 Genetic islands unique to Pseudomonas syringae. Genes encoding transposases are marked with an asterisk (*) and the asparaginyl tRNA gene is marked 'tAsn'. Black diamonds indicate genes encoding unique domain architectures [49].
Overall, this analysis suggests that a large number of the novel architectures present in P. syringae pathovar. tomato are uniquely associated with this species or pathovar of Pseudomonas, and that many of these genes have been acquired by horizontal gene transfer and are located in regions of the genome with a high potential for recombination and rearrangement.
Conclusions
Our initial observations, from the clustering of complete prokaryotic proteomes on the basis of domain content, motivated us to test whether any protein domains or domain architectures are specifically associated with a plant-associated lifefstyle. We identified nine protein domain families that are found in phylogenetically diverse plant-associated bacteria but not in non-plant-associated Bacteria (Table 1). Inevitably, there is an element of random chance in the species distribution of domain families; however, we observed that most of domains whose functions are at least partly known are implicated in the plant associated lifestyle. Therefore it seems possible that the two domains of unknown function (DUF811 and DUF1427) may also turn out to be significant for this lifestyle. Several domain families were also found only in plant pathogenic bacteria and in eukaryotes (Table 2). For example the RolB/RolC-like domain family is restricted to plant-associated bacteria and to plants of the genus Nicotiana, and is implicated in modulating auxin activity.
Having investigated patterns of presence or absence of domains within bacterial proteomes, we next identified which domains are most over-represented in the plant-pathogenic Proteobacteria as compared with the frequency of occurrence in all the sequenced Proteobacteria (Table 3). Amongst the most over-represented domains was the guanylate cyclase domain. This was largely due to the large number of guanylate-cyclase-like proteins encoded by B. japonicum and S. meliloti. Although this approach may have revealed some potential leads for further investigation, it should be remembered that this analysis was rather crude and susceptible to the biased phylogenetic distribution of the organisms for which complete genome sequence data are currently available. However, detailed analysis of the frequency distributions of protein domain families in various organisms may yield rewards.
As well as the repertoire of domains, another important aspect of a proteome is the repertoire of domain architectures; that is the combinations of domains found within a single protein. Just as for the repertoire of domains, the species distribution of a domain architecture might be explained by chance. Nevertheless, the proteins listed in Table 4 may be a good starting point for further investigation of bacterium-plant interactions.
Many of these protein identified in this study have N-terminal predicted signal peptide motifs, suggesting that they are secreted. Further experiments are required to determine whether proteins of unknown function will also have a role in plant-specific functions. Many proteins involved in bacteria-plant interactions, such as TTSS-secreted effectors have subtle or conditional phenotypes, and would not be identified in conventional mutant-phenotype screens. Assays to detect subtle differences in growth in planta or in disease development are labour-intensive. Bioinformatic analyses such as this one represent useful and informative tools for reducing experimental search space, particularly when combined with other post-genomic techniques such as microarray analyses.
We found relatively little evidence of lateral dissemination of niche-specific novel architectures between phylogenetically distinct divisions in the Proteobacteria, with less than 20 phytobacteria-specific domain architectures present in two or more divisions of the Proteobacteria. We did identify a number of domain architectures and domains that were uniquely conserved in both plant-associated prokaryotes and eukaryotes. The methodology used in this study makes no prior assumptions about the nature or cause of "uniqueness". Unique architectures identified using this approach include rare domains, novel domain combinations and architectures that are truncated relative to the majority of similar proteins (which may represent deletions and loss of function mutations). Some proteins will inevitability be included or excluded because of the limitations of current domain prediction technology. However, in addition to identifying protein candidates for further investigation, this type of analysis can be used to challenge and improve current models for domain prediction and expose errors and limitations of genome sequence data and protein prediction. For example, consider a case in which a protein is identified as having the "unique" architecture B~C~D. Additional examination of the protein may reveal that the protein has a similar sequence to proteins with the architecture A~B~C~D. The absence of the A domain may indicate a genuine alteration in structure and potentially in function, or a frameshift in the genome sequence data, or a functional "A" domain that fails to meet current predictive criteria. Each of these hypotheses can be tested by further research and experimentation, both in silico and in the lab.
Although our approaches to identifying candidate genes and proteins of significance to lifestyle have led to several potential leads and interesting hypotheses, there are some caveats. Firstly, evolution does not proceed exclusively through loss and gain of domains and domain shuffling; for example, protein innovation can also occur through mutation and divergence within domain families. Also, it is becoming increasingly apparent that an organism's physiology, behaviour and ecology depend as much on higher order 'systems level' phenomena as on the inventory of molecular components.
We chose to base our surveys of protein domains on the Pfam because this mature database is relatively comprehensive in its coverage (e.g. compared with SMART) and its data is of high quality. Furthermore, its data is distributed in a form that is ideally suited for constructing database queries such as those in this study. Another advantage is that in Pfam no two domains ever overlap in their coverage of a protein sequence, which significantly simplifies the analysis. However, it should be noted that Pfam is not absolutely infallible and some of its threshold values are rather stringent, leading to failure to identify some 'outlying' members of a domain family.
In summary, this study has described and applied a new approach for identifying architectural innovation and potentially important domains in proteins from genome sequence data. The data generated in this study have highlighted a large number of interesting and largely uncharacterised novel proteins and suggested new insights into the molecular basis of interactions between bacteria and their plant hosts, which will provide inspiration for future experimental research.
Methods
The Pfam relational database data files were downloaded from the Pfam website [46]. The census of domains and architectures were taken from Pfam release 16.0 (November 2004) using custom PERL scripts to wrap SQL queries against the Pfam relational database.
The complete bacterial genomes included in Pfam 16.0, and hence considered in this study, are listed in the supplementary data. We excluded from the analysis of domain architectures all protein sequences in UniProt [47] that are designated as fragments.
A file listing the presence or absence of each Pfam domain in each proteome can be found in the supplementary data. Each row in this file represented a vector used for the clustering of bacterial proteomes. Neighbour-joining was performed using PHYLIP [41]. Trees were visualised using ATV [51].
BLAST [42] searches were performed using the NCBI [48] and Expasy [49] web servers. Comparison between Pseudomonas genomes was aided by use of PseudoDB [50]. Transmembrane and signal peptide predictions were taken from Pfam, which in turn uses TMHMM [45] and SignalP [43]. It should be remembered that predictive methods often have difficulty distinguishing between signal peptides and N-terminal transmembrane helices [44].
Authors' contributions
DJS and GMP conceived the original study, carried out the bioinformatics analyses, and drafted the manuscript. JAD proposed extending the study to symbionts as well as pathogens. All the authors contributed to interpretation of the data and to writing the final manuscript.
Supplementary Material
Additional File 1
This table lists the 459 domain architectures that are found in one or more plant-associated bacteria but are absent from other bacteria for which complete sequence data is available.
Click here for file
Additional File 2
Prokaryotic genomes included in Pfam16.0 (and hence in this study).
Click here for file
Additional File 3
"domains.tab.gz" Species distribution of each of the 3,774 Pfam domains. This tab-delimited file has been compressed using gzip.
Click here for file
Acknowledgements
DJS is grateful to Lachlan Coin for early discussions about clustering of proteomes and over-representation of domains, which contributed to the conception of this work.
We thank Ray Dixon for helpful discussion. We are also indebted to the Pfam team for making their data readily available. Research at the Sainsbury Laboratory is funded by the Gatsby Charitable Foundation.
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| 15715905 | PMC554113 | CC BY | 2021-01-04 16:39:32 | no | BMC Genomics. 2005 Feb 16; 6:17 | utf-8 | BMC Genomics | 2,005 | 10.1186/1471-2164-6-17 | oa_comm |
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BMC PsychiatryBMC Psychiatry1471-244XBioMed Central London 1471-244X-5-111572536110.1186/1471-244X-5-11Research ArticleThe Bosnian version of the international self-report measure of posttraumatic stress disorder, the Posttraumatic Stress Diagnostic Scale, is reliable and valid in a variety of different adult samples affected by war Powell Steve [email protected] Rita [email protected] Department of Psychology, Ludwig-Maximilians-University, Leopoldstr. 13, 80802 Munich, Germany2005 23 2 2005 5 11 11 11 10 2004 23 2 2005 Copyright © 2005 Powell and Rosner; licensee BioMed Central Ltd.2005Powell and Rosner; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The aim of the present study was to assess the internal consistency and discriminant and convergent validity of the Bosnian version of a self-report measure of posttraumatic stress disorder (PTSD), the Posttraumatic Stress Diagnostic Scale (PTDS). The PTDS yields both a PTSD diagnosis according to the Diagnostic and Statistical Manual of Mental Disorders 4th edition (DSM-IV) and a measure of symptom severity.
Methods
812 people living in Sarajevo or in Banja Luka in Bosnia-Herzegovina, of whom the majority had experienced a high number of traumatic war events, were administered the PTDS and other measures of trauma-related psychopathology. The psychometric properties of the instrument were assessed using Cronbach's alpha and principal components analysis, and its construct validity was assessed via Spearman correlation coefficients with the other instruments.
Results
The PTDS and its subscales demonstrated high internal consistency. The principal components revealed by an exploratory analysis are broadly consistent with the DSM-IV subscales except that they reproduce some previously reported difficulties with the "numbing" items from the avoidance subscale. The construct validity of the PTDS was supported by appropriate correlations with other relevant measures of trauma related psychopathology.
Conclusion
The Bosnian version of the PTDS thus appears to be a time-economic and psychometrically sound measure for screening and assessing current PTSD. This self-report measure awaits further validation by interview methods.
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Background
To obtain a diagnosis of PTSD and an estimation of PTSD severity a wide range of measures either relying on interviews or self-report exist in many languages. However, most of the relevant validation studies for these instruments were carried out for English-language versions [1]. For many languages, validated instruments do not exist. A standard approach in this situation is to translate one of those English-language instruments which are well validated, to carry out a validation study for the translation and to compare the results of the validation study with the studies for the original.
Self-report instruments have several advantages as compared to interview measures. They are relatively economic in terms of administration and demand minimal clinician time. If clinicians are not familiar with psychiatric diagnostic procedures and especially the clinical diagnosis of PTSD, it is more advisable to use a psychometrically sound self-report measure which is less prone to mistakes than interview measures.
A good self-report measure for PTSD should allow a diagnosis of PTSD as well as an estimation of PTSD severity and should conform to the DSM-IV criteria for PTSD [2]. The English version of the Posttraumatic Stress Diagnostic Scale [PTDS; [3]] fulfils these criteria and has been shown to have adequate psychometric properties. The PTDS has been translated into a German version which also has adequate psychometric properties [4]. These two different language versions of the PTDS have been used in numerous studies [e.g. [4-9]]. Table 1 provides an overview over the internal consistency and the test-retest-reliability of the PTDS as published in the literature.
Table 1 Internal consistency and test-retest reliability of the PTDS and its DSM-III-R precursor the PSS-SR
Authors Samples Scales Cronbach's alpha Test-retest reliability
Foa, Riggs, Dancu, & Rothbaum (1993) [8] 44 women (rape and other non-sexual attack) Total score .91 .74 after one month (N = 29)
5 – 6 weeks after the event Reexperiencing .78 .66
Avoidance .80 .56
Hyperarousal .82 .71
Engelhard et al (2001) [7] 113 women after miscarriage Total score .87
Stieglitz, Frommberger, Foa, & Berger (2001) [9] 152 persons: Total score .85 and .86 .60 after six months
1. time point: a few days after accident Reexperiencing .75 and .82 .39
Avoidance .56 and .74 .53
2 time point: 6 months later Hyperarousal .75 and .64 .47
Foa, Cashman, Jaycox, & Perry (1997) [3] 284 victims of various traumatic experiences Total score .92 .83 (approx 2 weeks later)
Reexperiencing .78 .77
Avoidance .84 .81
Hyperarousal .84 .85
Kappa = .74 for PTSD diagnosis
In terms of convergent validity, Foa, Riggs, Dancu, and Rothbaum [8] compared Posttraumatic Symptom Scale scores (PSS-SR; the DSM-III-R version of the PTDS) with the diagnosis obtained by administering the Structured Clinical Interview for the DSM-III-R [SCID;[10]]. 86 % of the participants with a PTSD diagnosis according to DSM-III-R criteria were correctly identified with the self-report instrument. The sensitivity was 62% and the specificity 100%. The DSM-IV version of the PTDS achieved a sensitivity of .89 and a specificity of .75. Percentage agreement between SCID and PTDS diagnosis was 82 % and kappa was .65. Overall, the criterion validity of the PTDS with respect to SCID was encouraging. Table 2 provides an overview of convergent and divergent validity for the PTDS and some other self-report measures of trauma related psychopathology.
Table 2 Convergent and divergent validity of the PTDS and its DSM-III-R precursor PSS-SR
Authors PSS/PTDS Scales IES Total score IES Intrusion IES Avoidance BDI
Foa et al. (1993) [8] PSS Total score .81 .53 .80
Reexperiencing .81 .47 .66
Avoidance .71 .52 .73
Hyperarousal .70 .45 .75
Stieglitz et al. (2001) [9] PSS Total score .67 & .65 .61 & .57 .61 only at first measurement (a few days after the accident)
Reexperiencing .63 & .59 .53 & .47 .45
Avoidance .56 & .55 .50 & .51 .50
Hyperarousal .52 & .49 .47 & .45 .60
Foa et al. (1997) [3] Total score .78 .80 .66 .79
Reexperiencing .68 .77 .51 .67
Avoidance .75 .72 .69 .77
Hyperarousal .70 .74 .58 .73
The symptom items of the PTDS, which reflect more or less verbatim the corresponding items in the DSM-IV criteria, in empirical studies do not necessarily fall into the three groups explicit in DSM-III-R and DSM-IV. The results of a number of factor-analytic studies suggest that the avoidance symptoms load on two separate factors [11-13]. One factor captures wilful and effortful avoidance and the other factor captures involuntary strategies of "shutting down" the emotional system when effortful strategies fail, which thus may load together on the same factor as hyperarousal symptoms. This issue is to be borne in mind when examining the structure of instruments intended to measure PTSD symptoms according to DSM-IV.
Because of the many advantages of the PTDS we decided to use it for estimating rates of PTSD in a series of studies in different samples of war-traumatized inhabitants of Sarajevo and Banja Luka, Bosnia and Herzegovina. The results of these studies have been published elsewhere or are still in the process of being published [14-16].
The PTDS had to our knowledge never been used before in the area of former Yugoslavia; instead, many studies have used similar but more or less ad-hoc constructed checklist versions of the DSM-IV criteria. The introduction of the PTDS would therefore mean providing clinicians and researchers with a sound Bosnian version of an internationally accepted PTSD self-rating instrument. The goal of this paper is to report first results of the psychometric evaluation of the Bosnian PTDS.
Methods
Diagnostic assessment
Although all applied measures are questionnaires, not all subjects proved literate enough to complete them on their own. Therefore in some cases the interviewers had to read some of the questions to them and sometimes to reread or reformulate the questions. Thus the administration deviated slightly from the standard procedures.
The instrument under assessment was the Posttraumatic Stress Diagnostic Scale [3,17] which allows, as mentioned before, a diagnosis of PTSD as well as an estimation of symptom severity. The PTDS consists of four parts. Part 1 has 12 items in the original and asks about possible traumatic events (A1 criterion of DSM-IV). In part 2 the time of occurrence of the "most upsetting" event, together with the respondent's assessment of whether the event was life-threatening and whether it was accompanied by feelings of helplessness and intense fear are all evaluated (A2-criterion). Part 3 asks about symptoms of reexperiencing (5 items; criterion B), avoidance (7 items, criterion C), and arousal (5 items, criterion D). Part 4 explores the duration of the disturbance (criterion E) and the consequences of the symptomatology for important areas of functioning (criterion F). Since the original PTDS was designed for a civilian population in times of peace we replaced part 1 with a checklist of traumatic events specific to the war in Bosnia and Herzegovina 1992–5, the Checklist of War Related Experiences, CWE, the items of which are reproduced in Appendix 1. (The checklist also included other significant life events relevant to life in post-war Bosnia-Herzegovina. As these items are not relevant to this study, they are not discussed here.)
To obtain a Bosnian version we applied the procedures suggested by Vijver and Hambleton for the translations of psychological assessment measures [18]. That is, we performed an alternating procedure of translations and back-translations until no significant differences could be detected. In a second step we field-tested the resulting pilot versions to further check the appropriateness of the wording to the Bosnian language and the cultural context. The resulting modifications were then back-translated again.
The Impact of Event Scale [IES; [19]] is a questionnaire which assesses the frequency of intrusion and avoidance phenomena as a consequence of experiencing a particular event. In the more than 20 years since its publication it has very frequently been used to diagnose PTSD; however, that is neither the intended nor an appropriate use for it. The IES consists of 15 items each to be answered on a four-point scale assessing the frequency of the occurrence of stress reactions in the preceding week (0 = not at all; 1 = occasionally; 3 = sometimes; 5 = frequently). This means that total scores for the IES range between 0 and 75, with higher scores indicating more frequent intrusion and avoidance reactions. The IES has been applied in nearly every kind of traumatisation [for an overview, see [20]] and has been translated into many languages. The IES is one the most frequently used traumatic stress questionnaires internationally. The version used in the present study was almost identical to one which has been used in other studies in the region during and after the war and which has since been subject to a validation study [21] and found to have satisfactory factor structure and reliability.
The Symptom Checklist-90-R [SCL-90-R; [22]] is a 90 item self report questionnaire for measuring subjective psychological and somatic stress in the preceding seven days. Like the IES, the SCL-90-R is used widely internationally and has been used in a large number of research projects in a very wide variety of applications [for an overview, see [23]]. The SCL-90-R consists of nine scales and three global indices, of which the GSI, the Global Severity Index, is the most widely used.
Beck Depression Inventory (BDI)
The Beck Depression Inventory [BDI; [24]] is probably the best documented self-report method of measuring the intensity of depression [25,26]. By 1998 more than 2000 studies had been published using the BDI [27]. The current, revised, version consists of 21 items whose scores vary between 0 and 3 [24]. Zero indicates that the symptom is not present whereas three indicates the most extreme level of symptoms. Clients are instructed to report on how they felt in the preceding seven days.
Samples
The following data was collected between February 1998 and October 1999 in Sarajevo, Banja Luka and Prijedor, which are all in Bosnia-Herzegovina. Sarajevo is in the Federation of Bosnia and Herzegovina, namely that part of Bosnia and Herzegovina which has a predominantly Muslim and Catholic population, and Banja Luka and Prijedor are in the other part, the Republika Srpska, which is predominantly Serbian Orthodox. The samples were stratified by age and sex. The number of years of schooling was also recorded. All subjects participated voluntarily and gave fully informed consent. Table 3 shows sampling procedures, region, and numbers for each sub-sample included in the following analysis. Table 4 provides a description of the demographics.
Table 3 Overview of samples used
Sample Region Sampling procedure N
A 1998 Sarajevo randomised via maps of Sarajevo area 98
B 1998 Sarajevo admission to psychological treatment 114
C 1998 Sarajevo admission to medical treatment 99
D 1999 Sarajevo randomly selected repatriates to B&H from lists held by local councils 103
E 1999 Sarajevo randomly selected displaced or formerly displaced persons from lists held by local councils 97
F 1999 Banja Luka randomly selected subjects who stayed in the Banja Luka throughout the war, selected via maps of area 100
G 1999 Banja Luka randomly selected returned displaced persons, selected from lists of residents 100
H 1999 Prijedor randomly selected from lists of residents in collective centres 100
Table 4 Sample description
N Minimum Maximum Mean Std. Deviation
years of education 809 8.00 16.00 11.72 2.50439
age 812 16.00 68.00 37.89 13.78230
N % Missing
sex female 426 52.5 %
male 386 47.5 %
Total 812 100.0 % 0
employment status unemployed or waiting list 178 21.9 %
other (housewife, student) 360 44.3 %
employed 274 33.7 %
Total 812 100.0 % 0
family status single 363 44.8 %
married or long-term relationship 447 55.2 %
Total 810 100.0% 2
Other 70 8.7 %
religion Islam 383 47.3 %
Catholicism 45 5.6 %
Orthodox 311 38.4 %
Total 809 100.0 % 3
In total 812 persons participated. Inclusion criteria for all were a) age between 16 and 65, b) not suffering from a psychotic disorder and c) literate enough to answer the questionnaires with help. All subjects completed the PTDS and the SCL-90-R; therefore correlations for these subscales are based on the data of all the subjects. However for reasons of economy, in 1999 the full package of questionnaires including the BDI and IES were only administered to a random selection of participants in only the two Sarajevo sub-samples. All other participants in 1999 only answered a smaller package of questionnaires including the PTDS. Correlations between the PTDS and BDI and IES are therefore based on a smaller dataset.
In 20 cases an entire instrument was missing, as detailed in table 5. In the remaining cases, the number of individual missing values for individual items was small (much less than 5%), so it was deemed acceptable to form the total scores for the scales simply by multiplying the mean item score for each individual, allowing for any missing items, by the total number of items on each scale. So in the case of the inter-scale correlations the Ns are merely reduced by the number of completely missing questionnaires. In the case of the reliability analyses for the subscales of the PTDS, instruments with any missing items on the scale in question were excluded from the analyses, in each case slightly reducing the Ns.
Table 5 Details of which instruments were given to which sub-samples
BDI IES PTDS SCL
not given missing available not given missing available missing available missing available total
1998 samples, Sarajevo
non-displaced random sample 98 2 96 1 97 1 97 98
non-displaced medical treatment 1 98 4 95 5 94 1 98 99
non-displaced psychological treatment 114 1 113 114 114 114
1999 samples, Sarajevo
returnees from outside Former Yugoslavia 40 64 40 62 1 103 2 102 104
displaced or former displaced 21 76 21 1 75 97 97 97
1999 samples, Banja Luka and Prijedor
Banja Luka displaced or former displaced 100 100 100 100 100
Banja Luka non-displaced 100 100 100 100 100
Prijedor displaced in camps 100 100 100 100 100
Table Total 361 1 450 361 8 441 7 805 4 808 812
Interviewers
The medical and psychological samples were assessed through a total of 15 experienced counsellors/therapists, who were working at a variety of clinics and counselling centres in Sarajevo. All other samples were assessed by pairs of final year and third year students of Psychology at Sarajevo University and Banja Luka University. All interviewers were trained in the use of the questionnaires. Two pilot studies were performed to insure the appropriate use of the assessment. During the studies constant supervision for all interviewers was provided.
Statistical analysis
To obtain an estimation of internal consistency Cronbach's alpha was calculated for the total scores and the subscales of the PTDS. Convergent and divergent validity were estimated by using Spearman correlations between the scales. Spearman correlations were used because most of the distributions were not normal. For the principal components analysis, oblimin oblique rotation was used.
Results and discussion
The standardised Cronbach's alphas for the Bosnian PTDS were .93 for the total symptom score, .89 for the reexperiencing subscale, .84 for the avoidance subscale and .84 for the arousal subscale. The results correspond well with other published results.
The Spearman's correlations between the total scale and the subscales were all quite high at .89, .93 and .87 for re-experiencing, avoidance and hyperarousal respectively; re-experiencing correlated .74 and .67 with avoidance and hyperarousal; and the correlation between avoidance and hyperarousal was .72.
The item characteristics for the symptom items and subscale totals are shown in table 6. The characteristics are acceptable, with the lowest standard deviation being .77 for the item about not being able to remember details, which also had the lowest mean (.36 on a scale of 0 to 4).
Table 6 Item characteristics of the PTSD symptom items of the Bosnian PTDS
sex
female male total
Mean Standard Deviation Mean Standard Deviation Mean Standard Deviation
B1 intrusions 1.00 1.12 .76 1.00 .89 1.07
B2 bad dreams .73 1.02 .57 .92 .65 .98
B3 reexperiencing .70 .99 .53 .86 .62 .94
B4 upset after remembering 1.14 1.07 .89 1.01 1.02 1.04
B5 physical reaction after remembering .95 1.09 .62 .92 .79 1.02
C1 attempt not to think about it 1.14 1.16 .83 1.06 .99 1.12
C2 avoiding places people .86 1.13 .65 1.03 .76 1.09
C3 not being able to remember details .40 .80 .33 .74 .36 .77
C4 less interest in activities .66 .96 .51 .89 .59 .93
C5 detachment estrangement .65 1.00 .50 .93 .58 .97
C6 restricted affect .81 1.07 .50 .87 .66 .99
C7 foreshortened future .79 1.08 .58 .95 .69 1.02
D1 difficulty falling or staying asleep .92 1.11 .63 .96 .78 1.05
D2 irritability .70 .94 .55 .88 .62 .92
D3 difficulty concentrating .92 1.02 .68 .92 .80 .98
D4 hypervigilance .55 .88 .40 .77 .48 .83
D5 exaggerated startle response .75 .99 .42 .81 .60 .93
total score on subscale b (reexperiencing) 4.53 4.43 3.36 3.94 3.97 4.24
total score on subscale c (avoidance) 5.32 5.02 3.89 4.79 4.64 4.96
total score on subscale d (arousal) 3.82 3.77 2.67 3.44 3.28 3.66
total score on all symptom subscales 13.66 11.73 9.93 10.84 11.88 11.46
The items were scored on a scale of 0 (not at all or once a month) to 4 (5 or more times a week /almost always).
The items from the symptom subscales were submitted to a principal components analysis with oblimin oblique rotation. Factors with eigenvalues greater than 1 were retained. Items were considered as belonging to a factor if their loadings on that factor were above 0.4. (see table 7). The first solution had three factors explaining a total of 61.41% of the variance and was deemed to be satisfactory, so that no further solutions were sought. The first factor, which explains 47.64% of the variance, was labelled Arousal / Numbing. It contains all the items from the DSM-IV arousal scale and three DSM-IV avoidance items, two of which (detachment/estrangement and restricted affect) are also associated with numbing [11]. The second factor, explaining 7.85% of the variance, was labelled Intrusion and includes all the items from the DSM-IV intrusion scale together with one item ("attempting not to think about it") from the DSM-IV avoidance scale. The third factor, which explains 5.92% of the variance, was labelled Avoidance. It contains all the items from the DSM-IV avoidance scale except for two items which load on Arousal/Numbing. Every item loaded on at least one factor and only two items loaded on more than one factor (the item "attempt not to think about it" loaded on the Intrusion and Avoidance factors, and the item "detachment, estrangement" loaded on the Arousal/Numbing and Avoidance factors).
Table 7 Rotated factor pattern of the PTSD symptom items of the Bosnian PTDS
Loadings
Symptom Factor 1: Arousal / Numbing Factor 2: Intrusion Factor 3: Avoidance
b1 intrusions .031 -.824 .050
b2 bad dreams .127 -.779 -.049
b3 reexperiencing .189 -.704 -.047
b4 upset after remembering -.042 -.830 .079
b5 physical reaction after remembering .084 -.729 .074
c1 attempt not to think about it -.163 -.480 .544
c2 avoiding places people -.080 -.256 .666
c3 not being able to remember details .103 .014 .649
c4 less interest in activities .209 -.105 .511
c5 detachment, estrangement .567 .089 .438
c6 restricted affect .523 -.002 .397
c7 foreshortened future .596 .056 .326
d1 difficulty falling or staying asleep .460 -.361 .063
d2 irritability .652 -.199 -.031
d3 difficulty concentrating .732 -.123 -.017
d4 hypervigilance .753 -.072 -.122
d5 exaggerated startle response .746 -.065 -.003
Factor loadings greater than 0.40 are shown in bold underline.
In short, the three DSM-IV scales can be broadly identified, except that three DSM-IV avoidance items including two of the somewhat contentious numbing items load on the arousal scale, which replicates well the findings reported above [11-13].
Table 8 provides the correlations between the various other measures of psychopathology and the Bosnian PTDS. With samples of this size, correlations even as small as approximately .1 are significant, so all the correlations are highly significant and thus the significances are not reported here.
Table 8 Convergent and divergent validity of the Bosnian PTDS
IES Total IES Intrusion IES Avoidance BDI SCL GSI
PTDS Total Spearman's rho .709 .703 .619 .622 .568
N 439 438 439 444 802
Reexperiencing Spearman's rho .634 .687 .491 .485 .437
N 439 438 439 444 802
Avoidance Spearman's rho .651 .603 .610 .581 .515
N 439 438 439 444 802
Hyperarousal Spearman's rho .573 .574 .493 .596 .595
N 439 438 439 445 803
The correlations between the PTDS and the IES are somewhat lower than in the two American publications, closer to those in the German article. Re-experiencing on the PTDS correlates higher with intrusion than with avoidance on the IES, and avoidance on the PTDS correlates higher with avoidance on the IES than with intrusion on the IES, all of which are desirable results in that they support construct validity. The correlations between the re-experiencing and avoidance scales of the IES and the avoidance scale of the PTDS are quite similar, possibly indicating weak specificity of the latter, which was however also the case for all except the oldest of the three previous studies.
The correlation between the BDI total and the PTDS/PSS total is high, as reported in the literature. In fact the Bosnian version seems to differentiate a little better between PTSD and depression than do the American and German versions; nevertheless the specificity is still quite weak.
In the same way there are also quite high correlations with the SCL-90-R. Although the Bosnian version of the BDI and SCL have also not been adequately validated before, validating one new instrument against other instruments which are also not validated is not a meaningless affair but on the contrary the only possible procedure in a situation such as the one we (and our local and international researcher colleagues) found ourselves in, namely that very few world-standard instruments existed. If one does find, as we did, inter-instrument correlations similar to those for the corresponding instruments in other languages then that provides at least some provisional evidence for the psychometric quality and construct validity of all of those instruments.
One of the main uses of the PTDS is to provide a PTSD diagnosis in an economical way. As the PTDS assesses in questionnaire form all the information necessary for the diagnosis according to DSM-IV, the PTDS prevalences can be easily calculated and are in fact 24.72% for the whole sample, 31.37% for women and 17.40% for men.
The most important factor which restricts the interpretation of these results is that the PTDS was not compared with clinical interview, which would have been standard procedure in this kind of study. However, when we began the study there was no suitable validated interview available in the Bosnian language, which meant that we would have had to translate and extensively validate such an interview ourselves, and again we would have run into the problem of validating the interview against instruments which had also not been validated at that time. It also should be stressed that this study says very little about the cultural or contextual validity of the instrument or the construct PTSD which it is intended to measure.
On the other hand, the samples are quite large and taken together quite heterogeneous, and the selection methodologies in each case provided a reasonable approximation to randomness, so that all in all the data can be considered to be of good quality.
Conclusion
In conclusion it can be said that the psychometric properties of the Bosnian version of the PTDS are as good as those published for other languages. The internal consistencies are at least as good and the Bosnian version appears even to distinguish a little better than the American and German versions between PTSD as measured by the IES and depression as measured by the BDI. The principal components revealed by an exploratory analysis are broadly consistent with the DSM-IV subscales except that they reproduce some previously reported difficulties with the "numbing" items from the avoidance subscale; this issue might explain the poor specificity of the avoidance scale with respect to the IES subscales. None of the analyses revealed anything unusual or indicated problems either with the translation or with the application of the concepts inherent in the instrument to the post-war Bosnian population, all of which indicates that the Bosnian PTDS can be given the green light for further application in the future. Yet our results are only a necessary first step in the validation of the applied measures; a comparison with a validated translation of a Bosnian interview measure for PTSD still needs to be done.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
RR participated in the design of the study, and drafted the manuscript.
SP carried out the actual study and performed the statistical analysis.
Both authors worked on and approved the final manuscript.
Appendix 1
The war traumatic event items of the Checklist of War Events (which replaces the standard traumatic event checklist in the PTDS)
group 0: injury to self
Were you severely injured during the war?
group 1: sexual violence to self
Were you raped or sexually assaulted during the war?
During the war, were you sexually assaulted by a member of your close family who had been forced to do that?
During the war, were you sexually assaulted by a member of your close family who was not forced to do that?
group 2: torture to self
Were you tortured during the war?
group 3: other threat to self
During the war, were you in a situation in which you strongly believed you would be severely injured or killed?
During the war, did a bullet come so close to you that you could have been severely injured or killed?
During the war, did a bomb or grenade explode so close to you that you could have been severely injured or killed?
During the war, did anyone threaten to kill you or severely injure you?
Were you captured or held in a detention camp during the war?
During the war, were you without food or water for so long that you strongly believed you would die?
During the war, were you so cold that you strongly believed you would die?
During the war, did you stay in a cellar longer than 3 weeks without a break?
During the war, were you assaulted in a non-sexual way by a member of your close family who had been forced to do that?
During the war, were you assaulted in a non-sexual way by a member of your close family who had not been forced to do?
Were you in the army during the war?
During the war, were you seriously ill because of the war (e.g. heart attack)
group 4: witnessed: loved ones
Did you eyewitness a loved one being killed during the war?
Did you see dead body of a loved one who had been killed in the war? (excluding funerals)
Did you see a loved one being tortured or physically assaulted during the war?
Did you see a loved one being sexually assaulted during the war?
Did you touch a loved one who had been killed or wounded in the war?
During the war, did you see a loved one who was severely injured before he/she received medical help?
group 5: witnessed: others
Did you eyewitness somebody being killed (not a loved one) in the war?
Did you see the body of a person (but not a loved one) who had been killed in the war? (excluding funerals)
Did you see someone being tortured or physically assaulted during the war (but not a loved one)?
Did you see someone being sexually assaulted during the war (but not a loved one)?
Did you touch someone (but not a loved one) who had been killed or wounded in the war?
During the war, did you see a severely injured person (not a loved one) before they received medical help?
group 6: losses, nuclear family
Was your father killed in the war?
Was your mother killed in the war?
Was your spouse killed in the war?
Was a child of yours killed in the war?
Was a brother or sister of yours killed in the war?
group 7: losses, other loved ones
Was a close relative of yours killed in the war?
Was a close friend of yours killed in the war?
group 8: threat, violence, injury to loved ones
Was a loved one in the army during the war?
Was a loved one severely injured in the war?
Was a loved one raped or sexually assaulted in the war?
Was a loved one tortured in the war?
Was a loved one captured or held in a concentration camp during the war?
During the war, was a loved one seriously ill (e.g. cancer or heart attack) or had some chronic health problem?
group 9: other war events
Other traumatic event since 1991 due to war: 1
Other traumatic event since 1991 due to war: 2
Other traumatic event since 1991 due to war: 3
group 10: other events since 1991 not related to war
Did a loved one die in the war for reasons unrelated with the war?
(Other stressful and traumatic events since 1991 and unrelated to the war)
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
Our sincere thanks are due to the many people who were involved in this survey, above all to the citizens of Bosnia-Herzegovina who took the time to answer our sometimes distressing list of questions. Thanks are also due to the students of Sarajevo and Banja Luka Universities who carried out the interviews along with the counselors in Sarajevo, and to Vladimir Turjacanin, Jovan Savic and Branko Milosavljavic at the University of Banja Luka. Finally, we would particularly like to thank Edna Foa who developed the original English language version of the instrument.
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| 15725361 | PMC554114 | CC BY | 2021-01-04 16:33:03 | no | BMC Psychiatry. 2005 Feb 23; 5:11 | utf-8 | BMC Psychiatry | 2,005 | 10.1186/1471-244X-5-11 | oa_comm |
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BMC Fam PractBMC Family Practice1471-2296BioMed Central London 1471-2296-6-81572370810.1186/1471-2296-6-8Research ArticleOral vitamin B12 therapy in the primary care setting: a qualitative and quantitative study of patient perspectives Kwong Jeff C [email protected] David [email protected] Irfan A [email protected] Denise [email protected] Ross EG [email protected] Department of Family and Community Medicine, University of Toronto, 263 McCaul Street, Toronto, Ontario, M5T 1W7, Canada2 Department of Public Health Sciences, University of Toronto, McMurrich Building, 12 Queen's Park Crescent W., Toronto, Ontario, M5S 1A8, Canada3 Department of Medicine, University of Toronto, Suite RFE 3-805, 190 Elizabeth Street, Toronto, Ontario, M5G 2C4, Canada4 Department of Sociology, York University, 2060 Vari Hall, 4700 Keele Street, Toronto, Ontario, M3J 1P3, Canada5 Primary Care Research Unit, Sunnybrook and Women's College Health Sciences Centre, 2075 Bayview Avenue, #E-349, Toronto, Ontario, M4N 3M5, Canada2005 21 2 2005 6 8 8 31 8 2004 21 2 2005 Copyright © 2005 Kwong et al; licensee BioMed Central Ltd.2005Kwong et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Although oral replacement with high doses of vitamin B12 is both effective and safe for the treatment of B12 deficiency, little is known about patients' views concerning the acceptability and effectiveness of oral B12. We investigated patient perspectives on switching from injection to oral B12 therapy.
Methods
This study involved a quantitative arm using questionnaires and a qualitative arm using semi-structured interviews, both to assess patient views on injection and oral therapy. Patients were also offered a six-month trial of oral B12 therapy. One hundred and thirty-three patients who receive regular B12 injections were included from three family practice units (two hospital-based academic clinics and one community health centre clinic) in Toronto.
Results
Seventy-three percent (63/86) of respondents were willing to try oral B12. In a multivariate analysis, patient factors associated with a "willingness to switch" to oral B12 included being able to get to the clinic in less than 30 minutes (OR 9.3, 95% CI 2.2–40.0), and believing that frequent visits to the health care provider (OR 5.4, 95% CI 1.1–26.6) or the increased costs to the health care system (OR 16.7, 95% CI 1.5–184.2) were disadvantages of injection B12. Fifty-five patients attempted oral therapy and 52 patients returned the final questionnaire. Of those who tried oral therapy, 76% (39/51) were satisfied and 71% (39/55) wished to permanently switch. Factors associated with permanently switching to oral therapy included believing that the frequent visits to the health care provider (OR 35.4, 95% CI 2.9–432.7) and travel/parking costs (OR 8.7, 95% CI 1.2–65.3) were disadvantages of injection B12. Interview participants consistently cited convenience as an advantage of oral therapy.
Conclusion
Switching patients from injection to oral B12 is both feasible and acceptable to patients. Oral B12 supplementation is well received largely due to increased convenience. Clinicians should offer oral B12 therapy to their patients who are currently receiving injections, and newly diagnosed B12-deficient patients who can tolerate and are compliant with oral medications should be offered oral supplementation.
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Background
Intramuscular injections of vitamin B12 (cobalamin) have been the mainstay of B12 deficiency treatment for decades. However, because approximately 1% of orally ingested B12 is absorbed via simple diffusion throughout the gastrointestinal tract (i.e., independently of intrinsic factor)[1,2], oral replacement with high doses of cobalamin is both effective and safe, regardless of the etiology of B12 deficiency [1,3-12].
Oral B12 therapy would decrease physician burden, increase patient control over therapy, and avoid patient discomfort and inconvenience. Switching patients from B12 injections to oral therapy would also result in savings to the health care system [13]. While some commentators have argued that clinicians should switch to oral B12 therapy [13-15], little is known about patients' views concerning the acceptability and effectiveness of oral B12.
Efforts to switch patients who are well established on parenteral therapy (and who have previously been told they require lifelong injections) may fail without an understanding of what factors influence patient acceptability of oral therapy. Therefore, we combined qualitative and quantitative methods to test the hypothesis that patients offered oral B12 are willing to switch to oral therapy and to explore the reasons for their choice.
Methods
We administered a questionnaire to patients with B12 deficiency, offered the option of a six-month trial of oral replacement, and administered a follow-up questionnaire. Throughout the study, semi-structured interviews were conducted for the qualitative arm. We received ethics approval from the Sunnybrook and Women's College Health Sciences Centre Research Ethics Board. All patients provided informed consent.
Quantitative arm
Data collection
The study took place at two academic family practice units and a community health centre with diverse practice profiles in Toronto. We included all patients who received regular injections (i.e., every 1 to 3 months), regardless of age and etiology of B12 deficiency. We excluded patients if their last injection occurred more than three months before the start of patient recruitment, if they had left the practice, if their B12 therapy had been discontinued or if they had already switched to oral therapy, if they did not speak English and did not have access to a translator, or if they had been recently diagnosed with a serious illness. After applying these criteria, 133 patients were included in the study (Figure 1).
Figure 1 Flow diagram of patients in study.
The recruitment cover letter included one sentence describing the equivalency of oral B12 therapy to injections: "Studies have shown that vitamin B12 pills are just as effective and safe as B12 injections." Educational sessions on the effectiveness of oral therapy were held with the nursing staff (since they administer the injections) at all sites, and they were encouraged to pass this information on to their patients. As well, contact information for the study investigators was provided to the patients in case they had any questions.
We placed the initial questionnaire [see Additional file 1 ] in patients' charts to be completed at their next visit for a B12 injection. The initial questionnaire elicited demographic data, medication history, logistical aspects of B12 related visits, history of past B12 therapy, and attitudes about injection and oral therapy. Non-responders received up to two telephone reminders.
Patients willing to try oral therapy were given a six-month supply of B12 tablets (one 1000 μg tablet daily), and were offered testing of serum B12 levels at baseline (one month after their last injection) and after the six-month trial. Patients had the option to withdraw from the trial and return to injections at any time for any reason. A follow-up questionnaire was placed in the charts of patients who tried the pills. This questionnaire re-assessed patients' attitudes about the different forms of B12 therapy and asked whether they would continue on oral supplementation or return to injection therapy (see Additional file 2).
Statistical analyses
Where variables were not already dichotomous (e.g., for satisfaction with injections, the questionnaire listed "very satisfied," "satisfied," "neutral," "unsatisfied," and "very unsatisfied" as choices), we dichotomized the variable of interest (e.g., "satisfied" vs. "neutral/unsatisfied"). To ascertain the relationship between questionnaire responses and preference for oral therapy (as "willingness to switch" and "permanently switching") we performed bivariate and multivariate analyses. We used a p-value of 0.20 (for the continuity-adjusted χ2 and Fisher's exact tests) as the cut-off for inclusion of individual patient factors into multivariate logistic regression models. We then performed backwards stepwise regression to determine statistically significant relationships after adjustment. We generated both crude and adjusted odds ratios with 95% confidence intervals.
Qualitative arm
Participants and setting
One investigator (D.C.) conducted 17 semi-structured interviews in a private meeting room at the family practice unit. A purposive sample was selected amongst those willing to be interviewed to reflect diversity in terms of sex, age, willingness to switch, and final choice of therapy after the trial of B12 pills. These interviews were performed at various stages of the study (before, during, and after the trial of oral therapy). Amongst the 17 participants, four were selected from those not willing to switch, while of the 13 who were willing to try oral therapy, six were interviewed prior to, two during, and five after the trial of oral therapy.
Data collection and analysis
The interviews were audiotaped and transcribed. They lasted 20–30 minutes, with questions about the patient's knowledge and history of their B12 therapy, his or her relationship with health care providers, perceived advantages and disadvantages of both pills and injections, and attitudes about past and current B12 therapy. Analyses of the transcripts were performed independently by two investigators (J.K. and D.T-K.), using a three-step content analysis approach to identify and collate relevant themes [16]. Through a process of clarification, confrontation, and consensus, we reached agreement regarding the themes and sub-themes.
Results
Quantitative arm
For our initial questionnaire, we received responses from 86 out of the sample of 133 patients, for a response rate of 64.7%. Selected characteristics of the study population are presented in Table 1. Non-responders were younger and more likely to be female.
Table 1 Selected characteristics of study population and a comparison of responders vs. non-responders
Responders n = 86 Non-responders n = 47 p-value
Mean age – Yr ± SD 72 ± 15 65 ± 20 0.03
Female sex – No. (%) 48 (56) 37 (79) 0.009
Level of education completed
High school or less 28 (33)
Some post-secondary education 21 (25)
Bachelors degree or more 35 (42)
Annual household income
Less than $40K 40 (53)
$40–79K 20 (26)
$80K or greater 16 (21)
Self-reported perception of health
Above average 25 (29)
Average 40 (47)
Below average 20 (24)
Prescription medications taken
0 8 (10)
1 to 3 46 (55)
4 to 6 16 (19)
7 + 14 (17)
Monthly episodes of forgetting medications
0 45 (54)
1–2 26 (31)
3+ 12 (14)
Years on B12 therapy
0 to 2 19 (24)
3 to 5 25 (31)
6 to 10 17 (21)
11 to 19 13 (16)
20+ 6 (8)
Frequency of B12 injections
Less than once monthly 9 (10)
Once monthly 74 (86)
More than once monthly 3 (3)
Satisfaction with B12 injections
Satisfied 22 (26)
Neutral 59 (69)
Unsatisfied 5 (6)
Monthly visits to doctor for other reasons
0 49 (60)
1 23 (28)
2 + 10 (12)
Mode of travel to visit doctor
Personal vehicle 49 (58)
Public transit 23 (27)
Walk 9 (11)
Taxi 4 (5)
Travel time to visit doctor
< 15 min 27 (32)
15–29 min 29 (35)
30–44 min 20 (24)
45–59 min 5 (6)
60+ min 3 (3)
Patients at each study site
Sunnybrook Campus, SWCHSC* 51 (59)
Flemingdon Health Centre 19 (22)
Women's College Campus, SWCHSC* 16 (19)
* Sunnybrook and Women's College Health Sciences Centre
Willingness to switch to oral B12
Sixty-three of our 86 respondents reported a willingness to switch to oral therapy. A large number of patient factors had no clear statistical relationship to a "willingness to switch" to oral therapy (i.e., p > 0.20) and were excluded from the multivariate model: age, education, income, drug insurance coverage, study site, number of prescription medications, mode of travel to the clinic, years of past B12 therapy, number of non-B12 related visits per month, improvement in perceived well-being since initiating B12 injections, several perceived disadvantages of injections (risk of complications, travel/parking costs), and several perceived disadvantages of pills (take too many pills already, would have to pay for them, won't work as well as injections).
The following factors were included in the multivariate model: gender, time to clinic, satisfaction with past B12 injections, several perceived disadvantages of injections (shots are painful, frequent visits to see MD/nurse, cost to health care system), and one perceived disadvantage of pills (won't see MD/nurse as often). Factors associated with willingness to switch after multivariate adjustment were: being able to get to the clinic more quickly (i.e., in less than 30 minutes) (OR 9.29, 95% CI 2.16–39.97), and believing that injection therapy is disadvantageous due to the need for frequent visits to health care provider (OR 5.41, 95% CI 1.10–26.56) and the increased costs to the health care system (OR 16.68, 95% CI 1.51–184.22) (Table 2).
Table 2 Patient factors associated with willingness to switch to oral therapy on initial questionnaire.
No. (%) of subjects Unadjusted Adjusted*
Patient factor Willing to switch n = 63 Not willing to switch n = 23 OR (95% CI) p-value OR (95% CI) p-value
Time to clinic
0–29 minutes 49 (87) 7 (13) 9.34 <0.001 9.29 0.003
30+ minutes 12 (43) 16 (57) (3.14–27.78) (2.16–39.97)
Perceived disadvantages of injections
Frequent visits to see MD/nurse
Agree 38 (86) 6 (14) 4.31 0.010 5.41 0.038
Disagree 25 (60) 17 (40) (1.49–12.42) (1.10–26.56)
Cost to health care system
Agree 26 (93) 2 (7) 7.38 0.010 16.68 0.023
Disagree 37 (64) 21 (36) (1.59–34.23) (1.51–184.22)
* Adjusted for patient factors at least weakly associated (i.e., p < 0.20) with a willingness to switch to oral B12 therapy in bivariate analyses: gender, time to clinic, satisfaction with past B12 injections, perceived disadvantages of injections (shots are painful, frequent visits to see MD/nurse, cost to health care system), perceived disadvantage of pills (won't see MD/nurse as often)
Trial of oral therapy
Of the 63 patients who were willing to switch to oral B12 therapy, eight changed their minds before the trial started. Therefore, 55 patients were started on oral B12. Five dropped out and three were lost to follow-up, leaving 47 patients who completed the six-month trial. Reasons cited for discontinuing oral therapy included fatigue, neurological symptoms, and gastrointestinal intolerance.
Fifty-two patients returned the follow-up questionnaire. Over three-quarters (39 of 51 respondents) reported being satisfied or very satisfied with oral therapy. Only 8% of patients (4/51) perceived that they felt worse with pills, while 23.5% (12/51) felt better and the rest felt the same. Self-reported compliance was good; 48 patients or 92% reported forgetting to take the pills two times or less per month. Thirty-nine patients (71% of the 55 who actually switched) stated that they wished to permanently switch to B12 pills. Of the 35 patients who reported feeling the same with the pills as with injections, 28 (80%) chose pills.
Again, many patient factors had no association with a desire to permanently switch to oral B12. Patient factors that on bivariate analyses had a p-value of less than 0.20 and were consequently included in the multivariate model were: patient beliefs that frequent visits to see the doctor or nurse and the associated travel/parking costs are disadvantages of injections, and the belief oral B12 would add unnecessarily to an already large number of prescribed oral medications. Table 3 presents the factors significantly associated, after multivariate adjustment, with permanently switching to oral therapy: agreeing that disadvantages of injections included frequent visits to see the doctor/nurse (OR 35.41, 95% CI 2.90–432.70) and travel/parking costs (OR 8.66, 95% CI 1.15–65.30).
Table 3 Patient factors associated with permanently switching to oral therapy on follow-up questionnaire
No. (%) of subjects Unadjusted Adjusted*
Patient factor Choosing oral therapy n = 39 Choosing injection therapy n = 13 OR (95% CI) p-value OR (95% CI) p-value
Perceived disadvantages of injections
Frequent visits to see MD/nurse
Agree 27 (96) 1 (4) 27.00 <0.001 35.41 0.005
Disagree 12 (50) 12 (50) (3.14–231.87) (2.90–432.70)
Travel/parking costs
Agree 23 (92) 2 (8) 7.91 0.016 8.66 0.036
Disagree 16 (59) 11 (41) (1.54–40.60) (1.15–65.30)
* Adjusted for patient factors at least weakly associated (i.e., p < 0.20) with permanently switching to oral B12 therapy in bivariate analyses: perceived disadvantages of injections (frequent visits to see MD/nurse, travel/parking costs), perceived disadvantage of pills (take too many pills already)
Serum B12 levels
Baseline and post-intervention serum B12 levels were obtained for 39 of the 55 patients who switched to pills (Figure 2). The mean serum level increased from 387 to 698 pmol/L (p < 0.0001) (reference range: ≥ 180 pmol/L, unlikely to have B12 deficiency). Only one patient's serum level decreased but even she remained within the normal range.
Figure 2 Serum B12 concentrations before and after six months of oral B12 therapy for forty patients.
Qualitative arm
The mean age of interview participants was 71 (range 54–92) years, with 7 men and 10 women.
Perceived efficacy of B12 therapy
Some patients felt that the B12 injections they had been receiving in the past were effective: "I find it gives me more energy." Patients who expressed this view were often not willing to switch to oral therapy, or if they did try the pills, they eventually went back to injections. Others felt that the injections hadn't helped much: "There is a little feeling that, 'Oh, I'm taking this. I should feel better,' but I'm not sure if I do." These patients tended to switch permanently to oral therapy.
When asked about the anticipated efficacy of oral therapy, some patients, particularly those who were not willing to switch, were sceptical: "Well, I don't think it will work. If the doctors suggested that I needed to go on the injections, I'm certainly pretty sure that the pills are not going to work. Otherwise he'd have put me on pills, would he not?" Many others were unsure about the efficacy of oral therapy.
Reasons for switching to oral therapy
Nearly every patient cited convenience as an advantage of switching to B12 pill. Other advantages of oral therapy included savings to the health care system and ease of travel. Cited disadvantages of injections included decreased compliance due to the need for frequent visits and potential complications associated with injections. A number of patients decided to switch because they were interested in participating in a research study. Some patients acknowledged that oral therapy would benefit those who are averse to needles, however, needles did not bother most of the patients interviewed: "...needles don't bother me, because – well, I don't know. They just don't seem to bother me. I know some people are very concerned about it."
Satisfaction with oral therapy
For both patients interviewed during their trial of oral B12 and for three of the five patients interviewed afterward, all of whom permanently switched to oral therapy, there was a high level of satisfaction with oral B12. One patient even reported feeling better on oral B12: "Well, I'm more level. I don't feel at the end of the month that I'm running out of energy. I'm quite well aware of that." (see Additional file 3).
Reasons for staying with/switching back to injection therapy
Disadvantages of switching to oral therapy included having to take an additional oral medication, fear of side effects, concern about swallowing difficulties, the inconvenience of taking a medication daily, and the potential for losing contact with health care providers and the opportunity for minor drop-in consultations. Some patients were under the impression that injections would work more quickly and directly.
Two patients who switched back to injections after trying the pills were interviewed. These patients switched back due to side effects – one cited neurological and gastrointestinal symptoms (her post-trial serum B12 level was significantly increased from baseline) and the other cited decreased energy (his post-trial serum B12 level was not available). (see Additional file 4).
Discussion
We found that switching patients from injection to oral B12 is both feasible and acceptable to patients. Of those who responded to our initial questionnaire, nearly three-quarters were willing to try oral B12, and of those who did switch, most were satisfied and the majority wished to remain permanently on oral therapy. These patients believed that injections are disadvantageous because they are associated with too many visits to their health care provider and with higher patient costs (in terms of travel and parking expenses). In the qualitative arm of our study, most patients cited convenience as the reason for wanting to switch from injections to pills. We were also able to confirm findings from other studies indicating that oral B12 is biochemically equivalent to, if not better than, parenteral therapy.
One non-intuitive finding from the initial questionnaire assessing the factors associated with trying oral therapy (Table 2) was that those who were able to get to the clinic more quickly (i.e., in fewer than 30 minutes) were more likely to try switching. While one might expect that those who live closer to their clinic would benefit less from the convenience of not having to visit their health care provider as frequently, we speculate that perhaps these patients are more comfortable with trying oral therapy because they know that it is easy for them to access care if they need it, rather than having to rely on regular visits for their injections to see their primary care provider. Another interesting finding – that the perception of travel expenses being a disadvantage of the injections was not associated with being willing to try injections but was associated with permanently switching after the trial – suggests that perhaps patients did not realize the benefit of the saved travel costs until several visits had been averted.
By monitoring trough serum B12 levels (i.e., 1 month after their last injection) and again 6 months after oral therapy, we observed that one patient was a non-responder (i.e., serum level decreased), and 4 patients had serum levels that increased but remained below 295 pmol/L (a level considered by some to be borderline B12 deficient)[17]. This represents 13% out of the 39 patients who underwent testing, suggesting that follow-up testing may be warranted with serum B12 levels and, if available, functional tests such as serum homocysteine and methlymalonic acid. The sub-optimal response in these patients may be due to patient non-compliance or sub-optimal dosing (we used a 1-mg daily dose, rather than the 2-mg daily dose used by Kuzminski et al.) [8]. Further studies may be required to clarify the optimal dosing regimen for oral therapy.
The primary strength of our study was that we achieved triangulation through the use of both quantitative and qualitative methods. By using questionnaires, we were able to provide estimates such as switch rates and levels of patient satisfaction; with the qualitative arm, we were able to more thoroughly explore patient perspectives.
The main limitation of our study was the relatively small sample size for the quantitative arm, which led to large confidence intervals for the estimates of the relationship between patient factors and switching to oral B12. However, the sample was large enough to identify a number of patient factors associated with switching from injection to oral therapy and to determine that patient attitudes toward oral B12 therapy are greatly influenced by its convenience. A second limitation was that we did not assess the knowledge and attitudes of our patients' physicians and nurses, as they may have influenced patient's expectations and perceptions regarding the effective of oral B12 therapy. Finally, another limitation was the potential participant bias, evidenced by the differences between responders and non-responders in terms of age and sex. However, among the responders, neither age nor sex was significantly associated with switching to oral therapy, suggesting that any differences between participants and non-participants were likely irrelevant.
While there have been several studies examining physicians' perspectives on switching from intramuscular to oral therapy [18-21], we know of few studies that have examined patients' views. In a recent study that involved switching forty patients to oral therapy, the authors reported that 83% preferred the oral form [15]. However, no further details on patient perspectives were reported. Another study found that patients become very attached to receiving their injections; after identifying 48 patients who did not meet diagnostic criteria for B12 deficiency and providing educational sessions to encourage discontinuing injections, 38% stated they would leave the practice if denied their injections [22]. Explanations suggested by the authors include the reluctance to discontinue a therapy initiated by a trusted and respected physician, and the belief that intramuscular injections are more potent and more effective than medications taken orally. While the perception of increased potency of injections was reported by some patients in the qualitative arm of our study, this belief was supported by only 20% of respondents in the initial questionnaire (data not shown).
While oral B12 therapy may be acceptable for most patients, it may not be appropriate for those who will not be compliant with oral medications, such as patients with significant memory impairment or cognitive dysfunction, unless they have caregivers who could ensure compliance. Oral therapy also may not be appropriate for those with swallowing difficulties; for such patients, sublingual B12 therapy, which has been shown to be equally effective, may more appropriate [23].
Conclusion
In summary, the results of our study suggest that clinicians should offer oral B12 therapy to their patients who are currently receiving injections if they can tolerate and are compliant with oral medications. Most patients who switch from parenteral to oral therapy are satisfied and wish to stay permanently on oral therapy. They often cite the inconvenience and the travel-related expenses associated with frequent visits to the doctor as disadvantages of parenteral therapy. Therefore, oral B12 therapy is generally well-received by patients, and should be considered by clinicians as a superior alternative to the traditional injections for most patients.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
JK designed and coordinated the study, analyzed the qualitative and quantitative data, and drafted the manuscript. DC conducted the in-depth interviews and assisted in data collection. ID helped to develop the questionnaire and performed statistical analyses of the quantitative data. DTK analyzed the qualitative data. RU conceived the study and provided guidance on all aspects of the project. All authors participated in the development of the manuscript and gave approval to its final submission for publication.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Supplementary Material
Additional File 1
Initial questionnaire
Click here for file
Additional File 2
Follow-up questionnaire
Click here for file
Additional File 3
Reasons for switching to oral therapy
Click here for file
Additional File 4
Reasons for not switching to oral therapy
Click here for file
Acknowledgements
We would like to thank David Naylor for critically reviewing the manuscript, Paul Corey for providing statistical advice, Yasmine Rizwan and David Chong for coordinating data collection at the Women's College Hospital and Flemingdon Health Centre sites, respectively, Sean Carr for assistance with data entry, Shari Gruman for administrative support, the nurses and support staff at the three sites for making this project possible, and most of all, the patients who participated in the study.
This study was funded by a resident research grant from Physician Services Incorporated (PSI) Foundation. Based on peer-reviewed feedback during the grant review process, several changes were made to the study design. Natural Factors Nutritional Products Ltd donated the oral B12 tablets, but had no input in any part of the study.
This paper was presented at the College of Family Physicians of Canada (CFPC) Family Medicine Forum on October 24, 2003.
Dr. Upshur is supported by a New Investigator Award from the Canadian Institutes of Health Research (CIHR) and a Research Scholar Award from the Department of Family and Community medicine.
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| 15723708 | PMC554115 | CC BY | 2021-01-04 16:29:12 | no | BMC Fam Pract. 2005 Feb 21; 6:8 | utf-8 | BMC Fam Pract | 2,005 | 10.1186/1471-2296-6-8 | oa_comm |
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BMC PsychiatryBMC Psychiatry1471-244XBioMed Central London 1471-244X-5-121573056510.1186/1471-244X-5-12Research ArticleA Theory of Mind investigation into the appreciation of visual jokes in schizophrenia Marjoram Dominic [email protected] Howard [email protected] Patrick [email protected] Donald [email protected] David G Cunningham [email protected] Eve C [email protected] Stephen [email protected] Department of Psychiatry, University of Edinburgh, Edinburgh, UK2 University of Edinburgh Medical School, Edinburgh, UK2005 24 2 2005 5 12 12 29 9 2004 24 2 2005 Copyright © 2005 Marjoram et al; licensee BioMed Central Ltd.2005Marjoram et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
There is evidence that groups of people with schizophrenia have deficits in Theory of Mind (ToM) capabilities. Previous studies have found these to be linked to psychotic symptoms (or psychotic symptom severity) particularly the presence of delusions and hallucinations.
Methods
A visual joke ToM paradigm was employed where subjects were asked to describe two types of cartoon images, those of a purely Physical nature and those requiring inferences of mental states for interpretation, and to grade them for humour and difficulty. Twenty individuals with a DSM-lV diagnosis of schizophrenia and 20 healthy matched controls were studied. Severity of current psychopathology was measured using the Krawiecka standardized scale of psychotic symptoms. IQ was estimated using the Ammons and Ammons quick test.
Results
Individuals with schizophrenia performed significantly worse than controls in both conditions, this difference being most marked in the ToM condition. No relationship was found for poor ToM performance and psychotic positive symptomatology, specifically delusions and hallucinations.
Conclusion
There was evidence for a compromised ToM capability in the schizophrenia group on this visual joke task. In this instance this could not be linked to particular symptomatology.
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Background
Theory of Mind and schizophrenia
Theory of Mind (ToM) describes the ability to recognise that other people have minds containing beliefs and intentions and to be able to interpret these correctly. The term, first coined by Premack and Woodruff [1], is also referred to as mind-reading [2] or 'mentalising', when the correct inferences regarding the intentions and belief of others are used to predict and control behaviour [3].
ToM ability has been conceived as a capacity to represent epistemic mental states comprising an agent and an attitude to the truth of a proposition e.g. "Peter believes that it is raining" [4,5]. The truth of this proposition concerning the mental state of an agent (Peter, who believes it is raining) need not be affected by the truth of the embedded proposition (it is raining), which may be false [6,7]. In this way, Leslie and Roth [6] proposed that a major requirement for computing such representations is a mechanism that decouples the content of the proposition (it is raining) from reality. These special representations have come to be termed metarepresentations or M-representations.
It is widely reported that there are observed ToM deficits in schizophrenia from the numerous behavioural and neuroimaging studies that have been conducted investigating this phenomenon [e.g. [8]]. It is proposed that certain symptoms characteristic of schizophrenia may also reflect specific impairments in ToM abilities [see [9]], these being positive symptoms of delusions and hallucinations and chronic negative symptoms. Frith [10] also hypothesised that positive schizophrenic symptoms could result from impairment in metarepresentation. In more detail, Frith hypothesized that in certain cases of schizophrenia something may go wrong with the decoupling process involved in computing metarepresentations [11]. This might occur in two ways. Using the above scenario, firstly the content (it is raining) becomes detached from the rest of the proposition (Peter believes that...) and secondly, the content is perceived as a representation of the real world rather than someone's belief about it. This statement, unattached to any implication that it is a thought or belief of the patient, or another person, may then be misconstrued, e.g. as a third-person auditory hallucination. Different forms of hallucination may be experienced according to the precise propositions misperceived. Misinterpretation of the behaviour or intentions of others may manifest as the delusions of reference, misidentification and persecution, experienced by some individuals with schizophrenia. Indeed, it can be said that rather than an absence of ToM capabilities in these individuals there is actually an inappropriate and excessive use of basically intact theory of mind capabilities [9]. This follows since a basically intact theory of mind mechanism is needed, say, to infer other people's persecutory intentions (even when these are mistaken inferences) and there is an over-attribution of intentions of this type in persecutory-deluded people with schizophrenia [12,13]. Frith has also referred to a distinction between over-mentalising in schizophrenia and under-mentalising in autism [14].
Pictorial studies
Sarfati et al used a strictly pictorial task in which 3 picture cartoon sequences were shown depicting a character producing an action and the participants had to choose the fourth and final picture from a choice of three images [15]. Successful image choice depended on the understanding of the character's intent behind the action. They found that individuals with schizophrenia who had thought and speech disorganisation had a significant specific difficulty attributing mental states to others.
Sarfati et al then enlarged this experimental protocol by introducing a verbal dimension to the task [16]. There were now two answer conditions to the original 3 picture cartoon sequences relaying character intent: the pictorial condition identical to the above and a new verbal condition where the choice of endings were comprised of verbal sentences. Disorganised individuals with schizophrenia performed significantly worse than the other experimental groups. Interestingly, all the groups' performance improved in the verbal condition, but the presence of verbal material did not make the disorganised patient's performance similar to that of the other groups. Sarfati et al followed this work up by looking at the difference in performance on the same task before and after the introduction of the verbal answer condition [17]. They compared a schizophrenia group and a matched control group. The entire control group and half the schizophrenia group who did not perform at the best level in the pictorial answer condition, remediated with verbalization. In contrast to their a priori hypothesis, it was not schizophrenia patients with thought and language disorders who remediated in the verbal condition.
Langdon et al, used a task comprised of 4 card black and white cartoon picture sequences of four varieties: social script stories testing logical reasoning about people without needing to infer mental states, mechanical stories testing Physical cause and effect reasoning, false belief stories testing general mind reading abilities and capture stories testing inhibitory control. Cards were place face down in a square layout and participants had to turn the cards over and place them in the correct order to show a logical sequence of events. In order to control for possible contributory effects of executive dysfunction, inhibitory control was tested using capture picture-sequences and executive planning was tested using the Tower of London task.
In both studies, it was found that individuals with schizophrenia showed a selective ToM impairment which could not be completely explained by reasoning, planning deficits or poor inhibitory control [18,19].
Brüne showed individuals a muddled cartoon 4 picture sequence depicting a ToM scenario between characters [12]. The participants had to put the pictures into the correct sequence and then answer first and second order ToM questions related to the depiction. Where as first-order questions require acknowledgement of what one story character thinks about the world, second-order questions require acknowledgement of what one story character thinks about another story character's thoughts. The schizophrenia group was outperformed by the control group.
Corcoran et al used visual jokes to look at potential ToM deficits in schizophrenia [3]. Two sets of jokes were used: a Physical set of slapstick humour that did not require ToM capabilities to understand the joke contained within the picture and a ToM set in which an appreciation of the mental states of the characters (false belief and deception) were required. ToM deficits were found in individuals with schizophrenia exhibiting passivity phenomena (e.g. thought insertion/withdrawal) and behavioural disorders.
The primary interest of the current study was to examine the associations between specific schizophrenic symptoms and ToM capabilities using the cartoon method devised by Corcoran [3], but with a larger battery of visual jokes (over treble the number of picture stimuli). Patients with schizophrenia were compared to a closely matched group of healthy controls. It was anticipated, in keeping with Frith's model [11], and the data from Corcoran et al [3], that not only would the schizophrenia group perform significantly worse than the controls, but that the severity of positive symptoms, in particular hallucinations and delusions, would be most strongly associated with ToM impairment.
Methods
Participants
Forty participants aged from 19–65 years were recruited for this study. Twenty of these had a diagnosis of DSM IV schizophrenia [20]. These were either in-patients of an acute psychiatric ward who were clinically stable and awaiting discharge, or outpatients attending clinics at the Royal Edinburgh Hospital. They were all receiving antipsychotic medication. Antipsychotic medication dose at time of testing was recorded for each patient and using standard published tables was converted into daily chlorpromazine equivalent dosage [21,22]. Twenty healthy volunteers from various community and hospital sources were also recruited as a control group. An estimate of their current level of overall intellectual function was made using the Quick Test [23]. Demographic characteristics for both the experimental and control groups are shown in Table 1 and the clinical details of psychiatric participants can be seen in Table 2.
Table 1 The demographic characteristics-mean (SD) – of the subject groups
Group n (m:f) Age Estimated IQ Years of Education
Schizophrenia 20 (12:8) 39.8 (11.6) 97 (9.5) 13.3 (2.9)
Control 20 (11:9) 39.8 (13.2) 100 (7.7) 13.5 (2.5)
Table 2 Clinical details of the patients with schizophrenia
Age of onset Duration of illness(yrs) Number of admissions Medication
Mean (sd) Mean (sd) Mean (sd) Typical Atypical
Antipsychotics
28.4 (10.6) 10.9 (11) 8.85 (13.2) 40% 60%
Symptom assessment
To assess their present symptomatology, the schizophrenia patients were assessed on the Krawiecka Standardized Scale for Rating Chronic Psychotic Patients [24]. Symptoms present over the previous week, or signs at interview, are assigned a score on a five-point scale (where 0 = absent, 1 = mild, 2 = moderate, 3 = marked, 4 = severe). Ratings are given for four positive symptoms (coherently expressed delusions, hallucinations, incoherence and irrelevance of speech and incongruity), two negative symptoms (poverty of speech and flattened behaviour) and three non specific symptoms (depression, anxiety and psychomotor retardation). As a result, the maximum scores obtainable were 16 for positive symptoms, 8 for negative symptoms and 12 for non specific symptoms. The Krawiecka scores were also used to investigate in more detail the effect specific positive symptomatology had on ToM capabilities: the scores out of four given for delusions and hallucinations were used in this analysis.
All participants in this study gave written, informed consent.
The task
Sixty-three single-image cartoon jokes, printed on A4 cards were generously provided by the authors of previous studies [25]. Thirty-one of these were designated to be 'theory of mind cartoons'. Understanding the humour in these jokes required the attribution of ignorance, false belief or deception to one of its characters and therefore, an analysis of their mental state. The other 32 jokes were Physical ("slapstick") or behavioural in nature and subsequently did not require ToM capabilities for their correct interpretation. All of the images were caption-less. Examples of each type are shown in Figure 1.
Figure 1 (a) An example of the Physical jokes subset. (b) An example of the ToM jokes subset.
It was explained to the subjects that they would be shown cartoons intended to be funny. The two complete sets of cartoons were then shown to each subject in turn. The order in which they were presented was alternated so that half the participants viewed the ToM cartoons first, and half viewed the ToM cartoons second.
The subjects were shown each joke one by one and instructed to indicate to the observer when they believed they had understood its meaning. This response time was then recorded to the nearest second using a stopwatch. The participants then gave a short explanation of their interpretation of the joke's meaning. Responses were scored 1 for a correct answer and 0 for an incorrect answer. For a theory of mind answer to be correct, appropriate mental state language had to be used. Furthermore, participants were asked to subjectively grade each cartoon image for humour and difficulty on a scale of 1–5, where 1 was not funny or very easy and 5 very funny or very difficult respectively.
Simple Physical descriptions of the scenario were required for the Physical joke responses to be scored correct. An example of acceptable responses can be viewed in Table 3.
Table 3 Examples of acceptable and unacceptable replies to jokes featured in Fig 1
(a) Physical Joke
Acceptable responses
'The man is using the swing like a giant Newton's Cradle'
'The children are swinging against each other, like one of those desk toys'
Unacceptable responses
'The man is happy because the children are on swings'
'The man wants to send him on the end flying off the swing, so he gets hurt'
(b)Theory of mind joke
'The man thinks that someone is putting a gun in his back, but it's a guitar'
'The couple don't realise that they are making the man think he is being robbed'
Unacceptable responses
'The couple are waiting for a bus and the man is jumping to reach something'
'The couple are trying to push the man over with the guitar so that they can get on the bus first'
Tests were all performed in quiet, distraction-free rooms.
Statistical analysis
Data analysis was performed using SPSS for Windows Version 11.0.
General linear model repeated measures ANOVA was used to determine the significance of any difference in the Physical versus ToM scores seen between the groups. General linear model ANCOVA controlling for Physical joke score was used to investigate the selectivity of any group difference in ToM capabilities. Linear regression analysis was used to relate Physical and ToM scores to Krawiecka sub-totals for positive, negative and non specific symptoms, individual Krawiecka symptoms, medication dose and joke block presentation order. Independent two-tailed t-tests were used to compare the group score differences in the two conditions (when carrying out simple contrasts following the general linear model repeated measures ANOVA), the average subjective ratings for humour and difficulty assigned to the stimuli by the participants, and the average response times to get the jokes.
Results
Patients with schizophrenia compared to controls
Using general linear model, repeated measures ANOVA, highly significant main effects were found for repeated measure (i.e. joke type: F = 112.9, p < 0.0001) and group (F = 42.6, p < 0.0001) as well as a significant interaction of group by joke (F = 10.3, p = 0.003). Table 4 summarises this.
Table 4 Performance on Physical and ToM jokes between the study groups
Physical jokes score mean (sd) ToM jokes score mean (sd)
Schizophrenia Group 23.3(4.5) 12.7(6.2)
Controls 28.2(2.94) 22.6(2.4)
Follow-up t-tests comparing individuals with schizophrenia to controls were highly significant for both the ToM condition (p < 0.0001) and the Physical condition (p < 0.001).
Additionally, within both the patient and control groups, scores were significantly worse for ToM jokes than Physical jokes (p < 0.0001 for both groups). However, the significant interaction showed that the difference of 10.6 for the patient group was greater than that for the controls (5.6). Using the general linear model, ANCOVA, controlling for Physical joke score, a significant group difference on ToM joke scores was still evident, F = 19.5, p < 0.05.
The two groups were well matched for age, IQ and sex, and any difference between them was shown to be insignificant by independent 2-tailed t-test (p > 0.1). It was unnecessary, therefore, to perform regression analyses to co-vary for these factors.
Subjective joke ratings and response times and order of joke set presentation
It was found via independent T-test analysis that there was no significant difference between the schizophrenia patients and control participants' subjective ratings for humour and difficulty or between the average response times of correct responses (p > 0.05). Results are summarized in Table 5.
Table 5 Subjectivity scores and response times
Picture Condition Average humour score Average difficulty score Average time for correct responses
Controls Physical 2.3 (.48) 1.9 (.62) 5.04 (2.2)
ToM 2.4 (0.35) 1.9 (.57) 5.2 (2.9)
Schizophrenia Group Physical 2.4 (.47) 2.4 (.68) 7.2 (2.5)
ToM 2.6 (0.42) 2.4 (.66) 6.8 (2.7)
NB: Values are means; standard deviations in parentheses.
Furthermore, linear regression indicated that the order of presentation of the joke sets had no significant effect on ToM or Physical joke scores.
Symptoms
Correlations were run to investigate the relationships between performances on ToM and Physical jokes and different symptom scores (assessed on the Krawiecka five-point scale). These data are displayed in Table 6.
Table 6 Krawiecka symptom scores in patients with schizophrenia and their association with performance on ToM and Physical joke conditions.
N Mean Krawiecka Score SD Correlation with ToM* Correlation with Physical*
Positive symptoms 20 5.0 3.2 -0.029 0.36
Negative Symptoms 20 1.6 1.8 -0.108 0.015
Non specific Symptoms 20 1.6 1.8 0.100 0.157
Delusions 20 2.5 1.6 0.153 -0.083
Hallucination 20 1.9 1.7 -0.053 0.173
Depression 20 0.65 0.875 0.306 0.222
Incoherence of Speech 20 0.3 0.657 -0.194 0.097
Poverty of Speech 20 0.45 0.826 -0.186 -0.102
* None of these correlations reach significance
As stated, performance was not significantly reduced in association with increasing severity of positive or negative symptoms as a whole or delusions and hallucinations specifically.
The features of depression, incoherence and poverty of speech were also analysed to see if they could be having an effect on the patients ToM and Physical joke performance but there were no significant findings.
The converted equivalent daily chlorpromazine patient medication doses were correlated to performance and also found to be non significant for both cartoon conditions.
Discussion
Schizophrenia subjects compared to controls
This study showed that individuals with schizophrenia and normal IQ had a poorer understanding of both types of jokes (and at least a reduced ability to relay their humorous intent) than matched healthy controls. This is to be expected, as schizophrenia patients have previously been reported to show poor appreciation of humour [3]. It seems unlikely that this is explained by depression as regression analysis showed it not to be significantly related to poor ToM performance.
However, the difference between the Physical and ToM joke scores was significantly greater for schizophrenia patients, than controls. This implies that it is some aspect of the schizophrenia disease process that is associated with ToM impairment in the patient group, rather than a general difficulty with appreciation of humour.
If the schizophrenia group had a poorer understanding of the jokes then we would expect this to be reflected in the subjective gradings for humour and difficulty. As shown in table 5, the schizophrenia group actually graded the jokes non-significantly higher for both humour and difficulty. Furthermore, despite both groups performing significantly worse in the ToM condition than in the Physical condition, they both graded the two joke sets as equally difficult. Possible explanations for this could be that people were instructed that the cartoons were meant to be funny and so consequently may have stated that a joke was humorous even if they didn't find a joke funny. The subjective gradings of the jokes did not necessarily require a correct understanding of the joke for a numerical value for humour and difficulty to be assigned. Everyone could give numerical gradings for a joke but not everyone could correctly describe the jokes or use the relevant mentalising language in their joke description.
It was found that both groups found the ToM jokes significantly more difficult than the Physical ones. The former were certainly more detailed and by their very nature were comprised of characters in ToM scenarios. It could be that these jokes were more difficult to understand, but there was no significant difference between the response times of the two joke types for either group. Poor verbal report of mentalistic terms may be an intrinsic feature of schizophrenia and this could have resulted in this schizophrenia group's poor performance on this set of jokes.
Language and thought are intrinsically linked and the question arises as to whether disordered verbalisation in schizophrenia is a speech disturbance only or part of a disorder in thinking [26]. Likewise, the observed ToM deficit seen in this study could reflect a lack of response in mentalistic terms, related either to a specific deficit in inferential skills or to a more general inability to verbalise others mental states [16]. As regards our patients' verbalisation skills, they all scored none or low Krawiecka scores for the symptoms of poverty of speech and incoherence/irrelevance of speech. We therefore believe that their poor performance was the result of a compromised ToM function rather than a general verbalisation expression deficit.
This data suggests that, as predicted, schizophrenia patients have problems in interpreting the thoughts of others, supporting the findings of previous work [3]. The closely matched demographic characteristics of the two groups, suggests that problems in 'mentalising' evident in schizophrenia, are not simply attributable to the influence of factors such as age, sex and, importantly, IQ.
There is however an alternative interpretation to these results. The individuals with schizophrenia may not be showing a domain -specific difficulty with ToM function but rather may be performing differentially more poorly than the control group on the more difficult ToM condition, such that the observed deficit could reflect a differential sensitivity to increased task difficulty.
Symptom specific findings
When the obtained totals for positive Krawiecka symptoms were analysed it was found that there was not a significant relationship between higher positive symptomatology and poor ToM performance, contrary to what had been predicted. Closer scrutiny of individual positive symptoms also revealed that neither delusions, hallucinations nor speech incoherence were significantly linked to an impaired ToM performance. Previous studies have shown paranoid delusions to be significantly related to poor ToM performance, in both first and second order ToM tasks and in both verbal and pictorial paradigms [[27,28] 32]. Interestingly, Langdon et al [14], also using a pictorial paradigm, found no evidence linking poor mentalising capabilities to positive symptoms.
These findings might be attributed to several individuals who despite scoring the maximum Krawiecka score (4) for delusions, hallucinations or both, performed similarly to controls in the ToM condition.
Alternatively, perhaps the nature of our patients' delusions and hallucinations may not be those specifically implicated in ToM impairment. Unfortunately, our sample size was too small to allow further investigation of patients with different types of delusion. Unlike the findings of previous research, negative features of schizophrenia were not associated with ToM capabilities. However, the mean Krawiecka scores for these features were low within the subject group, and our number of subjects was relatively small.
Limitations and further work
This study was limited especially for symptom sub-groups analyses, by its relatively small sample size, although we did find disease effects. With a large sample, further symptom-specific sub-groups could be made (e.g. different types of delusions or hallucinations, formal thought disorder, different aspects of negative symptomatology, etc). Furthermore, another control group of non-schizophrenia, psychiatric patients may have been useful to explore more closely the role of diagnosis as opposed to symptoms. One of our previous studies used a psychiatric control group of patients with a psychotic affective disorder and found that positive psychotic symptomatology was linked to poor ToM performance and was not diagnosis specific [29]. This implies that ToM deficits are not necessarily specific to schizophrenia but could be related to psychoses and specifically to the positive symptoms of delusions and hallucinations. Although, as acknowledged above, we found no evidence for such an association in the present study.
We believe that the Physical cartoons themselves acted as an adequate internal control. If the schizophrenia group had performed as poorly on the Physical cartoons as they did on the ToM cartoons, this could imply either a general verbalization deficit or a general cognitive impairment. Since this was not the pattern found, our results count against a domain-general interpretation of this type. Furthermore, as mentioned previously, regression analysis showed no significant effect of language impairment, as assessed using the Krawiecka symptoms of poverty of speech and incoherence of speech, on ToM joke performance. ANCOVA also showed that the group differences on the ToM jokes could not be accounted for by the group differences on the Physical jokes. This was taken as evidence for an observable and selective compromise of ToM capacity within the schizophrenia group.
However, an unrelated cognitive neuropsychological task could have been implemented testing another cognitive domain (e.g. executive function, working memory) and this could have been used to further elaborate whether the observed compromised ToM function was a specific deficit or secondary to general cognitive impairment [see for example, 18–19 who used the Tower of London task in this way].
Further research is then required in ToM and schizophrenia to see whether the presence of schizophrenia itself is enough to impair ToM capabilities or whether ToM impairment is due, instead, to presence of particular symptoms or presence of some general neuropsychological deficit. A further question that we did not address at all in the present study was whether the ToM deficits observed in schizophrenia could be state (related to fluctuating symptom severity) or trait in nature.
Conclusion
The schizophrenia group performed significantly worse in both the Physical and ToM conditions on this visual joke task than the matched control group. The performance on the ToM condition was significantly worse and is taken as evidence for a compromised ToM capability in the schizophrenia group which is in keeping with previous research. In this instance poor ToM performance could not be significantly linked to any particular symptomatology as had been hypothesised.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
DM conceived and designed the study, collected neuropsychological test data and drafted the manuscript. HT helped implement the study and collect neuropsychological test data and co wrote first draft. DMac and DCO were involved in the psychiatric ratings of the patients and revisions of later drafts. PM advised on statistical analysis and helped to write the corresponding sections. SL supervised clinical aspects of study and revised later drafts and ECJ revised final draft and approved this version to be published.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We would like to thank Margaret Ferguson for her help in obtaining the patients psychiatric case histories and Dr Helen Gallagher for kindly sharing her cartoon images with us. Finally, we would like to thank all the participants in the study who kindly volunteered their time.
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| 15730565 | PMC554116 | CC BY | 2021-01-04 16:33:03 | no | BMC Psychiatry. 2005 Feb 24; 5:12 | utf-8 | BMC Psychiatry | 2,005 | 10.1186/1471-244X-5-12 | oa_comm |
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Med ImmunolMedical Immunology1476-9433BioMed Central London 1476-9433-4-21574061910.1186/1476-9433-4-2CommentaryThe Future of Smallpox Vaccination: is MVA the key? Slifka Mark K [email protected] Vaccine and Gene Therapy Institute, Oregon Health & Sciences University, 505 NW 185th Avenue, Beaverton, OR 97006, USA2005 1 3 2005 4 2 2 27 2 2005 1 3 2005 Copyright © 2005 Slifka; licensee BioMed Central Ltd.2005Slifka; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Eradication of the smallpox virus through extensive global vaccination efforts has resulted in one of the most important breakthroughs in medical history, saving countless lives from the severe morbidity and mortality that is associated with this disease. Although smallpox is now extinct in nature, laboratory stocks of this virus still remain and the subject of smallpox vaccination has gained renewed attention due to the potential risk that smallpox may be used as a biological weapon by terrorists or rogue states. Despite having the longest history of any modern vaccine, there is still much to be learned about smallpox vaccination and the correlates of protection remain to be formally defined. This Commentary will discuss the strengths and weaknesses of traditional smallpox vaccination in comparison with immunization using modified vaccinia virus Ankura (MVA), a non-replicating virus with a strong safety record but weakened immunogenicity.
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Introduction
Smallpox (Variola major) is a virus that no longer exists in the wild, but during its reign it caused 20–30% mortality in previously unvaccinated individuals and often left survivors with deeply pitted scars for life [1]. The last case of smallpox in the U.S. occurred in 1949 and the last case of naturally occurring smallpox in the world occurred in Somalia in 1977. Smallpox has no known animal reservoir, so in the absence of any more natural cases of human smallpox being recorded after 1977, the virus was considered fully eradicated in 1980 [1]. Despite extinction in nature, smallpox virus stocks still reside in secure locations within the U.S. and Russia but it is impossible to know if other undeclared stocks of smallpox remain in other countries [2]. Moreover, in the age of genetic engineering it is possible that more virulent strains of smallpox or other potentially dangerous orthopoxviruses could be developed and unleashed in an effort of bioterrorism. Although the potential for developing a pathogen more lethal than wild smallpox is theoretically possible [2,3] it would by no means be a simple task to undertake and the outcome would likewise be uncertain [4,5]. Nevertheless, smallpox is considered a potential risk to national security and efforts are underway to prepare the United States and several other countries for a deliberate release of smallpox as a biological weapon.
The first line of defense against smallpox is vaccination. Smallpox vaccination is highly effective at protecting against lethal infection and even if only partial protective immunity is attained, this often still results in survival and decreased viral spread to others (Chapter 4, pages 189–90 of [1]). The smallpox vaccine was discovered by Edward Jenner, who was the first to prove that infection by cowpox resulted in protective cross-reactive immunity against smallpox [6]. Dr. Jenner not only demonstrated that cowpox could induce protective immunity, but in answer to critics of his day who argued that this form of immunity would only be short-lived, he demonstrated full protection against smallpox in several individuals at 25, 27, 31, 38, and even 53 years after cowpox infection [6,7]. It is not a coincidence that such long-term time points were examined. As Dr. Jenner noted, "I have purposely selected several cases in which the disease [i.e. cowpox] had appeared at a very distant period previous to the experiments made with variolous matter, to show that the change produced in the constitution is not affected by time." [6].
Following the elegant studies initiated by Edward Jenner, the world was eventually freed of the scourge of smallpox following a massive global eradication campaign [1] and although hugely successful, there were still many questions that were left unanswered. These questions represent the topics of this commentary. For instance, by then end of the 1960's it was realized that smallpox vaccination was the cause of a substantial number of adverse events and resulted in a lethal infection in approximately one out of one million people who received this live viral vaccine. In Germany, an extremely safe attenuated strain of vaccinia, known as modified vaccinia virus, Ankura (MVA) was developed [8,9], but it was only used as primary vaccination followed by traditional smallpox vaccination. Moreover, its efficacy against smallpox was never directly tested due to the eradication of smallpox shortly thereafter and the question remains as to whether it would induce full or only partial immunity when faced against fully virulent smallpox. Although the virology, pathology, and epidemiology of smallpox are well described [1], there is a relative dearth of information regarding the immunology of smallpox and smallpox vaccination. Most importantly, there is currently no consensus on the immunological correlates of protection, making it difficult to implement rationale vaccine design when it not established which immunological benchmarks are necessary for full or even partial protection. Recent quantitative analysis of the cellular and humoral immune response following smallpox vaccination, coupled with historical evidence of protective immunity, are beginning to shed light on this important subject.
Discussion
The question of safety following smallpox vaccination
The current smallpox vaccine was prepared prior to 1982 under standards that today, would be unlikely to be approved by the FDA. To produce the vaccine, the torso of bovine calves were shaved, and their skin scarified (scratched) with an inoculum containing vaccinia virus. After the infection has reached a point in which a great deal of exudate was observed, the purulent lymph material was scraped from the infected cow, clarified, and lyophilized in the presence of antibiotics. The inoculum is later reconstituted with diluent containing 0.25% phenol to further decrease bacterial contamination (<200 viable bacterial/mL after reconstitution; see Dryvax package insert). Unlike most other vaccines that are administered by subcutaneous or intramuscular injection, vaccinia virus replicates poorly under these conditions and optimal vaccination occurs by scarification of the virus inoculum onto the skin surface [10]. Viral replication on the skin surface typically results in a vesicular or pustular lesion that is described as a "take" which later crusts over and sloughs off, leaving behind a small scar. Since the time of Edward Jenner, the presence of a vesicular or pustular lesion has remained the gold-standard measurement of successful vaccination.
The success of smallpox vaccination does not come without unwanted consequences. The most common side effects of smallpox vaccination are fever and other flu-like symptoms. More serious adverse events include inadvertent inoculation (529 cases/106 doses), generalized vaccinia (242 cases/106 doses), eczema vaccinatum (39 cases/106 doses), vaccinia necrosum (1.5 cases/106 doses), encephalitis (12 cases/106 doses), or death (~1 death/106 doses) [11]. The mortality rate is highest for infants that are <1 year of age (5 deaths/106 doses) whereas the mortality rate for older children and adolescents aged 1–4 or 5–19 is approximately 0.5 deaths/106 doses (see also [12,13] and Dryvax package insert). It is rare for adults to die after smallpox vaccination; of 68 deaths attributed to smallpox vaccination over a 9-year period of evaluation, only 8/68 (12%) of cases occurred in adults. Five of the eight adults were over the age of 60 and 4/8 of the lethal cases occurred in adults who were diagnosed with terminal cancer at the time of vaccination [11].
Myopericarditis is a recently identified adverse event that occurs at a rate of ~124 cases/106 smallpox vaccinations [14,15]. In 2003, there were three fatal heart attacks that were temporally related to smallpox vaccination and this triggered critical evaluation of any vaccine-related cardiac events thereafter. It was later realized that all three heart attack victims (age 55, 55, and 57) had pre-existing risk factors for cardiac disease including hypertension, hyperlipidemia, and smoking. At autopsy, none of the three heart attack victims showed signs of myo/pericarditis but the deaths were instead linked directly to ischemic events [16]. A retrospective study analyzing the number of cardiac deaths among approximately 80,000 death certificates issued near the time of a massive smallpox vaccination effort in New York City in 1947 has also brought new insight into the dangers of smallpox vaccine-induced myocarditis[16]. Following an imported case of smallpox, about 6.4 million people of all ages were vaccinated in a one-month period. Analysis of the frequency of cardiac deaths before, during, and after the vaccination campaign failed to show a statistical increase in these events. Moreover, analysis of 64 recently identified cases of myocarditis in the U.S. military smallpox vaccination program found that approximately 80% of patients reported no long term sequelae and 100% of patients demonstrated objective normalization of echocardiography, electrocardiography, laboratory testing, graded exercise testing, and functional status [15].
To overcome some of the problems associated with the current calf lymph smallpox vaccine (Dryvax), an improved tissue culture-derived vaccine has been recently developed [17]. Unlike Dryvax, this new vaccine (designated ACAM1000) is produced under sterile GMP conditions, so bacterial contamination is avoided. Also, unlike Dryvax which contains a heterologous mixture of virus variants with some differing in their neurovirulence [17,18], ACAM1000 is clonally derived (triple plaque-purified) and tested extensively for low neurovirulence in animal studies. Thus, it is hopeful that ACAM1000 vaccine will have reduced risk of encephalitis, one of the major risk factors following smallpox vaccination. It remains to be seen whether or not myopericarditis or other potentially serious adverse events will be affected by the use of this new vaccine.
Both Dryvax and ACAM1000 represent replication-competent smallpox vaccines and since these vaccines are based on the use of live viruses, there is always an inherent risk of severe adverse events or death (albeit rare) in vaccinees that have unknown or undisclosed immunodeficiencies at the time of vaccination. To overcome the hazards of replicating viruses, a highly attenuated strain of vaccinia, designated Modified Vaccinia virus Ankara (MVA) was developed by growing the virus for >500 passages on chicken embryo fibroblasts (CEF) and following the loss of about 15% of its parental genome, it no longer was capable of replicating in most mammalian cells, including human cells [19]. Other strains of non-replicating orthopoxviruses have also been developed, including NYVAC, which was derived from vaccinia virus following a deletion of 18 genes – including those encoding virulence factors and human host range replication, and ALVAC, an attenuated viral vector that is based on canarypox, an avipoxvirus that grows only in avian species. In one study, recombinant NYVAC and recombinant ALVAC expressing JEV proteins were found to be well tolerated but more reactogenic than the commercially available formalin-inactivated JEV vaccine [20]. Of these attenuated poxvirus vaccine strains, MVA is the one with the most extensive history of safety in humans. Beginning in 1968, >100,000 people in Germany were vaccinated with MVA (followed by traditional smallpox vaccination) and although it was well tolerated, MVA was not used alone and since there were no smallpox outbreaks at that time, its efficacy in the face of an actual smallpox outbreak has not been tested. With an excellent safety profile in humans and in animal models of immunodeficiency, recombinant MVA expressing candidate immunogens from a variety of infectious agents (e.g. HIV, HPV, and malaria) or tumor antigens (e.g. melanoma) have now reached Phase I and Phase II clinical trials [21,22]. However, the role of MVA in the future of smallpox vaccination has yet to be decided and will likely be determined by the outcome of clinical trials that directly compare the immunogenicity of MVA to either Dryvax or ACAM1000, two vaccines that are likely to provide the required high levels of protective immunity that will be necessary in the event of an accidental or deliberate smallpox outbreak.
Quantitative analysis of vaccine efficacy
Edward Jenner developed the first test for vaccine efficacy when he immunized subjects with cowpox and then later challenged them with smallpox by inoculation. If a subject showed no secondary smallpox lesions indicative of systemic spread, then the individual was believed to have protective immunity. He noted that cowpox-vaccinated individuals who developed a pox-like lesion at the vaccination site were fully protected against smallpox challenge. To this day, most studies still use the identification of a "take" (vesicular or pustular lesion) as evidence of successful vaccination. However, there are rare cases of revaccinated subjects who present with vesicle formation after smallpox vaccination, but show no detectably boosted cellular or humoral immunity [23] and M.K. Slifka, unpublished results). For this reason, it is important that pre- and post-vaccination serum antibody and peripheral T cell responses be monitored in individuals who plan to work with virulent orthopoxviruses or who would be expected to enter a hot zone in the case of a smallpox outbreak.
Quantitative immunology is beginning to gain acceptance as a measurement of vaccine efficacy, although the Jennerian vesicle at the site of smallpox vaccination still remains the primary endpoint of successful vaccination in most studies [17,23-25]. One of the reasons why quantitative immunology is more important now than ever before is that with some vaccines such as MVA, there is no vesicle formed due to the route of immunization – so unless immunological measurements are made, then vaccine immunogenicity cannot be determined or compared. Humoral immunity following smallpox vaccination was measured in the 1960's and 1970's by means of neutralizing assays (primarily against the IMV form of vaccinia or smallpox) and today, humoral immune responses are quantitated by analysis of neutralizing activity against IMV or EEV forms of vaccinia or by the use of ELISA assays using whole-virus lysate and/or individual vaccinia IMV or EEV proteins. Moreover, the vaccinia-specific memory B cell response has also been recently studied [26] providing the first direct quantitation of this memory cell subset. Quantitation of the antiviral T cell response mounted after smallpox vaccination was not an option during the smallpox era because the tools and technology were not available for analysis of cellular immunity. In contrast, today there are now several sophisticated techniques that can be used to monitor antiviral T cell responses directly ex vivo including vaccinia-specific IFNγ ELISPOT assays [17,23,26-28], intracellular cytokine staining analysis (ICCS) [29-33], or peptide/MHC Class I tetramer staining [28,30]. Each of these techniques has high sensitivity and high specificity and each has both advantages and disadvantages. For instance, the IFNγ ELISPOT assay provides a highly sensitive calculation of IFNγ-producing cells, but one drawback is that it only allows detection of one cytokine at a time and the phenotype of the IFNγ-producing subset (CD4, CD8 or possibly NK cells) must be determined by purifying each population independently prior to the assay. The advantage of ICCS is that antiviral T cells can be quantitated based on the production of more than one cytokine and the phenotype of the responding lymphocyte subset is directly determined by flow cytometry. The main disadvantage of ICCS is that a relatively large number of cells are required in order to detect rare populations of virus-specific T cells. The advantage of using peptide/MHC tetramers is that CD8+ T cells can be quantitated regardless of their cytokine profiles, but the main disadvantages with this approach include the lack of identified CD4+ T cell/MHC Class II epitopes, the requirement for knowledge of the MHC haplotype of the subject, and the inability to measure the total antiviral T cell response, which may be directed against any number of immunodominant and subdominant peptide epitopes.
Direct quantitation of the antiviral immune response induced by smallpox vaccination has been critical for several recent advances in orthopoxvirus immunobiology – especially since smallpox has been eradicated and other human orthopoxvirus outbreaks are too small and sporadic (e.g. cowpox [34,35] or monkeypox [36,37]) to be feasible for field studies of protective efficacy. For example, Weltzin et al. [17] not only compared the neutralizing titers induced by Dryvax vs. ACAM1000 vaccines, but also compared vaccinia-specific T cell responses by IFNγ ELISPOT as well as by cytolytic T cell assays and proliferation assays that, although less quantitative than the ELISPOT assay, are nevertheless important for analysis of antiviral T cell functions. These techniques allowed the investigators to demonstrate non-inferiority of the new tissue culture-derived smallpox vaccine compared to the Dryvax vaccine that is currently in use.
Another study by Earl et al. [38], used quantitative immunology to compare vaccine efficacy of Dryvax, MVA followed by a Dryvax booster, and MVA followed by an MVA booster in a cynomolgus monkey (Macaca fascicularis) model of monkeypox infection. Using ELISA assays and neutralizing assays to measure vaccinia-specific antibody responses, and ICCS to measure antiviral T cell responses, the authors showed that antiviral immunity appeared similar between these three groups. Likewise, each of these groups were protected against lethal monkeypox challenge, although primates that only received MVA plus an MVA booster showed partial protection with 6/6 animals presenting with 1–36 monkeypox lesions (compared to >500 monkeypox lesions in the unvaccinated controls). This indicates that two MVA vaccinations are required to induce partial immunity whereas MVA followed by Dryvax immunization or a single Dryvax immunization each provides full immunity. The protective efficacy of a single dose of MVA is unknown, but based on the results of the Earl et al. study [38] wherein two doses were required to elicit partial immunity, it appears likely that a single dose of MVA would be of only low protective value in the face of a virulent orthopoxvirus infection. This is not surprising since MVA, NYVAC, and ALVAC (all replication-deficient vaccines), typically require booster doses to be administered in order to elicit optimal immune responses. Moreover, several prime-boost strategies (including DNA vaccination followed by MVA booster) are being tested in order to overcome the low immunogenicity of MVA alone [21]. This is different from most replicating viruses [39] including vaccinia, which require only one immunization (or infection) to induce optimal and often lifelong immunity [5,26,32,40]. Approximately half of the U.S. population has been vaccinated against smallpox and continue to maintain pre-existing antiviral immunity [26,32] and this may have an impact on the efficacy of MVA vaccination. For instance, studies involving the closely related, non-replicating NYVAC strain have found that pre-existing immunity significantly effected the outcome of vaccination [20]. In this particular study, immunization with recombinant NYVAC expressing JEV proteins failed to induce protective antibodies in 0/5 vaccinia pre-immune individuals and although 5/5 vaccinia-naive subjects seroconverted after NYVAC-JEV immunization, the resulting neutralizing titers were substantially lower than that observed in subjects who received the standard formalin-inactivated JEV vaccine [20]. Oddly, the authors noted that both the NYVAC-JEV and the ALVAC-JEV vaccines (2 doses administered 28 days apart) failed to induce a detectable anti-vaccinia neutralizing response.
It is difficult to speculate the efficacy of MVA vaccination of humans as a method of protection against smallpox. It is possible that booster doses of MVA would provide strong enough immunity to at least protect against lethal smallpox, similar to its ability to protect primates from lethal monkeypox [38]. On the other hand, clinical trials could indicate that MVA vaccination alone may be too inconsistent or only induce low levels of immunity that would be considered inferior to live smallpox vaccination with calf-lymph or tissue culture-derived vaccine preparations. Unlike live smallpox vaccination, which can be used as an effective post-exposure treatment against the lethal consequences of smallpox [1,5], it is unlikely that the low immunogenicity of MVA would be capable of fulfilling this role. Moreover, in the event of a smallpox outbreak there would not be enough time to administer two or more doses of MVA if people were at a high risk of exposure. Under these circumstances, use of live viral vaccines would be critical for ring vaccination or mass vaccination scenarios. This is not to say that MVA would not have a potential role in biodefense strategies. For instance, in a pre-event scenario, one could foresee the use of MVA followed by vaccination with a live viral vaccine such as Dryvax or its equivalent. Under these circumstances, MVA would likely induce partial immunity that would reduce the adverse events that are associated with traditional smallpox vaccination. Moreover, MVA is the vaccine of choice in immunocompromised individuals with suppressed immune systems (cancer patients, organ-transplant patients, AIDS patients, etc.) who would otherwise be contra-indicated for administration of the live viral vaccines. More studies will be needed to determine the immunogenicity and protective efficacy of MVA and other related non-replicating vaccines in terms of their potential to counter a smallpox outbreak.
The need for formal definition of protective immunity and the correlates of immunity
There is more than one definition of protective immunity against smallpox. There is protection against infection, protection against disease, and protection against death. Of these, one might argue that protection against lethal infection is the ultimate definition of protective immunity. However, protection against infection and protection against disease not only reduce the morbidity of an outbreak, but these high levels of protective immunity are also associated with reduced virus spread to others (Chapter 4, pages 189–90 of [1]). Protection against infection is the most rare level of protective immunity since this requires that an infection be blocked at the point of entry. A meta-analysis of 10 epidemiological studies on smallpox noted that on average, the virus only infected ~4% of previously vaccinated household contacts (Chapter 4, pages 189–90 of [1]). However, these results were based on whether or not the vaccinated contacts showed disease symptoms and did not necessarily prove that they were never infected per se. Further analysis indicated that approximately 10% of previously vaccinated household contacts of smallpox patients were actually infected with smallpox as demonstrated by isolation of infectious virus from pharyngeal mucosa, but only 4/34 (12%) of these subjects developed the clinical symptoms of smallpox [41]. Moreover, another study showed that about 50% of previously vaccinated, disease-free contacts demonstrated serological results indicative of a recent orthopoxvirus infection [42]. This suggests that most instances of "protection against infection" may not be complete protection. Instead, many of the individuals thought to have had protection against infection may have actually been infected with smallpox but didn't know it because they were clinically asymptomatic. Similar to these historical studies, during the U.S. monkeypox outbreak in 2003 [37] we have identified three previously unreported cases of monkeypox in subjects who had received smallpox vaccination many years earlier and were unaware that they had become infected with monkeypox because they were spared any recognizable disease symptoms (M.K. Slifka, unpublished data).
A major issue in the smallpox field is that there is no consensus on what is exactly required for protective immunity against this disease. In the age of quantitative immunology, we are beginning to find clues that might help answer this age-old question. In a study of >300 subjects, the levels of vaccinia-specific serum antibody and antiviral T cell responses were determined from 30 days to up to 75 years after smallpox vaccination [32]. Antiviral antibody responses were maintained essentially for life, whereas antiviral CD4+ and CD8+ T cell responses declined with a half-life of approximately 8–15 years, with CD4+ T cell memory being more stable than CD8+ T cell responses. Similar duration of antibody production was demonstrated by other recent studies as well as older literature [24,26,40]. Moreover, the gradual loss of T cell memory was also confirmed [26], as was the differential loss of CD8+ T cell memory over CD4+ T cell memory [33]. Based on historical analysis of vaccine-mediated protection against lethal smallpox (dating back to the age of Edward Jenner), indicates that protective immunity is often lifelong [40]. One might argue that if protective immunity against smallpox had an absolute requirement for antiviral CD4+ or CD8+ T cells, then protective immunity would not be life-long but would instead be more likely to decline at the same rates as T cell memory. This suggests that in humans, humoral immunity might play a more important role in protection against lethal infection than cellular immunity [5,40]. Smallpox disease symptoms become more pronounced with increased time since vaccination [43], and it likely that the combination of intact cellular and humoral immunity together provide the most robust antiviral immunity. Recent vaccinia studies in mice using either antibodies to deplete T cell subsets or mouse strains that are genetically deficient in CD4+ T cells or CD8+ T cells (or both) have indicated that, as long as there is strong humoral immunity against vaccinia, T cell memory is dispensable for protective immunity [44-46]. This result corresponds well with other studies in which protection against vaccinia or smallpox has been clearly demonstrated by the adoptive transfer of immune serum in humans or by transfer of monoclonal neutralizing antibodies in animal models [5,40]. On the other hand, adoptive transfer of virus-specific T cells and experiments in mice that are genetically deficient in B cells, also indicate that in the absence of pre-existing antibody responses, memory T cells can play an important role in protection. This indicates that cellular and humoral immunity have overlapping roles in protective immunity and one may compensate for a deficiency in the other.
Based on the overlapping roles for T cell and B cell memory on protective immunity, is there any chance that a consensus can be reached in regard to defining an immunological correlate of immunity? One way to address this issue is to examine historical studies in which an immunological correlate was identified. In this regard, there are two independent studies in which investigators showed that subjects with vaccinia-specific neutralizing antibody titers of >1:20 [47] or ≥ 1:32 [48] were fully protected against smallpox. The latter study was the largest of the two and showed that 3/15 (20%) of subjects with titers below 1:32 contracted smallpox whereas 0/127 (<1%) of subjects with antibody titers of ≥ 1:32 contracted the disease. The only caveat to these studies is that the subjects also received post-exposure vaccination at the same time that serum samples were drawn, so the full protection afforded to the subjects with high pre-existing neutralizing titers may have been due to their high antibody titers, or the combination of strong pre-existing antibody titers in the context of post-exposure vaccination. A neutralizing titer of 1:32 is equivalent to a vaccinia ELISA titer of 944 Elisa Units (EU) or approximately 4 International Units (IU) of the WHO/NIBSC International Smallpox Serum Standard [32]. Interestingly, about 50% of subjects vaccinated in the distant past maintain neutralizing titers of 1:32 or greater for life and this coincidentally is the same proportion of vaccinated smallpox contacts who demonstrated fully protective immunity when exposed to smallpox-infected family members [42]. This leads one to speculate that 4 IU may constitute a protective level of serological immunity against smallpox. This is a testable hypothesis and to determine if this is a correlate of protection, it will be important to perform adoptive transfer of VIG or its equivalent into non-human primates (resulting in antiviral serum antibody levels of approximately 1:32) and determine if they are protected against a lethal orthopoxvirus infection, such as following monkeypox challenge. This experiment would prove or disprove the hypothesis that a serological correlate of protective immunity exists and may lay the foundation for future vaccine design. Of note, the protective level of yellow fever immunity (log10 ≥ 0.7 neutralizing titer) was also established in non-human primates by simply vaccinating groups of animals with different doses of the yellow fever vaccine, quantitating vaccine-induced antibody levels, and then challenging them with a lethal dose of yellow fever virus [49]. This serological correlate of protection (which ignores the role of vaccine-induced T cell responses) has been established as the benchmark of protective yellow fever virus-specific immunity for over 30 years and demonstrates the potential for using animal models to correlate protective immunity in humans.
Conclusion
Traditional smallpox vaccination has lead to the global eradication of smallpox but continues to be used today in an effort to thwart the potential use of smallpox as a biological weapon. Since this vaccine employs the use of a live virus, there is an inherent risk of adverse events, although these are generally quite rare. New generation smallpox vaccine candidates include MVA and other non-replicating poxviruses and although they demonstrate a high degree of safety, their immunogenicity appears to be substantially lower than traditional smallpox vaccination with live vaccinia virus. The role of MVA and traditional smallpox vaccination (or a combination thereof) in future vaccination campaigns has yet to be determined. However, developing a consensus on the definition of what is required for protective immunity and defining an immunological correlate of immunity would aid in the evaluation of current and future vaccine approaches.
List of Abbreviations
MVA modified vaccinia virus Ankura
GMP good manufacturing practices
HIV human immunodeficiency virus
CEF chicken embryo fibroblasts
HPV human papilloma virus
FDA Federal Drug Administration
JEV Japanese Encephalitis Virus
IMV Intracellular mature virus
EEV Extracellular enveloped virus
ELISA Enzyme-linked immunosorbent assay
IFNγ Interferon-gamma
ELISPOT Enzyme-linked immunosorbent Spot assay
ICCS Intracellular cytokine staining
MHC Major histocompatibility complex
VIG Vaccinia immune globulin
WHO World Health Organization
NIBSC National Institute of Biological Standards and Control
EU Elisa Units
IU International Units
Competing Interests
OHSU and Dr. Slifka have a financial interest in Najít Technologies, Inc., a company that may have a commercial interest in the results of this research and technology. This potential conflict was disclosed to the OHSU Conflict of Interest in Research Committee and an approved management plan was implemented.
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| 15740619 | PMC554756 | CC BY | 2021-01-04 16:39:09 | no | Med Immunol. 2005 Mar 1; 4:2 | utf-8 | Med Immunol | 2,005 | 10.1186/1476-9433-4-2 | oa_comm |
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Med ImmunolMedical Immunology1476-9433BioMed Central London 1476-9433-4-21574061910.1186/1476-9433-4-2CommentaryThe Future of Smallpox Vaccination: is MVA the key? Slifka Mark K [email protected] Vaccine and Gene Therapy Institute, Oregon Health & Sciences University, 505 NW 185th Avenue, Beaverton, OR 97006, USA2005 1 3 2005 4 2 2 27 2 2005 1 3 2005 Copyright © 2005 Slifka; licensee BioMed Central Ltd.2005Slifka; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Eradication of the smallpox virus through extensive global vaccination efforts has resulted in one of the most important breakthroughs in medical history, saving countless lives from the severe morbidity and mortality that is associated with this disease. Although smallpox is now extinct in nature, laboratory stocks of this virus still remain and the subject of smallpox vaccination has gained renewed attention due to the potential risk that smallpox may be used as a biological weapon by terrorists or rogue states. Despite having the longest history of any modern vaccine, there is still much to be learned about smallpox vaccination and the correlates of protection remain to be formally defined. This Commentary will discuss the strengths and weaknesses of traditional smallpox vaccination in comparison with immunization using modified vaccinia virus Ankura (MVA), a non-replicating virus with a strong safety record but weakened immunogenicity.
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Introduction
Smallpox (Variola major) is a virus that no longer exists in the wild, but during its reign it caused 20–30% mortality in previously unvaccinated individuals and often left survivors with deeply pitted scars for life [1]. The last case of smallpox in the U.S. occurred in 1949 and the last case of naturally occurring smallpox in the world occurred in Somalia in 1977. Smallpox has no known animal reservoir, so in the absence of any more natural cases of human smallpox being recorded after 1977, the virus was considered fully eradicated in 1980 [1]. Despite extinction in nature, smallpox virus stocks still reside in secure locations within the U.S. and Russia but it is impossible to know if other undeclared stocks of smallpox remain in other countries [2]. Moreover, in the age of genetic engineering it is possible that more virulent strains of smallpox or other potentially dangerous orthopoxviruses could be developed and unleashed in an effort of bioterrorism. Although the potential for developing a pathogen more lethal than wild smallpox is theoretically possible [2,3] it would by no means be a simple task to undertake and the outcome would likewise be uncertain [4,5]. Nevertheless, smallpox is considered a potential risk to national security and efforts are underway to prepare the United States and several other countries for a deliberate release of smallpox as a biological weapon.
The first line of defense against smallpox is vaccination. Smallpox vaccination is highly effective at protecting against lethal infection and even if only partial protective immunity is attained, this often still results in survival and decreased viral spread to others (Chapter 4, pages 189–90 of [1]). The smallpox vaccine was discovered by Edward Jenner, who was the first to prove that infection by cowpox resulted in protective cross-reactive immunity against smallpox [6]. Dr. Jenner not only demonstrated that cowpox could induce protective immunity, but in answer to critics of his day who argued that this form of immunity would only be short-lived, he demonstrated full protection against smallpox in several individuals at 25, 27, 31, 38, and even 53 years after cowpox infection [6,7]. It is not a coincidence that such long-term time points were examined. As Dr. Jenner noted, "I have purposely selected several cases in which the disease [i.e. cowpox] had appeared at a very distant period previous to the experiments made with variolous matter, to show that the change produced in the constitution is not affected by time." [6].
Following the elegant studies initiated by Edward Jenner, the world was eventually freed of the scourge of smallpox following a massive global eradication campaign [1] and although hugely successful, there were still many questions that were left unanswered. These questions represent the topics of this commentary. For instance, by then end of the 1960's it was realized that smallpox vaccination was the cause of a substantial number of adverse events and resulted in a lethal infection in approximately one out of one million people who received this live viral vaccine. In Germany, an extremely safe attenuated strain of vaccinia, known as modified vaccinia virus, Ankura (MVA) was developed [8,9], but it was only used as primary vaccination followed by traditional smallpox vaccination. Moreover, its efficacy against smallpox was never directly tested due to the eradication of smallpox shortly thereafter and the question remains as to whether it would induce full or only partial immunity when faced against fully virulent smallpox. Although the virology, pathology, and epidemiology of smallpox are well described [1], there is a relative dearth of information regarding the immunology of smallpox and smallpox vaccination. Most importantly, there is currently no consensus on the immunological correlates of protection, making it difficult to implement rationale vaccine design when it not established which immunological benchmarks are necessary for full or even partial protection. Recent quantitative analysis of the cellular and humoral immune response following smallpox vaccination, coupled with historical evidence of protective immunity, are beginning to shed light on this important subject.
Discussion
The question of safety following smallpox vaccination
The current smallpox vaccine was prepared prior to 1982 under standards that today, would be unlikely to be approved by the FDA. To produce the vaccine, the torso of bovine calves were shaved, and their skin scarified (scratched) with an inoculum containing vaccinia virus. After the infection has reached a point in which a great deal of exudate was observed, the purulent lymph material was scraped from the infected cow, clarified, and lyophilized in the presence of antibiotics. The inoculum is later reconstituted with diluent containing 0.25% phenol to further decrease bacterial contamination (<200 viable bacterial/mL after reconstitution; see Dryvax package insert). Unlike most other vaccines that are administered by subcutaneous or intramuscular injection, vaccinia virus replicates poorly under these conditions and optimal vaccination occurs by scarification of the virus inoculum onto the skin surface [10]. Viral replication on the skin surface typically results in a vesicular or pustular lesion that is described as a "take" which later crusts over and sloughs off, leaving behind a small scar. Since the time of Edward Jenner, the presence of a vesicular or pustular lesion has remained the gold-standard measurement of successful vaccination.
The success of smallpox vaccination does not come without unwanted consequences. The most common side effects of smallpox vaccination are fever and other flu-like symptoms. More serious adverse events include inadvertent inoculation (529 cases/106 doses), generalized vaccinia (242 cases/106 doses), eczema vaccinatum (39 cases/106 doses), vaccinia necrosum (1.5 cases/106 doses), encephalitis (12 cases/106 doses), or death (~1 death/106 doses) [11]. The mortality rate is highest for infants that are <1 year of age (5 deaths/106 doses) whereas the mortality rate for older children and adolescents aged 1–4 or 5–19 is approximately 0.5 deaths/106 doses (see also [12,13] and Dryvax package insert). It is rare for adults to die after smallpox vaccination; of 68 deaths attributed to smallpox vaccination over a 9-year period of evaluation, only 8/68 (12%) of cases occurred in adults. Five of the eight adults were over the age of 60 and 4/8 of the lethal cases occurred in adults who were diagnosed with terminal cancer at the time of vaccination [11].
Myopericarditis is a recently identified adverse event that occurs at a rate of ~124 cases/106 smallpox vaccinations [14,15]. In 2003, there were three fatal heart attacks that were temporally related to smallpox vaccination and this triggered critical evaluation of any vaccine-related cardiac events thereafter. It was later realized that all three heart attack victims (age 55, 55, and 57) had pre-existing risk factors for cardiac disease including hypertension, hyperlipidemia, and smoking. At autopsy, none of the three heart attack victims showed signs of myo/pericarditis but the deaths were instead linked directly to ischemic events [16]. A retrospective study analyzing the number of cardiac deaths among approximately 80,000 death certificates issued near the time of a massive smallpox vaccination effort in New York City in 1947 has also brought new insight into the dangers of smallpox vaccine-induced myocarditis[16]. Following an imported case of smallpox, about 6.4 million people of all ages were vaccinated in a one-month period. Analysis of the frequency of cardiac deaths before, during, and after the vaccination campaign failed to show a statistical increase in these events. Moreover, analysis of 64 recently identified cases of myocarditis in the U.S. military smallpox vaccination program found that approximately 80% of patients reported no long term sequelae and 100% of patients demonstrated objective normalization of echocardiography, electrocardiography, laboratory testing, graded exercise testing, and functional status [15].
To overcome some of the problems associated with the current calf lymph smallpox vaccine (Dryvax), an improved tissue culture-derived vaccine has been recently developed [17]. Unlike Dryvax, this new vaccine (designated ACAM1000) is produced under sterile GMP conditions, so bacterial contamination is avoided. Also, unlike Dryvax which contains a heterologous mixture of virus variants with some differing in their neurovirulence [17,18], ACAM1000 is clonally derived (triple plaque-purified) and tested extensively for low neurovirulence in animal studies. Thus, it is hopeful that ACAM1000 vaccine will have reduced risk of encephalitis, one of the major risk factors following smallpox vaccination. It remains to be seen whether or not myopericarditis or other potentially serious adverse events will be affected by the use of this new vaccine.
Both Dryvax and ACAM1000 represent replication-competent smallpox vaccines and since these vaccines are based on the use of live viruses, there is always an inherent risk of severe adverse events or death (albeit rare) in vaccinees that have unknown or undisclosed immunodeficiencies at the time of vaccination. To overcome the hazards of replicating viruses, a highly attenuated strain of vaccinia, designated Modified Vaccinia virus Ankara (MVA) was developed by growing the virus for >500 passages on chicken embryo fibroblasts (CEF) and following the loss of about 15% of its parental genome, it no longer was capable of replicating in most mammalian cells, including human cells [19]. Other strains of non-replicating orthopoxviruses have also been developed, including NYVAC, which was derived from vaccinia virus following a deletion of 18 genes – including those encoding virulence factors and human host range replication, and ALVAC, an attenuated viral vector that is based on canarypox, an avipoxvirus that grows only in avian species. In one study, recombinant NYVAC and recombinant ALVAC expressing JEV proteins were found to be well tolerated but more reactogenic than the commercially available formalin-inactivated JEV vaccine [20]. Of these attenuated poxvirus vaccine strains, MVA is the one with the most extensive history of safety in humans. Beginning in 1968, >100,000 people in Germany were vaccinated with MVA (followed by traditional smallpox vaccination) and although it was well tolerated, MVA was not used alone and since there were no smallpox outbreaks at that time, its efficacy in the face of an actual smallpox outbreak has not been tested. With an excellent safety profile in humans and in animal models of immunodeficiency, recombinant MVA expressing candidate immunogens from a variety of infectious agents (e.g. HIV, HPV, and malaria) or tumor antigens (e.g. melanoma) have now reached Phase I and Phase II clinical trials [21,22]. However, the role of MVA in the future of smallpox vaccination has yet to be decided and will likely be determined by the outcome of clinical trials that directly compare the immunogenicity of MVA to either Dryvax or ACAM1000, two vaccines that are likely to provide the required high levels of protective immunity that will be necessary in the event of an accidental or deliberate smallpox outbreak.
Quantitative analysis of vaccine efficacy
Edward Jenner developed the first test for vaccine efficacy when he immunized subjects with cowpox and then later challenged them with smallpox by inoculation. If a subject showed no secondary smallpox lesions indicative of systemic spread, then the individual was believed to have protective immunity. He noted that cowpox-vaccinated individuals who developed a pox-like lesion at the vaccination site were fully protected against smallpox challenge. To this day, most studies still use the identification of a "take" (vesicular or pustular lesion) as evidence of successful vaccination. However, there are rare cases of revaccinated subjects who present with vesicle formation after smallpox vaccination, but show no detectably boosted cellular or humoral immunity [23] and M.K. Slifka, unpublished results). For this reason, it is important that pre- and post-vaccination serum antibody and peripheral T cell responses be monitored in individuals who plan to work with virulent orthopoxviruses or who would be expected to enter a hot zone in the case of a smallpox outbreak.
Quantitative immunology is beginning to gain acceptance as a measurement of vaccine efficacy, although the Jennerian vesicle at the site of smallpox vaccination still remains the primary endpoint of successful vaccination in most studies [17,23-25]. One of the reasons why quantitative immunology is more important now than ever before is that with some vaccines such as MVA, there is no vesicle formed due to the route of immunization – so unless immunological measurements are made, then vaccine immunogenicity cannot be determined or compared. Humoral immunity following smallpox vaccination was measured in the 1960's and 1970's by means of neutralizing assays (primarily against the IMV form of vaccinia or smallpox) and today, humoral immune responses are quantitated by analysis of neutralizing activity against IMV or EEV forms of vaccinia or by the use of ELISA assays using whole-virus lysate and/or individual vaccinia IMV or EEV proteins. Moreover, the vaccinia-specific memory B cell response has also been recently studied [26] providing the first direct quantitation of this memory cell subset. Quantitation of the antiviral T cell response mounted after smallpox vaccination was not an option during the smallpox era because the tools and technology were not available for analysis of cellular immunity. In contrast, today there are now several sophisticated techniques that can be used to monitor antiviral T cell responses directly ex vivo including vaccinia-specific IFNγ ELISPOT assays [17,23,26-28], intracellular cytokine staining analysis (ICCS) [29-33], or peptide/MHC Class I tetramer staining [28,30]. Each of these techniques has high sensitivity and high specificity and each has both advantages and disadvantages. For instance, the IFNγ ELISPOT assay provides a highly sensitive calculation of IFNγ-producing cells, but one drawback is that it only allows detection of one cytokine at a time and the phenotype of the IFNγ-producing subset (CD4, CD8 or possibly NK cells) must be determined by purifying each population independently prior to the assay. The advantage of ICCS is that antiviral T cells can be quantitated based on the production of more than one cytokine and the phenotype of the responding lymphocyte subset is directly determined by flow cytometry. The main disadvantage of ICCS is that a relatively large number of cells are required in order to detect rare populations of virus-specific T cells. The advantage of using peptide/MHC tetramers is that CD8+ T cells can be quantitated regardless of their cytokine profiles, but the main disadvantages with this approach include the lack of identified CD4+ T cell/MHC Class II epitopes, the requirement for knowledge of the MHC haplotype of the subject, and the inability to measure the total antiviral T cell response, which may be directed against any number of immunodominant and subdominant peptide epitopes.
Direct quantitation of the antiviral immune response induced by smallpox vaccination has been critical for several recent advances in orthopoxvirus immunobiology – especially since smallpox has been eradicated and other human orthopoxvirus outbreaks are too small and sporadic (e.g. cowpox [34,35] or monkeypox [36,37]) to be feasible for field studies of protective efficacy. For example, Weltzin et al. [17] not only compared the neutralizing titers induced by Dryvax vs. ACAM1000 vaccines, but also compared vaccinia-specific T cell responses by IFNγ ELISPOT as well as by cytolytic T cell assays and proliferation assays that, although less quantitative than the ELISPOT assay, are nevertheless important for analysis of antiviral T cell functions. These techniques allowed the investigators to demonstrate non-inferiority of the new tissue culture-derived smallpox vaccine compared to the Dryvax vaccine that is currently in use.
Another study by Earl et al. [38], used quantitative immunology to compare vaccine efficacy of Dryvax, MVA followed by a Dryvax booster, and MVA followed by an MVA booster in a cynomolgus monkey (Macaca fascicularis) model of monkeypox infection. Using ELISA assays and neutralizing assays to measure vaccinia-specific antibody responses, and ICCS to measure antiviral T cell responses, the authors showed that antiviral immunity appeared similar between these three groups. Likewise, each of these groups were protected against lethal monkeypox challenge, although primates that only received MVA plus an MVA booster showed partial protection with 6/6 animals presenting with 1–36 monkeypox lesions (compared to >500 monkeypox lesions in the unvaccinated controls). This indicates that two MVA vaccinations are required to induce partial immunity whereas MVA followed by Dryvax immunization or a single Dryvax immunization each provides full immunity. The protective efficacy of a single dose of MVA is unknown, but based on the results of the Earl et al. study [38] wherein two doses were required to elicit partial immunity, it appears likely that a single dose of MVA would be of only low protective value in the face of a virulent orthopoxvirus infection. This is not surprising since MVA, NYVAC, and ALVAC (all replication-deficient vaccines), typically require booster doses to be administered in order to elicit optimal immune responses. Moreover, several prime-boost strategies (including DNA vaccination followed by MVA booster) are being tested in order to overcome the low immunogenicity of MVA alone [21]. This is different from most replicating viruses [39] including vaccinia, which require only one immunization (or infection) to induce optimal and often lifelong immunity [5,26,32,40]. Approximately half of the U.S. population has been vaccinated against smallpox and continue to maintain pre-existing antiviral immunity [26,32] and this may have an impact on the efficacy of MVA vaccination. For instance, studies involving the closely related, non-replicating NYVAC strain have found that pre-existing immunity significantly effected the outcome of vaccination [20]. In this particular study, immunization with recombinant NYVAC expressing JEV proteins failed to induce protective antibodies in 0/5 vaccinia pre-immune individuals and although 5/5 vaccinia-naive subjects seroconverted after NYVAC-JEV immunization, the resulting neutralizing titers were substantially lower than that observed in subjects who received the standard formalin-inactivated JEV vaccine [20]. Oddly, the authors noted that both the NYVAC-JEV and the ALVAC-JEV vaccines (2 doses administered 28 days apart) failed to induce a detectable anti-vaccinia neutralizing response.
It is difficult to speculate the efficacy of MVA vaccination of humans as a method of protection against smallpox. It is possible that booster doses of MVA would provide strong enough immunity to at least protect against lethal smallpox, similar to its ability to protect primates from lethal monkeypox [38]. On the other hand, clinical trials could indicate that MVA vaccination alone may be too inconsistent or only induce low levels of immunity that would be considered inferior to live smallpox vaccination with calf-lymph or tissue culture-derived vaccine preparations. Unlike live smallpox vaccination, which can be used as an effective post-exposure treatment against the lethal consequences of smallpox [1,5], it is unlikely that the low immunogenicity of MVA would be capable of fulfilling this role. Moreover, in the event of a smallpox outbreak there would not be enough time to administer two or more doses of MVA if people were at a high risk of exposure. Under these circumstances, use of live viral vaccines would be critical for ring vaccination or mass vaccination scenarios. This is not to say that MVA would not have a potential role in biodefense strategies. For instance, in a pre-event scenario, one could foresee the use of MVA followed by vaccination with a live viral vaccine such as Dryvax or its equivalent. Under these circumstances, MVA would likely induce partial immunity that would reduce the adverse events that are associated with traditional smallpox vaccination. Moreover, MVA is the vaccine of choice in immunocompromised individuals with suppressed immune systems (cancer patients, organ-transplant patients, AIDS patients, etc.) who would otherwise be contra-indicated for administration of the live viral vaccines. More studies will be needed to determine the immunogenicity and protective efficacy of MVA and other related non-replicating vaccines in terms of their potential to counter a smallpox outbreak.
The need for formal definition of protective immunity and the correlates of immunity
There is more than one definition of protective immunity against smallpox. There is protection against infection, protection against disease, and protection against death. Of these, one might argue that protection against lethal infection is the ultimate definition of protective immunity. However, protection against infection and protection against disease not only reduce the morbidity of an outbreak, but these high levels of protective immunity are also associated with reduced virus spread to others (Chapter 4, pages 189–90 of [1]). Protection against infection is the most rare level of protective immunity since this requires that an infection be blocked at the point of entry. A meta-analysis of 10 epidemiological studies on smallpox noted that on average, the virus only infected ~4% of previously vaccinated household contacts (Chapter 4, pages 189–90 of [1]). However, these results were based on whether or not the vaccinated contacts showed disease symptoms and did not necessarily prove that they were never infected per se. Further analysis indicated that approximately 10% of previously vaccinated household contacts of smallpox patients were actually infected with smallpox as demonstrated by isolation of infectious virus from pharyngeal mucosa, but only 4/34 (12%) of these subjects developed the clinical symptoms of smallpox [41]. Moreover, another study showed that about 50% of previously vaccinated, disease-free contacts demonstrated serological results indicative of a recent orthopoxvirus infection [42]. This suggests that most instances of "protection against infection" may not be complete protection. Instead, many of the individuals thought to have had protection against infection may have actually been infected with smallpox but didn't know it because they were clinically asymptomatic. Similar to these historical studies, during the U.S. monkeypox outbreak in 2003 [37] we have identified three previously unreported cases of monkeypox in subjects who had received smallpox vaccination many years earlier and were unaware that they had become infected with monkeypox because they were spared any recognizable disease symptoms (M.K. Slifka, unpublished data).
A major issue in the smallpox field is that there is no consensus on what is exactly required for protective immunity against this disease. In the age of quantitative immunology, we are beginning to find clues that might help answer this age-old question. In a study of >300 subjects, the levels of vaccinia-specific serum antibody and antiviral T cell responses were determined from 30 days to up to 75 years after smallpox vaccination [32]. Antiviral antibody responses were maintained essentially for life, whereas antiviral CD4+ and CD8+ T cell responses declined with a half-life of approximately 8–15 years, with CD4+ T cell memory being more stable than CD8+ T cell responses. Similar duration of antibody production was demonstrated by other recent studies as well as older literature [24,26,40]. Moreover, the gradual loss of T cell memory was also confirmed [26], as was the differential loss of CD8+ T cell memory over CD4+ T cell memory [33]. Based on historical analysis of vaccine-mediated protection against lethal smallpox (dating back to the age of Edward Jenner), indicates that protective immunity is often lifelong [40]. One might argue that if protective immunity against smallpox had an absolute requirement for antiviral CD4+ or CD8+ T cells, then protective immunity would not be life-long but would instead be more likely to decline at the same rates as T cell memory. This suggests that in humans, humoral immunity might play a more important role in protection against lethal infection than cellular immunity [5,40]. Smallpox disease symptoms become more pronounced with increased time since vaccination [43], and it likely that the combination of intact cellular and humoral immunity together provide the most robust antiviral immunity. Recent vaccinia studies in mice using either antibodies to deplete T cell subsets or mouse strains that are genetically deficient in CD4+ T cells or CD8+ T cells (or both) have indicated that, as long as there is strong humoral immunity against vaccinia, T cell memory is dispensable for protective immunity [44-46]. This result corresponds well with other studies in which protection against vaccinia or smallpox has been clearly demonstrated by the adoptive transfer of immune serum in humans or by transfer of monoclonal neutralizing antibodies in animal models [5,40]. On the other hand, adoptive transfer of virus-specific T cells and experiments in mice that are genetically deficient in B cells, also indicate that in the absence of pre-existing antibody responses, memory T cells can play an important role in protection. This indicates that cellular and humoral immunity have overlapping roles in protective immunity and one may compensate for a deficiency in the other.
Based on the overlapping roles for T cell and B cell memory on protective immunity, is there any chance that a consensus can be reached in regard to defining an immunological correlate of immunity? One way to address this issue is to examine historical studies in which an immunological correlate was identified. In this regard, there are two independent studies in which investigators showed that subjects with vaccinia-specific neutralizing antibody titers of >1:20 [47] or ≥ 1:32 [48] were fully protected against smallpox. The latter study was the largest of the two and showed that 3/15 (20%) of subjects with titers below 1:32 contracted smallpox whereas 0/127 (<1%) of subjects with antibody titers of ≥ 1:32 contracted the disease. The only caveat to these studies is that the subjects also received post-exposure vaccination at the same time that serum samples were drawn, so the full protection afforded to the subjects with high pre-existing neutralizing titers may have been due to their high antibody titers, or the combination of strong pre-existing antibody titers in the context of post-exposure vaccination. A neutralizing titer of 1:32 is equivalent to a vaccinia ELISA titer of 944 Elisa Units (EU) or approximately 4 International Units (IU) of the WHO/NIBSC International Smallpox Serum Standard [32]. Interestingly, about 50% of subjects vaccinated in the distant past maintain neutralizing titers of 1:32 or greater for life and this coincidentally is the same proportion of vaccinated smallpox contacts who demonstrated fully protective immunity when exposed to smallpox-infected family members [42]. This leads one to speculate that 4 IU may constitute a protective level of serological immunity against smallpox. This is a testable hypothesis and to determine if this is a correlate of protection, it will be important to perform adoptive transfer of VIG or its equivalent into non-human primates (resulting in antiviral serum antibody levels of approximately 1:32) and determine if they are protected against a lethal orthopoxvirus infection, such as following monkeypox challenge. This experiment would prove or disprove the hypothesis that a serological correlate of protective immunity exists and may lay the foundation for future vaccine design. Of note, the protective level of yellow fever immunity (log10 ≥ 0.7 neutralizing titer) was also established in non-human primates by simply vaccinating groups of animals with different doses of the yellow fever vaccine, quantitating vaccine-induced antibody levels, and then challenging them with a lethal dose of yellow fever virus [49]. This serological correlate of protection (which ignores the role of vaccine-induced T cell responses) has been established as the benchmark of protective yellow fever virus-specific immunity for over 30 years and demonstrates the potential for using animal models to correlate protective immunity in humans.
Conclusion
Traditional smallpox vaccination has lead to the global eradication of smallpox but continues to be used today in an effort to thwart the potential use of smallpox as a biological weapon. Since this vaccine employs the use of a live virus, there is an inherent risk of adverse events, although these are generally quite rare. New generation smallpox vaccine candidates include MVA and other non-replicating poxviruses and although they demonstrate a high degree of safety, their immunogenicity appears to be substantially lower than traditional smallpox vaccination with live vaccinia virus. The role of MVA and traditional smallpox vaccination (or a combination thereof) in future vaccination campaigns has yet to be determined. However, developing a consensus on the definition of what is required for protective immunity and defining an immunological correlate of immunity would aid in the evaluation of current and future vaccine approaches.
List of Abbreviations
MVA modified vaccinia virus Ankura
GMP good manufacturing practices
HIV human immunodeficiency virus
CEF chicken embryo fibroblasts
HPV human papilloma virus
FDA Federal Drug Administration
JEV Japanese Encephalitis Virus
IMV Intracellular mature virus
EEV Extracellular enveloped virus
ELISA Enzyme-linked immunosorbent assay
IFNγ Interferon-gamma
ELISPOT Enzyme-linked immunosorbent Spot assay
ICCS Intracellular cytokine staining
MHC Major histocompatibility complex
VIG Vaccinia immune globulin
WHO World Health Organization
NIBSC National Institute of Biological Standards and Control
EU Elisa Units
IU International Units
Competing Interests
OHSU and Dr. Slifka have a financial interest in Najít Technologies, Inc., a company that may have a commercial interest in the results of this research and technology. This potential conflict was disclosed to the OHSU Conflict of Interest in Research Committee and an approved management plan was implemented.
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| 15737238 | PMC554757 | CC BY | 2021-01-04 16:36:40 | no | Retrovirology. 2005 Feb 28; 2:13 | latin-1 | Retrovirology | 2,005 | 10.1186/1742-4690-2-13 | oa_comm |
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Virol JVirology Journal1743-422XBioMed Central London 1743-422X-2-161573056210.1186/1743-422X-2-16ResearchPlant viral intergenic DNA sequence repeats with transcription enhancing activity Velten Jeff [email protected] Kevin J [email protected] Christopher I [email protected] USDA-ARS, Plant Stress and Water Conservation Laboratory, 3810 4th St., Lubbock, TX 79415, USA2 Department of Biology, Colorado State University, Fort Collins, CO 80523, USA2005 24 2 2005 2 16 16 14 12 2004 24 2 2005 Copyright © 2005 Velten et al; licensee BioMed Central Ltd.2005Velten et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The geminivirus and nanovirus families of DNA plant viruses have proved to be a fertile source of viral genomic sequences, clearly demonstrated by the large number of sequence entries within public DNA sequence databases. Due to considerable conservation in genome organization, these viruses contain easily identifiable intergenic regions that have been found to contain multiple DNA sequence elements important to viral replication and gene regulation. As a first step in a broad screen of geminivirus and nanovirus intergenic sequences for DNA segments important in controlling viral gene expression, we have 'mined' a large set of viral intergenic regions for transcriptional enhancers. Viral sequences that are found to act as enhancers of transcription in plants are likely to contribute to viral gene activity during infection.
Results
DNA sequences from the intergenic regions of 29 geminiviruses or nanoviruses were scanned for repeated sequence elements to be tested for transcription enhancing activity. 105 elements were identified and placed immediately upstream from a minimal plant-functional promoter fused to an intron-containing luciferase reporter gene. Transient luciferase activity was measured within Agrobacteria-infused Nicotiana tobacum leaf tissue. Of the 105 elements tested, 14 were found to reproducibly elevate reporter gene activity (>25% increase over that from the minimal promoter-reporter construct, p < 0.05), while 91 elements failed to increase luciferase activity. A previously described "conserved late element" (CLE) was identified within tested repeats from 5 different viral species was found to have intrinsic enhancer activity in the absence of viral gene products. The remaining 9 active elements have not been previously demonstrated to act as functional promoter components.
Conclusion
Biological significance for the active DNA elements identified is supported by repeated isolation of a previously defined viral element (CLE), and the finding that two of three viral enhancer elements examined were markedly enriched within both geminivirus sequences and within Arabidopsis promoter regions. These data provide a useful starting point for virologists interested in undertaking more detailed analysis of geminiviral promoter function.
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Background
Traditionally, analyses of viral promoter structure-function relationship have involved directed deletion or disruption of promoter structure, followed by determination of resulting changes in transcription, if any, resulting from the alterations [1]. A relatively small subset of the promoter elements identified in this way have been subsequently isolated and tested for their ability to influence transcription when inserted into alternative, well defined, basal promoters [2]. As an alternative to so-called 'promoter bashing' approaches to the study of promoter structure, we have instead chosen to 'mine' specific regions of viral DNA for sequence elements that, when combined with a minimal plant promoter, are able to enhance transcription of a reporter gene in planta.
To test the enhancer mining approach we chose to examine a collection of geminivirus and nanovirus intergenic sequences obtained from GenBank. There are a relatively large number of available sequences for these DNA viruses and due to conserved genomic organization they contain easily identifiable intergenic regions [3]. Additionally, several studies have demonstrated in planta promoter activity using isolated or modified geminivirus or nanovirus intergenic sequences [4-21]. Although some areas of sequence similarity exist within the intergenic regions of the geminiviruses [22], very few of these common sequence elements have been experimentally shown to contribute to transcriptional activity. We specifically avoided using any test for evolutionary conservation of candidate elements, hoping to identify unique elements that may not necessarily be shared by large groups of related viruses. For this first broad screen, the experimental rational used made two basic assumptions; 1} that viral intergenic regions contain an enrichment of DNA transcriptional regulatory elements; and 2} that important regulatory sequence elements are often duplicated within promoters, either directly repeated, or as inverted copies of sequence segments [22].
The described enhancer mining of viral sequences is not intended to be a comprehensive analysis of viral promoter structure since by design it is limited to identification of promoter elements that up-regulate gene expression and that make use of endogenous plant transcription factors available within the un-infected test plant. However, based upon their iteration, location within intergenic regions, and ability to enhance transcription in planta, any elements identified using this approach are likely to contribute to regulation of in vivo viral gene expression during plant infection. By allowing relatively large numbers of viral sequences to be examined using a defined system, the approach has the potential of generating data useful in comparing positively acting viral promoter elements within and between viral families. In addition, identification of elements that are active in planta in the absence of viral infection provides results pertinent to understanding virus-host interactions at the level of gene control. Finally, the resulting list of active and inactive viral sequences provides a valuable starting points for subsequent, more detailed, analysis of transcription regulation of individual viruses.
Results
Search for candidate elements
The initial search for sequence repeats was performed on the major intergenic regions of 29 different geminivirus or nanovirus genomic sequences (Figure 1 and Additional file 1). The search was arbitrarily halted after 105 candidate repeats were identified and was not intended to provide a comprehensive representation of all duplicated sequences within any of the viral sequences examined. Although generated using different search criteria than those employed by Arguello-Astorga et al [22], the resulting collection of geminivirus sequence repeats contains some sequences similar or identical to the described "iterons" (it should be noted that functional testing of nearly all of the "iterons" listed has not yet been reported in the literature).
Figure 1 Viral enhancer elements. All viral repeats that produced greater than a 25% increase in 35S min activity are listed. For each active element the accession number, relative enhancement (with standard error), repeat length, repeat separation, source virus (and genus) and viral sequence are shown. Adaptor sequences are listed in the header of the sequence column and with imperfect repeats in bold and partial palindromes within repeats underlined.
Functional testing of elements
Of the 105 repeats tested (Figure 1 and Additional file 1), 14 (13%) reproducibly resulted in increases of at least 25% above that of the 35S min construct (p < 5% by Student's T-test, the T-test was used only as a guide since by the nature of the assay used, individual data sets are small) (Figure 1 and Additional file 1). The remaining 91 (87%) failed to produce any measurable enhancement of reporter gene activity (see Additional file 1). All the positive elements identified by the in vivo assay were subsequently tested using an in vitro dual-luciferase® system from Promega Corp. and produced levels of enhancement very similar to those obtained using the in vivo assay (the enhancement values and standard error reported in Figure 1 and Additional file 1 include both in vivo and in vitro data normalized to 35S min = 1.0). The observed enhancement of promoter activity (~2 fold) is relatively modest compared to other viral transcriptional enhancers that have been isolated and tested (e.g., G-box [23] and AS-1 [24] type elements enhance 35S min activity 8–10 fold using this assay, data not shown). This outcome may reflect limitations of the original search parameters (only repeated elements were tested). However, several of the geminiviral elements identified in this screen have been subsequently found to display clear and unique synergistic effects when combined or multimerized (Cazzonelli, Burke and Velten, manuscript in preparation), supporting their potential to contribute to viral gene regulation during infection.
Since all assays were performed on tobacco plants that had been neither infected with any of the viruses screened, nor transfected with any viral components, it is unlikely that elements strictly dependent upon virally encoded regulatory factors, or factors not native to N. tobacum, would be identified. In addition, the screen was limited to those elements that increase gene expression, and no effort was made to confirm data suggesting that an element might be a 'repressor' (e.g., the 11 elements that show 'enhancement' values less than, or equal to, one third of the 35S min activity, see Additional file 1). Considering these limitations, the finding that 13% of the sequences tested produced measurable up-regulation of transcription supports the original assumption that basic transcription regulatory elements are enriched within repeated sequences from the viral intergenic regions. Despite having tested approximately equal numbers of inverted sequence repeats (IR) and direct sequence repeats (DR), 11 of 14 active elements were members of the DR set, with the remaining 3 positives being palindromic (inverted repeats with no sequence between the repeats). This is somewhat surprising since many of the iterated DNA sequence elements within geminivirus intergenic regions are found as both direct and inverted repeats [22], and as such could have been present in either the DR or IR set of elements. Although the numbers tested are small, and the screen was performed using a single plant species, these results suggest that directly repeated sequences within geminivirus and nanovirus intergenic repeats have a higher probability of positively influencing transcription levels than do the inverted sequence structures. It is possible that this bias may reflect the presence within the intergenic region of DNA elements responsible for viral replication [25], including a conserved inverted repeat structure with a ubiquitous central-loop sequence [26]. Seven of the IR elements tested in this study are part of predicted replication hairpin structures (see Additional file 1) and did not, in this test system, result in any measurable enhancement of reporter gene expression.
Manual alignment of all the active DR sequences produced three classes of related elements and several unique individuals (Figure 3). Five of the 14 positive DR elements contain an already identified geminiviral transcription control element, the "conserved late element" or CLE {GTGGTCCC, [22,27]}. The CLE sequence had been previously shown to affect expression from a minimal 35S promoter, and to be up-regulated by the viral AC2 gene product [27]. The two remaining grouped elements include a pair of "CT" rich repeats (DR08 and DR13) and two related, nearly-palindromic direct repeats from beet curly top virus (BCTV, elements DR19 and DR30). Despite the lack of an exact G-box core sequence {ACGT, [28]}, the nearly palindromic structure of the DR19 and DR30 elements {aaACTTc} is reminiscent of duplicated G-box type geminiviral elements noted by Arguello-Astorga et al [22] and later proposed as functional components within tomato golden mosaic virus (TGMV) and subterranean clover stunt virus (SCSV) promoters [11,20]. When scanned against the online PlantCARE promoter element database {[29,30]} no clear consensus emerges regarding similarity of the discovered viral elements with characterized plant cis regulatory elements (the most common hits were against light or stress responsive elements, although that may simply represent the distribution of plant elements contained within the database).
Figure 3 Alignment of active repeat elements. Each directly repeated element is offset (at the "/") to align both copies of the repeat. Related elements are additionally aligned as paired repeat alignments. Bases that differ within paired repeats are in lowercase bold and palindromic sub-elements within the repeats are indicated by arrows. Areas of the alignments used to determine a consensus sequence are boxed.
Element occurrence in viral and Arabidopisis sequence databases
Short of directed mutagenesis of each identified viral element, followed by analysis of resulting 'mutant' virus function within infected plants, it is difficult to directly determine what contribution each of the identified enhancer elements makes to viral gene regulation. Computer analysis of an element's frequency of occurrence in defined DNA sequence databases provides an alternative mechanism for gaining insight into likely biological function for short sequence elements [31]. For example, the occurrence frequency of functionally important promoter elements is higher within DNA sequences upstream from gene coding regions, compared to the frequency within non-regulatory sequences [31]. Since the element enrichment approach works best when applied to relatively short, core consensus sequences [31], viral element searches were limited to those viral enhancers that showed a clear core consensus (CLE, BCTV DR19/30, CT-rich, Figure 3).
The viral enhancers identified in this work were found to function within un-infected test plants, indicating that the viral elements can make use of intrinsic plant transcription factors (not virally encoded) and may, therefore, be similar or identical to endogenous plant promoter elements. In order to test for enhancement of viral enhancer sequences within higher plant promoters, the PatMatch page of the TAIR web site [32] was used to access sub-datasets of the A. thaliana genomic sequence that are exclusive to annotated coding sequences {CDS} and three upstream sequence lengths {-3000, -1000, -500 bp, measured from each CDS start codon}. Each of the sub-datasets was searched for the viral elements (CLE, BCTV DR19/30, CT-rich) and, as controls, several well defined plant promoter element consensus sequences (the "G-Box" {CACGTG}, a common plant promoter element that is associated with members of the pZIP family of transcription factors [33,34], and two less prevalent plant promoter elements, the drought response element ('DRE', RCCGAC [35]) and abscisic acid response element (ABRE-like, ACGTGKM) [35]).
Performing similar oligonucleotide frequency searches for element enrichment within viral promoters was complicated by the lack of comprehensive annotation of viral sequence entries within the GenBank database. Without clear annotation of intergenic and coding sequences within the viral GenBank entries, it was impossible to directly perform the same sort of 'upstream sequence' (in this case, viral intergenic regions) versus 'coding sequence' frequency comparisons that were possible using the fully annotated Arabidopsis genome sequence and PatMatch. As an alternative, screens were performed to determine frequencies of occurrence for viral enhancers (and control plant elements) within a sequence database consisting of all geminivirus or nanovirus GenBank entries as of May 13, 2004 [36], and the results compared with those obtained scanning the same sequences against the Arabidopsis PatMatch datasets. The searched viral sequence database has the potential for bias due to the existence of a numerous entries containing only coding regions or only intergenic sequences, as well as some duplication of sequences in separate entries. Any such bias should, however, similarly affect the baseline frequency values resulting from searches using the 18 matched random oligonucleotides (in parenthesis, Table 1), thus all element enrichments are considered relative to the random oligo values. It was decided to perform the searches using the full geminiviral plus nanoviral database, since limiting the viral entries to only those containing fully annotated, complete viral sequences would have greatly reduced the number of different viruses examined.
Table 1 Element occurrence frequencies within viral and Arabidopsis sequence databases
Element Identifier Element Sequence Occurrence frequency from each database. Values are relative to Arabidopsis CDS = 1.00 (Mean of 18 matched oligomer frequencies)
Arabidopsis -3000 to -1001 Arabidopsis -1000 to -501 Arabidopsis -500 to -1 Arabidopsis CDS Gemini + nanovirus
Previously Identified Promoter Elements from A. thaliana (*also confirmed as geminiviral element)
ABRE-like ACGTGKM 1.65 (1.72) 1.78 (1.8) 3 (1.44) 1 (1.59) 2.45 (1.28)
DRE RCCGAC 1.86 (1.75) 1.81 (1.55) 2.13 (1.46) 1 (1.13) 1.09 (0.85)
G-box* CACGTG 2.28 (1.79) 2.57 (1.58) 4.35 (1.47) 1 (1.41) 3.81 (1.43)
Consensus Gemini/Nanoviral Sequence Elements (**not a promoter element)
CLE GTGGNCCC 3.15 (3.51) 3.62 (2.99) 3.9 (2.79) 1 (1.56) 17.36 (2.81)
DR08/13 TCTCTCTCTA 3.15 (0.46) 3.6 (0.4) 7.75 (0.35) 1 (0.51) 6.92 (0.53)
BCTV DR19/30 AAACTTC 0.7 (0.62) 0.69 (0.64) 0.68 (0.66) 1 (0.72) 0.64 (0.52)
GV rep-stem** CGCGNCCA 2.52 (3.51) 2.2 (2.99) 2.26 (2.79) 1 (1.89) 17.11 (3.42)
The results of the searches are displayed in Table 1. Each frequency value (cHits/Mbp) represents the number of hits per million base pairs, corrected for the database base composition using empirically determined G/C and A/T ratios for each of the databases examined (see Materials and Methods). To facilitate comparison, the resulting cHits/Mbp from the Arabidopsis upstream databases (-3000 to -1001, -1000 to -501, and -500 to -1 bp) were normalized relative to the value obtained for each element's occurrence within the A. thaliana coding sequence database (CDS value set to 1.0). In addition to the predicted frequency values, in each case, the element's observed frequency was also compared to a value generated using the average of 18 random oligomers having the same length and base composition as the element tested (in parenthesis, Table 1). The test sequences for plant ABRE-like and G-box elements showed clear enrichment within the upstream Arabidopsis sequences, especially within the -1 to -500 region (ABRE-like element = 3.0 time the CDS value, vs 1.44 for random sequences and G-box = 4.35 vs 1.47 for random sequences, all as normalized cHits/Mbp). Results for the DRE element were less convincing (2.13 vs 1.46 in the -1 to -500 dataset) and likely reflect lower functional usage of this element within the Arabidopsis genome [35].
As expected, the CLE consensus sequence (GTGGNCCC) was found to be markedly enriched within the viral database, occurring 6 times more frequently than the mean of 18 random 8-mers of identical base composition (CLE = 17.36 normalized cHits/Mbp vs 2.81 from matched random sequences). This frequency is similar to that found (17.11 vs 3.42) using a short sequence of identical base composition and length that matches a highly conserved replication stem-loop sequence (CGCGNCCA), a component that is evolutionarily conserved within the geminivirus population [37]. Enhancement of CLE within Arabidopsis promoters is less obvious (CLE = 3.9 in the -1 to -500 database vs 2.79 for random sequences). The observed relatively small CLE enrichment is consistent with reports of a low frequency of occurrence for a CLE-like "TCP domain" binding consensus sequence (Gt/cGGNCCC) within Arabidopsis promoters [38]. It is possible that TCP domain-containing transcription factors contribute to the observed CLE enhancer activity since Arabidopsis promoters containing the TCP domain consensus binding element were found to function in transgenic tobacco and to show reduced activity after mutation of the element's core sequence [38].
The test sequences for plant element occurrence within the viral database (ABRE-like = 2.4 vs 1.28 and G-box = 3.81 vs 1.43, DRE = 1.09 vs 0.85) provide further indication of the technique's utility. The G-box viral frequency is consistent with a previous report that a G-box element contributes to transcriptional regulation from the major intergenic region of Tomato Golden Mosaic Virus {TGMV, ([20]}. The ABRE-like element enrichment in the viral database may indicate that viruses make use of biotic and abiotic stress-induced up-regulation [39] of genes driven by ABRE-containing promoters, a possibility open to additional research.
Of the remaining viral elements tested against the Arabidopsis and viral databases (Table 1), only the DR08/13 TC-rich sequence showed clear enrichment in both plant promoter and viral sequences (Arabidopsis -1 to -500 = 7.75 vs 0.35 and viral = 6.92 vs 0.53). Similar TC-rich regions have been reported within plant promoter regions [40,41], but we are unaware of any published report that confirms enhancer activity associated with an isolated TC-rich element, either viral or plant in origin.
Discussion
Except for the CLE elements, none of the active elements identified in this work have been experimentally reported as regulatory components of viral promoters. This is likely a reflection of both the limited number of geminivirus and nanovirus promoters that have been examined in detail [4,5,11,12,14,20,27,42,43], and the alternative approach of examining individual isolated elements used in this study. The mapped promoter components within the intergenic region of Tomato golden mosaic virus (TGMV) sub-genome A (TGMV-A) [14,20] provide a useful benchmark for comparison of results from this enhancer screen. Application of the repeated sequence screen to the TGMV (component B) intergenic region identified a single TGMV Direct repeat, DR38, and a single palindrome (PAL20), both of which were found to be inactive in our assay. This is consistent with published work that indicates most of the defined regulatory sequences within the TGMV-A intergenic region appear to occur as single copies [14,20]. The screen of intergenic repeats reported in this paper did, however, identify the CLE element, one copy of which has been shown to be part of the TGMV-A rightward promoter [14,20]. It is clear that testing only repeated elements will not identify all components of a promoter region, and when focusing on a specific promoter, testing of non-repeated elements (perhaps identified by evolutionary conservation) should be combined with other techniques such as insertion scanning [44]. Recently a collection of plant-functional promoters and terminators were isolated from the set of 7 Subterranean clover stunt virus (SCSV1-SCSV7) sub-genomic circles. The collection of sequence repeats tested in this study included 11 inverted or direct repeats from SCSV circles, only one of which (DR08 from SCSV2) showed any enhancing activity. It will be interesting to see how these tested repeated elements behave when examined in the context of the remainder of the SCSV promoter components.
Conclusion
This screen of viral intergenic repeats was undertaken to specifically identify general transcriptional enhancing elements contained within intergenic regions of a subset of geminivirus and nanovirus genomes. The screen was successful in demonstrating transcriptional enhancer activity from one proven viral promoter element and several previously unidentified elements. The occurrence of the repeated elements within intergenic regions, combined with the clear enrichment within viral sequences and Arabidopsis upstream sequences for at least the CLE and TC-rich (DR08/13) classes of elements, strongly supports participation of the enhancers in viral gene expression.
The technique of testing isolated elements represents an alternative to normal promoter-by-promoter dissection and provides a useful tool for screening promoter regions for potential functional elements that have been implicated by any number of possible criteria (e.g. copy number, evolutionary conservation, comparison of promoters with similar function, microarray data, etc.). Although the number of elements tested is relatively small and, so far, only representative of promoters from the geminiviruses and nanoviruses classes of plant viruses, there is a clear trend suggesting that directly repeated elements (including those containing small internal palindromic sequences) are more likely to play significant roles in the enhancement of transcription than inverted repeats. This work represents one of the first attempts to directly screen for individual plant promoter elements that are isolated from their native promoter context. It is therefore, difficult to gauge the actual contribution of any of the elements identified to viral gene regulation and biological activity. These results do, however, provide a useful starting point for more detailed analyses of not only geminivirus and nanovirus promoters, but also overall plant promoter structure-function relationships.
Methods
Identification of sequence repeats
The search for repeated DNA sequences was performed by visual inspection of computer-generated dot matrix comparisons (criteria: ≥ 66% identity, 10 base window, GeneWorks v2.5.2, Oxford Molecular Group Inc.). Dot matrices generated using each viral plus strand plotted against itself were used to identify direct repeats while inverted repeats were found by plotting each plus strand against its complement.
Production of sequence repeat test constructs
The identified repeats were synthesized as DNA cassettes containing the duplicated elements in their original orientation, either directly repeated with spacer sequence ('DR', 41 elements), inversely repeated with spacer sequence ('IR', 45 elements), or palindromic inverted repeats without spacer ('PAL', 20 elements). In order to limit the tested component to only the repeated elements themselves, any sequence occurring between the viral repeats (ranging from 0 to 146 bp, median separation = 9 bp) was replaced with a 10 bp randomized stuffer sequence (GAAGATAATC). The resulting cassettes were inserted immediately upstream from a minimal promoter (-46 to +1 relative to transcription start, 35S min) reporter system derived from the cauliflower mosaic virus (CaMV) 35S promoter fused to an intron-modified firefly luciferase (FiLUC) gene (Figure 2, [45]). The resulting test constructs were generated as part of a modified pPZP211 [46] binary plant transformation vector (Figure 2) and were introduced into the Agrobacteria tumefaciens strain, EHA105 [47] by electroporation [48]. The final Agrobacteria strains each contain, in addition to the test plasmids, a second, compatible, binary transformation vector expressing an intron-modified version of the Renilla reniformis luciferase gene (RiLUC) [49] under control of the constitutive Super-promoter [50]. The FiLUC and RiLUC enzymes can be independently assayed, making the co-transferred constitutive RiLUC gene a useful marker for gene transfer and for normalization of FiLUC values between individual elements [45].
Figure 2 T-DNA map of plasmid 35S min (in pPZP212). T-DNA borders: RB = right border, LB = left border, FiLUC = firefly luciferase, Nost = nopaline synthase transcription terminator, PClSV = Peanut chlorotic streak virus promoter, Bar = phosphinothricin acetyl transferase, 35St = transcription terminator for the Cauliflower mosaic virus (CaMV) 35S transcript. DNA sequence insert shows the minimal 35S promoter from CaMV, from -46 to +1 (transcription start). Upstream from the minimal 35S promoter are the restriction sites (underlined: HindIII; BamH, overlined: XbaI; KpnI) used to insert test sequences and downstream is the start codon from the luciferase coding region (bold ATG).
Lucifrease assays
Agrobacteria harboring the test and normalization binary plasmids were grown at 28°C in LB media containing the appropriate antibiotic selection (25 μg/mL kanamycin sulfate or 100 μg/mL spectinomycin) until an OD600 of 0.8 was achieved. The resulting cultures were centrifuged at 3000 rpm for 15 minutes, washed and re-suspended in an equal volume of infiltration media (50 mM MES, 0.5% glucose, 2 mM NaPO4, 100 μM Acetosyringone) before being mechanically infused (5 ml syringe) into multiple individual tobacco (N. tobacum, cv. SR1) leaves (2–4 leaves per test construct). Assays were performed in groups of 4–8 constructs and the resulting luciferase activities (both FiLUC and RiLUC) determined after 3–4 days using an in vivo floating leaf-disk assay developed for this enhancer screen [45]. Test constructs were assayed from 1 to 6 times, with each assay consisting of 2–4 disks (3 mm diameter) per infusion. The disks used in vivo assays were each measured for light production in separate wells of a white-walled 96 well microtiter plate (FLUOstar Optima luminometer® from BMG Lab Technologies Inc.) and all elements that tested positive in the in vivo assay were subsequently confirmed using the in vitro dual-luciferase® from Promega Corp (assays performed according to the manufacturers instructions, separate leaf disks from the same leaf infusions were used for the in vivo assays). Each test group included an infusion containing the 35S min construct (lacking any viral test element). In order to compare the various assay systems, all activities were normalized to the activity of the 35S min construct included within each assay set (35S min activity arbitrarily set to 1.0).
Determining DNA sequence element frequency in viral and Arabidopsis databases
Since the element enrichment approach works best when applied to relatively short, core consensus sequences [31], database searches were limited to those viral enhancers that displayed a clear core consensus (CLE, BCTV DR19/30, CT-rich, Figure 3). Results from the viral enhancer searches were compared to values obtained using previously reported plant promoter elements (DRE, ABRE-like, and G-box), and a short DNA sequence that is part of a highly conserved geminiviral replication loop stem sequence (CGCGNCCA) that is identical in base composition and length to the CLE consensus (Table 1). The short sequence elements were each tested for their frequency of occurrence within a set of DNA sequence databases. One database consists of all entries for geminiviruses plus nanoviruses ([36], as of May, 2004) and all others are from the A. thaliana genomic sequence at the TAIR, PatMatch web site [32]. The geminivirus/nanovirus BLAST searches were set for short exact matches (the statistical significance threshold set to 1000 and word size set at the element's length), returning the number of occurrences of exact matches for the full length element within the database. The TAIR PatMatch searches (default settings: Max hits, 7500; both strands; mismatch = 0; minimum hits/seq = 1; maximum hits/seq = 100) were performed against sub-datasets representing Arabidopsis coding sequences {"GI CDS (- introns, - UTRs)"}, and various lengths of upstream regions {"Locus Upstream Sequences", -1 to -500, -1 to -1000 and -1 to -3000}. Results from the -500 search were subtracted from the -1000 results, to generate hits from -501 to -1000 and -1000 results subtracted from the -3000 data to calculate hits from -1001 to -3000. In order to allow direct comparison between searches in different databases, using sequence elements of differing length and base composition, the number of database hits was corrected for the size of the database (number of hits divided by the database size in mega-base pairs {Mbp}) and base composition (hits/Mbp divided by the predicted number of hits per Mbp using upon the element sequence and base composition of each search database). The dataset base compositions were determined from downloaded sequence files and are: A. thaliana CDS: A/T = 55.8%, G/C = 44.2%; A. thaliana upstream (-1 to -500): A/T = 67.43%, G/C = 32.57%; A. thaliana upstream (-501 to -1000): A/T = 66.24%, G/C = 33.76%; viral: A/T = 56.2%, G/C = 43.8%. The resulting frequency of occurrence is a corrected number of hits per mega-base pairs (cHits/Mbp). For ease of comparison between elements, all of the cHits/Mbp values have been normalized to the corresponding cHits/Mbp number from the A. thaliana CDS database (set arbitrarily to 1.0). Correction of the element's frequency using the calculated random probability of occurrence does not account for the possible impacted by intrinsic base-order bias that may occur within each sequence database, specifically the coding region database. These biases can potentially shift cHits/Mbp numbers markedly from those calculated using simple random base composition frequencies. To help confirm the significance of any observed enhancement in an elements frequency, mean cHits/Mbp values for 18 randomly generated sequences that match each test sequence for base composition and length were determined to provide a baseline value for comparison to that of the test element (shown in parenthesis, Table 1). A total of 18 sequences were used to produce the reported baseline as mean cHits/Mbp values were found to routinely level off at n values of between 8–12 random sequences examined (data not shown).
Competing interests
A patent application is being considered for synthetic plant promoters containing some of the elements described in this article.
Disclaimer
Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.
Authors' contributions
JV conceived of the study, participated in its design and coordination and drafted the manuscript. KM performed much of the search for short repeats within viral sequences and contributed to development of protoplast-based reporter gene assays. CIC generated and tested all the elements examined and developed the in vivo assay used to quantify enhancer activity. All authors read and approved the final manuscript.
Supplementary Material
Additional File 1
Excel worksheet listing viral elements that fail to enhance expression
Click here for file
Acknowledgements
We are very grateful for the helpful comments on the manuscript generously provided by Dr. John Stanley, Dr. Bruno Gronenborn and Dr. Mel Oliver. Dr. Scot Dowd's assistance was indispensable in the setup and analysis of the viral GenBank database. This work benefited greatly from the expert technical assistance of Mr. David Wheeler.
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| 15730562 | PMC554758 | CC BY | 2021-01-04 16:39:00 | no | Virol J. 2005 Feb 24; 2:16 | utf-8 | Virol J | 2,005 | 10.1186/1743-422X-2-16 | oa_comm |
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J NeuroinflammationJournal of Neuroinflammation1742-2094BioMed Central London 1742-2094-2-71573056110.1186/1742-2094-2-7ReviewInvolvement of β-chemokines in the development of inflammatory demyelination Banisor Ileana [email protected] Thomas P [email protected] Bernadette [email protected] SLRHC, Columbia University, New York, NY, USA2 Thomas Jefferson University, Philadephia, PA, USA2005 24 2 2005 2 7 7 13 1 2005 24 2 2005 Copyright © 2005 Banisor et al; licensee BioMed Central Ltd.2005Banisor et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The importance of β-chemokines (or CC chemokine ligands – CCL) in the development of inflammatory lesions in the central nervous system of patients with multiple sclerosis and rodents with experimental allergic encephalomyelitis is strongly supported by descriptive studies and experimental models. Our recent genetic scans in families identified haplotypes in the genes of CCL2, CCL3 and CCL11-CCL8-CCL13 which showed association with multiple sclerosis. Complementing the genetic associations, we also detected a distinct regional expression regulation for CCL2, CCL7 and CCL8 in correlation with chronic inflammation in multiple sclerosis brains. These observations are in consensus with previous studies, and add new data to support the involvement of CCL2, CCL7, CCL8 and CCL3 in the development of inflammatory demyelination. Along with our own data, here we review the literature implicating CCLs and their receptors (CCRs) in multiple sclerosis and experimental allergic encephalomyelitis. The survey reflects that the field is in a rapid expansion, and highlights some of the pathways which might be suitable to pharmaceutical interventions.
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Introduction
Multiple sclerosis (MS) is a disabling disease of the central nervous system (CNS) with features of autoimmunity and neurodegeneration. Although the identity of primary antigenic determinant(s) is uncertain, an interaction between β-chemokine ligands and their receptors plays a central role in the recruitment and retention of inflammatory cells in the CNS. Thus, both the disease relevant chemokine ligands and their receptors represent potential therapeutic targets in MS.
Chemokines are a group of small, structurally related chemoattractant molecules that regulate cell trafficking through interactions with a set of receptors [1]. Evidence suggests that the migration of autoreactive immune cells via the blood-brain barrier (BBB) is an early and critical process during the development of inflammatory CNS lesions of experimental allergic encephalomyelitis (EAE) and MS, and that this transmigration is regulated by chemokines produced at the blood-brain barrier (BBB) and in the CNS. Subcellular signals induced by the binding of chemokines to their G-protein-coupled receptors leads to an increased avidity of integrins on leukocytes to their corresponding receptors on endothelial cells, followed by a facilitated migration of leukocytes towards the chemokine gradient in the CNS [2,3].
In addition to chemotaxis, chemokines are also involved in the regulation of T cell differentiation, apoptosis, cell cycle, angiogenesis and metastatic processes. Further, chemokines can control the generation of soluble inflammatory products such as free radicals, nitric oxide, cytokines and matrix metalloproteases [1,4]. Considering the predominantly T helper type 1 (TH1) mediated process of inflammatory demyelination and the TH2 driven suppression of inflammation, the differential effects of various chemokines on TH1 or TH2 polarization may have particular significance. The currently known, approximately 50 chemokine genes in humans are divided into four subfamilies on the basis of characteristic patterns of cysteine residues close to the N-terminal end of the products. The CC chemokine ligand family (CCL) (also known as β-chemokines or Small Cytokine Group A – SCYA in mice) is characterized by two adjacent cysteines, while the CXC (SCYB) and CX3C (SCYD or fractalkine) chemokine families have one or three intervening amino acids, respectively, between the two cysteines. In the XC family (SCYC or lymphotactin), only one cysteine is present [1]. All four classes of chemokines play important roles in the immune inflammatory network, but because of the complexity of interactions, here we only discuss the CC chemokine family. In humans, there are 27 CC chemokines, most of which including CCL2, CCL7, CCL11, CCL8, CCL13, CCL1, CCL5, CCL16, CCL14, CCL15, CCL23, CCL18, CCL3 and CCL4, respectively, are encoded as a cluster within chromosome 17q11. The genes for CCL27, CCL19 and CCL21 are located within chromosome 9p13, while CCL17 and CCL22 are encoded at 16q13. The remaining CCL genes can be found on chromosome 2 and 7 [1].
A functional classification was also proposed to distinguish between lymphoid and inflammatory chemokines [1,5]. Lymphoid or homeostatic chemokines (e.g. CCL21, CCL25, CXCL13) are constitutively expressed and control physiologic trafficking of cells of the adoptive immune system during hematopoiesis and immunosurveillance. Inflammatory or induced chemokines (e.g. CCL2, CCL3, CCL5, CCL7, CCL8, etc...) are transcriptionally regulated during inflammation and mediate the recruitment of inflammatory cells to target tissues.
The effects of chemokines are mediated by G-protein coupled receptors with seven-transmembrane-domains. Chemokine receptors tend to bind multiple chemokine ligands and vice versa. However, the biologically most efficient interaction often occurs between a receptor and its primary ligand (e.g. CCL2 – CCR2). The receptor binding involves high affinity interactions and signal transduction initiated by the dissociation of G-protein complex into Gα and Gβγ subunits. Gα induces the activation of the phosphoinositidine 3-kinase pathway, while the Gβγ subunits activate phospholipase C and induce Ca2+ influx and protein kinase C activation. The involvement of MAP kinases as well as JAK/STAT signaling also has been shown [6]. As of today, 10 CC chemokine receptors (CCRs), 6 CXCRs, one CX3CR1 and one XCR1 are known [1,6].
This review focuses on the immunomodulatory effects of the β-chemokine or CCL family in EAE and MS. CC chemokines predominantly are involved in the recruitment of monocytes / macrophages and dendritic cells (monocyte chemoattractant proteins -MCP-1 [CCL2], MCP-2 [CCL8], MCP-3 [CCL7], MCP-4 [CCL13] and macrophage inflammatory proteins – MIP-1α [CCL3] and MIP-1β [CCL4]), and to lesser degrees, T lymphocytes and NK cells (MCP and MIP chemokines, regulated upon activation normal T cell expressed and secreted cytokine [RANTES]) or occasionally other cell types (e.g. eosinophil chemotactic protein – eotactin [CCL11]) into inflammatory lesions of MS.
Genetic evidence for the involvement of β-chemokines in multiple sclerosis
A meta-analysis of raw genotype data from three genome scans in MS families revealed the highest nonparametric linkage (NPL) score = 2.58 at 17q11 [7]. Among several candidate genes (e.g. NOS2A, OMG, NF1), a cluster of evolutionarily closely related β-chemokine genes [CCL2, CCL7, CCL11, CCL8, CCL13, CCL1, CCL5, CCL16, CCL14, CCL15, CCL23, CCL18, CCL3 and CCL4, respectively] is encoded within a 1.85 Mb segment of 17q11.2-q12. Our recent linkage disequilibrium mapping confined the susceptibility regions to 3–30 kb haplotypes defined by single nucleotide polymorphisms (SNP) within the genes of CCL2, CCL11-CCL8, CCL8-CCL13, CCL13 and CCL3 [8]. A second study is under way to confirm and further refine the MS relevant haplotypes, and then, to identify the specific disease causing nucleotide variants in an independent set of families.
Within the orthologous mouse chromosome 11, two quantitative trait loci (QTL), eae6 and eae7 were identified. While these loci control the severity and duration of EAE, eae7 is also a susceptibility locus for the monophasic remitting / non-relapsing subtype of the disease [9]. Sequence polymorphisms within the genes of Scya1 (TCA-3 or CCL1), Scya2 (MCP-1 or CCL2) and Scya12 (MCP-5 or CCL12) in eae7 showed striking segregations among mouse strains resistant or susceptible to EAE.
Using an advanced intercross line in combination with congenic strains, Jagodic et al. [10] fine mapped eae18 and identified two adjacent QTLs, eae18a and eae18b, on the rat chromosome 10 in a myelin-oligodendrocyte glycoprotein (MOG)-induced, chronic relapsing EAE. The eae18b locus is also orthologous to human chromosome 17q11 and encodes a cluster of β-chemokine genes.
The recognition of β-chemokine genes as susceptibility and quantitative trait loci in mouse and rat EAE along with the human data revealing the β-chemokine gene cluster as a susceptibility locus in MS, strongly suggest the involvement of β-chemokine variants in the development of inflammatory demyelination.
CCL and CCR molecules in inflammatory demyelination
Experimental allergic encephalomyelitis
EAE is a valuable model for studying the effector arm of immune response in inflammatory demyelination. It can be induced in susceptible strains of inbred and outbred species by active immunization with myelin related proteins and their peptides (myelin basic protein – MBP, proteolipid lipoprotein – PLP, myelin oligodendrocyte glycoprotein – MOG) emulsified in Freund's complete adjuvant along with intravenous Pertussis toxin, or with a passive transfer of myelin antigen specific T cell lines into naïve recipients. Using various immunization protocols, acute and chronic relapsing (CR-EAE) models have been developed. In both the active immunization and the passive transfer models of EAE, the efferent arm of immune response involves the migration of monocytes / macrophages, dendritic cells and activated myelin-antigen-specific T lymphocytes from the blood circulation into the CNS, where a reactivation of specific lymphocytes by myelin-antigen-presenting dendritic cells, macrophages and residential microglia takes place, and the sequential development of perivascular and parenchymal inflammation is followed by demyelination and neuronal degeneration.
Encephalitogenic T lymphocytes have CD4+ TH1 phenotype characterized by the production of interleukin (IL)-2 and interferon-γ. TH2 lymphocytes producing IL4, IL5, IL6 and IL10 cytokines are involved in the counter-regulation of TH1 effects, and promote clinical recovery. The TH1 / TH2 polarization is regulated by cytokines and chemokines. The transmigration of immune competent cells via the blood-brain barrier is aided by a temporal and spatial regulation of adhesion molecules on T lymphocytes and their counterparts on endothelial cells, and of chemokine ligands and their receptors in the residential CNS and hematogenous mononuclear cells.
One of the most comprehensively studied CC chemokines in inflammatory demyelination is MCP-1 (CCL2). MCP-1 (CCL2) influences both innate immunity through its chemoattractant effect on monocytes / macrophages, and adaptive immunity through its effect on T cell polarization towards the TH2 subtype [11]. CCL2 primarily acts via the CCR2 receptor [1].
MIP proteins have both chemotactic and proinflammatory effects, but also promote homeostasis [6]. The MIP-1 family includes MIP-1α (CCL3), MIP-1β (CCL4), MIP-1δ (CCL9/10) and MIP-1γ (CCL15) that are produced by macrophages, microglia, astrocytes, dendritic cells and lymphocytes. These MIP-1 molecules act via CCR1, CCR3 and CCR5 expressed by lymphocytes and monocytes. MIP-1 proteins also regulate immune response by modulating T cell differentiation. The CCL3 and CCR5 interaction promotes polarization towards the TH1 subtype.
Our understanding concerning the role of these CC chemokines and their receptors in inflammatory demyelination was greatly advanced by studies in the EAE model. In mice, the increased expression of MCP-1 (CCL2) by CNS immune cells is closely associated with the clinical activity of EAE [12-14]. Some studies, however, suggest that the presence of leukocytes is necessary for the production of CCL2 by astrocytes, as the expression of CCL2 prior to the accumulation of inflammatory mononuclear cells has not been observed in the CNS. Substantial MCP-1 (CCL2) expression may only occur in the late phase of acute disease and in the relapsing phases of CR-EAE. It was therefore postulated, that CCL2 is involved in the amplification rather than in the initiation of EAE [4]. In contrast, the MIP-1α (CCL3) expression correlates with the severity of acute disease and also is elevated during relapses in CR-EAE. RANTES (CCL5) is expressed in the CNS throughout the course, but does not correlate with the severity of acute or CR-EAE [13].
Jee et al [15] compared the histological features and MCP-1 (CCL2) and CCR2 expression levels in the lesions of Lewis rats during the acute attack of monophasic EAE and during the first two clinical events of CR-EAE. In concert with the mouse data [4,13], not only higher numbers of macrophages infiltrated the spinal cord during the first and second attacks of CR-EAE as compared to those at the peak of acute EAE in these rats, but the expression of MCP-1 (CCL2) was also significantly higher in the lesion of CR-EAE as compared to that of acute EAE. Similarly, CCR2, the main receptor for CCL2, was expressed by astrocytes, macrophages and T cells in higher amounts during CR-EAE than at the peak of acute EAE. This observation confirmed the role of CCL2 – CCR2 interaction in the development of relapses.
Youseff et al [16] observed an increased mRNA transcription not only for MCP-1 (CCL2), but also for MIP-1α (CCL3) and MIP-1β (CCL4) at the onset of EAE in rat brains. MIP-1α (CCL3) and MCP-1 (CCL2) declined in two days even though the clinical disease further progressed. MIP-1β (CCL4) mRNA declined in correlation with the clinical recovery. RANTES (CCL5) mRNA, in contrast, increased in the brains only after recovery. The full length, reverse transcribed and PCR amplified DNA product for each of these four CCL molecules was transferred into a plasmid vector and injected as naked DNA vaccine into rats. Both the transcription of a relevant chemokine and the induced antibody response against it was monitored. The in vivo immune response to these CCL molecules differentially influenced the evolution of EAE. MIP-1α (CCL3) and MCP-1 (CCL2) DNA vaccines prevented EAE, while MIP-1β (CCL4) aggravated the disease and RANTES (CCL5) did not have an effect on the course of EAE. This study emphasizes the importance of CCL2 and CCL3 in the development of active EAE in rats.
CCL1 also attracted attention in EAE. Teutscher et al [9] identified eae7 encoding CCL1 and other chemokines as a susceptibility locus and QTL in murine EAE. SNPs in CCL1 differentially segregated in mouse strains susceptible or resistant to EAE. mRNA molecules for both CCL1 and its receptor CCR8 were detected in spinal cord lesions of EAE, in correlation with the expression of tumor necrosis factor (TNF)-α by inflammatory leukocytes [17-19]. As both CCL1 and CCR8 were detected in microglia, an autocrine signaling mechanism was postulated. CCR8 (-/-) mice showed marked delay in the onset and reduced severity of EAE as compared to controls. Leukocyte infiltration in the spinal cord was not diminished in the CCR8 (-/-) mice, suggesting that that a defective microglial activation might have altered the clinical phenotype.
Recent studies addressed the role of chemokines at the blood-brain barrier. Using intravital fluorescence videomicroscopy, Vajkoczy et al [20] demonstrated that the interaction between encephalitogenic T cells and endothelial cells of the BBB involves α4-integrin (VLA-4) which mediates a G-protein-independent capture (arrest) followed by G-protein-dependent adhesion strengthening of circulating T cells to VCAM-1 on endothelial cells. Postulating the involvement of chemokines in the integrin-mediated arrest of autoreactive T cells at the BBB, the investigators [3] subsequently aimed to identify the specific chemokines by performing in situ hybridization and immunohistochemistry on brain and spinal cord sections of mice with EAE. Constitutive expression of the lymphoid chemokine called EBV-induced molecule 1 ligand chemokine (ELC) / CCL19 in a subpopulation of CNS venules and induced expression of the secondary lymphoid chemokine (SLC) / CCL21 in inflamed CNS venules was detected. CCR7, the common receptor for these two chemokines was expressed on a subpopulation of cells in the perivascular cuffs. Encephalitogenic T cells in vitro showed expression of CCR7 and CXCR3, the alternative receptor for CCL21, and chemotaxed towards both CCL19 and CCL21 in a concentration-dependent and a Pertussis toxin-sensitive manner similar to naïve T cells. Functional deletion of CCR7 and CXCR3 or immune blockade of CCL19 and CCL21 reduced the binding of encephalitogenic T cells to inflamed venules in frozen brain sections. Altogether, these data suggest that CCL19 and CCL21 are expressed in cerebral endothelial cells and are involved in α4-integrin mediated adhesion strengthening of autoreactive T cells and subsequently of other inflammatory cells to the endothelial layer of the BBB. These molecular interactions may lead to permanent inflammatory cell immigration into the CNS in chronic autoimmune disease.
CCL20 or MIP-3α (exodus-3 / LARC) is a chemokine active on dendritic cells and lymphocytes that express CCR6 [1]. Serafini et al [21]demonstrated the occurrence of dendritic cells in the spinal cord of mice immunized with the PLP139–151 peptide. Although dendritic cells were present during early acute, chronic and relapsing EAE, most prominent infiltration of spinal cord by mature dendritic cells was noted in relapsing disease. In all stages of EAE, CCL20 and CCR6 were upregulated in the CNS. This study emphasizes the importance of dendritic cells in antigen presentation and T cell restimulation, and links the immigration of dendritic cells to the expression of CCL20 in the CNS during EAE.
CCL22 or macrophage-derived chemokine (MDC) is chemoattractant for monocytes, dendritic and NK cells, and T lymphocytes of the TH2 subtype. MDC / CCL22 acts via CCR4 which is preferentially detected on TH2 type, memory and regulatory T cells [22]. While MDC / CCL22 is considered to be predominantly involved in TH2 mediated immunity, Columba-Cabezas et al [22] demonstrated mRNA expression for MDC / CCL22 in the CNS of mice with relapsing-remitting and chronic-relapsing EAE induced by PLP139–151 or whole spinal cord homogenate. Immunohistochemistry demonstrated that MDC / CCL22 is produced by infiltrating leukocytes and residential microglia, while CCR4 is expressed by infiltrating leukocytes. In vitro activation of microglia resulted in secretion of bioactive MDC / CCL22 that induced chemotaxis of TH2 lymphocytes. This study concludes that MDC / CCL22 produced by microglia may play a role in a TH1 mediated CNS inflammation by inducing the homing of TH2 regulatory cells into the lesion site.
To further clarify the role of chemokine receptors involved in EAE, Fife et al [23] examined CCR expression in normal (unprimed), PLP139–151 primed non-activated, PLP139–151 primed and reactivated lymph node derived T cells, and CNS-isolated CD4+ T cells from SJL mice receiving PLP139–151 specific, in vitro reactivated T cells. Normal resting CD4+ T cells and primed non-activated T cells expressed mRNA for CCR1, CCR2, CCR3, CCR5, CCR6, CCR7 and CCR8. In vitro activated T cells expressed in higher amounts most of the CCRs found in normal T cells as well as CCR4. After passive transfer of encephalitogenic activated T cells in naïve recipients, the donor derived encephalitogenic cells and the host-derived CD4+ T cells isolated only from the CNS lesions but not from spleen expressed mRNA for CCR1. This latter observation was confirmed at protein level, and appeared to be specific for acute EAE. Neutralization of the CCR1 ligand CCL3 (MIP-1α) diminished the inflammatory infiltrate in the CNS.
The effects of anti-chemokine treatments in the mouse EAE is summarized by Elhofy et al [24] and Karpus et al [25] and is in consensus with data in the rat model. Although various strains and protocols were used, overall anti-RANTES (CCL5) had no effect in these models, anti-MIP-1α (CCL3) decreased the severity of acute EAE and anti-MCP-1 (CCL2) reduced the severity of both acute EAE and the relapses in CR-EAE. However, it is important noting that in some respect, these observations are model specific. While the impact of anti-CCL5 immune treatment was unremarkable in the autoantigen-induced forms of EAE, antibody treatment targeting CCL5 in a mouse hepatitis virus-induced inflammatory demyelination model resulted in diminished leukocyte infiltration and reduced neurological disability [26].
Genetic manipulations of the murine model provide further insights in the characterization of CCL / CCR molecules in EAE. In mice with the CCL2 transgene under the control of the lck (which directs the expression of transgene to cortical thymocytes) or MBP promoters (which directs the expression of transgene to the CNS), a spontaneous infiltration of monocytes / macrophages in the thymus and CNS was observed, respectively [27]. LPS injection induced higher CCL2 expression in the brain and markedly enhanced the mononuclear cell (MNC) infiltrate. The relationship between LPS treatment, CCL2 expression and MNC recruitment into the CNS remains partially understood, and seems to involve a complex immune regulatory mechanism rather than just a selective effect mediated by the upregulation of the CCL2 transgene. Nevertheless, these transgenic mice were clinically normal both before and after LPS injection. More recently, Elhofy et al [28] examined TH1 lymphocytes in a PLP-induced EAE model using a transgenic mouse strain that constitutively expressed low CCL2 levels in the CNS under the control of the astrocyte-specific glial fibrillary acidic protein promoter. CCL2 transgenic mice developed milder EAE than the littermate controls, despite similar numbers of CD4 and CD8 T cells in the CNS infiltrates and an increased number of monocytes in the CNS of the CCL2 transgenic animals. Functional studies revealed that encephalitogenic T cells from the CCL2 transgenic mice produced significantly less interferon-γ and proliferated less in the presence of PLP peptides than those of the non-transgenic controls. Increased CCL2 expression in the CNS also resulted in a decreased IL-12 receptor expression by PLP-specific T cells. Thus in this model, the overexpression of CCL2 in the CNS resulted in a suppression of the TH1 response and a milder clinical phenotype of EAE, despite the enhanced effect on monocytes.
The CCL2 knock out (-/-) mice showed resistance to EAE and significantly decreased macrophage infiltration in the CNS following active immunization with MOG35–55 peptide. While T cells from CCL2 (-/-) mice transferred EAE to wild type mice, wild type T cells did not induce EAE in CCL2 (-/-) recipient mice. These observations suggest a key role for CCL2 in the recruitment of macrophages into the CNS and thus, in the pathogenesis of EAE [29,30,4]. The array of ligands for CCR2 includes MCP-1 (CCL2), MCP-2 (CCL8), MCP-3 (CCL7) or MCP-5 (CCL12). As CCL2 (-/-) mice did not show a compensatory upregulation of MCP-2 (CCL8), MCP-3 (CCL7) or MCP-5 (CCL12) mRNA molecules, MCP-1 (CCL2) is likely to be the main ligand for CCR2 in mice with EAE.
The clinical phenotype of CCR2 (-/-) genotype was similar to that of the CCL2 (-/-) genotype, characterized by a reduced macrophage infiltration in the spinal cord and a decreased susceptibility to actively (MOG35–55) induced acute EAE in the studies by Fife et al [31] and Izikson et al [32]. T cells from CCR2 (-/-) immunized mice produced similar levels of interferon-γ and IL2 as those from controls, and were capable of transferring EAE in a naïve recipient. In contrast, T cells from wild type mice did not cause EAE in a CCR2 (-/-) recipient [31]. However, these observations again appeared to be model specific. Gaupp et al [33] reported that, even though the disease was milder or delayed, three CCR2 (-/-) mouse strains retained susceptibility to EAE in their experiments. Histological analyses revealed an abundance of neutrophils in lesions of the CCR2 (-/-) mice in contrast to the monocyte abundance in EAE lesions of wild-type mice. The development of compensatory immune mechanisms for the lack of CCR2 was evidenced by the increased mRNA expression for other CCL and CCR molecules (most notably IL8 and its receptor involved in neutrophil recruitment). This study emphasizes that promiscuity of chemokines and their receptors may overcome the deletion of a single CCR receptor with a resultant mild modification of the clinical and more profound modification of the histological phenotype.
Further studies demonstrated an approximately 50% reduction of clinical EAE activity in the CCR1 (-/-) mice, likely involving the altered migration of monocytes and lymphocytes [34]. In contrast to the observed EAE suppression in the CCR1 (-/-) and CCR2 (-/-) models, the CCR5 knockout mice had the same disease severity as the wild-type controls [35]. These studies underscore the importance of CCR1 and CCR2 in the development of inflammatory demyelination and give support to novel alternative strategies targeting these CCR molecules. Such strategies include the development of small functional CCR antagonists, amongst which the most significant progress has been made with CCR1 antagonists [36,37]. CCR1 antagonist compounds were shown to inhibit CCL3 and CCL5 induced migration of MNCs in a dose dependent manner, and to reduce clinical EAE in rat [36-38].
In sum, CCL and CCR data from rodent EAE models using inbred, transgenic and knockout strains along with data from chemokine-specific antibody treatments or CCL DNA immunization in EAE suggest that concentration gradients of CCL2 and CCL3 decreasing from the CNS to the peripheral circulation are involved in the spatially and temporally regulated recruitment of mononuclear cells into the CNS which correlates with the course of clinical disease. CCL2 may play a more significant role during relapses than during the induction phase of the disease. CCL5 is expressed by mononuclear cells in the perivascular space during the recovery phase of an acute event, and may therefore be involved in the regulation of recovery rather than in the initiation of the disease. In addition, CCL19 and CCL21 are expressed by endothelial cells of the BBB, and are involved in the strengthening of leukocyte adhesion to inflamed venules followed by homing of encephalitogenic T lymphocytes to the CNS. CCL20 can control the recruitment of dendritic cells into lesions, whereas CCL22 may be involved in a TH2 mediated regulatory process during EAE. CCL1 is likely playing an important role in the autocrine regulation of activation of macrophages and microglia in EAE lesions. Thus, the functional involvement of CCL chemokines during EAE is not only restricted to a well orchestrated recruitment of dendritic cells, monocytes, macrophages, T effector and regulatory cells into the CNS, but also includes a temporal and spatial regulation of TH1 (CCL3, CCL5) or TH2 (CCL2, CCL22) polarization, and monocyte, macrophage and microglial activation (CCL1, CCL2, CCL7, CCL8). Their receptors, the CCRs play equally important roles in these processes. Experimental evidence now suggests that CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8 and CXCR3 on hematogenous mononuclear cells recognize these chemoattractant and regulatory molecules to induce cell differentiation, adhesion or migration of distinct inflammatory cells in peripheral lymphoid organs, at the BBB and in the CNS during the course of EAE. Even taking into consideration the complex and promiscuous nature of the CCL – CCR network, certain pathways may be associated with distinct biological function amenable to intervention. Targeting CCR molecules either by monoclonal antibodies or by small functional antagonists has become a novel and realistic strategy in the treatment and prevention of autoimmune diseases.
Multiple sclerosis
The complexity of disease pathogenesis, difficulties accessing the site of pathology and the descriptive nature of studies explain why the available CCL / CCR data are less comprehensive in MS as compared to those in EAE. Nevertheless, new observations support the generally accepted views that MS is a predominantly TH1 lymphocyte mediated disease, and CCL – CCR molecules play a significant part in the regulation of intercellular interactions in the peripheral lymphoid organs, at the BBB and in the CNS. In addition to defining chemotaxis, CCL-CCR interactions are involved in TH1 / TH2 polarization and regulation in MS. Recent studies also raise the possibility that distinct molecular mechanisms with characteristic CCL-CCR kinetics correlate with the development of histological subtypes of the disease.
CCRs in the multiple sclerosis brain
A recent review of chemokines and their receptors [39] suggests that every CC chemokine receptor (CCR1-CCR5) interact with multiple CCLs and vice versa. Five CCRs (CCR1, CCR2, CCR3, CCR5 and CXCR3) were detected on infiltrating monocytes, macrophages and lymphocytes in MS lesions. In contrast, several members of the CCL family [CCL2 = MCP-1, CCL3 = MIP-1α, CCL4 = MIP-1β, CCL5 = RANTES, CCL7 = MCP-3, CCL8 = MCP-2] were expressed in astrocytes, microglia and other inflammatory cells within MS lesions.
While control brain specimens had only scarce appearance of CCR positive (microglial) cells throughout the CNS, foamy macrophages, microglia, perivascular lymphocytes and occasionally, astrocytes were positive for CCR2, CCR3 and CCR5 in chronic active plaques [39,40]. In other studies, CCR1, CCR2, CCR3 and CCR5 were detected on mononuclear cells and macrophages in demyelinating plaques [41,42].
Trebst et al [43] investigated the kinetics of CCR expression. In early demyelinating lesions, CCR1+/CCR5+ hematogenous monocytes and CCR1-/CCR5- microglial cells were detected. In later stages, macrophages became CCR1-/CCR5+, while microglia upregulated CCR5. This observation suggest that CCR1+/CCR5+ hematogenous monocytes enter into the CNS and stay there in the presence of appropriate ligands. During evolution of lesions, these cells down-regulate CCR1 while retain the CCR5 expression. A more recent study [44] reveals that this distinct temporal pattern, namely the decrease in CCR1+ and increase in CCR5+ cells, may be restricted to the histological type II demyelinating lesions characterized by mononuclear cell infiltration and immunoglobulin plus complement deposition, and is not seen in type III lesions characterized by oligodendrocytopathy and apoptosis [45].
CCR2 may also play a key role in the lesion development based on more indirect information. CCR2 is the main, but not exclusive, functional receptor for CCL2 [4], and as discussed above, the CCL2 – CCR2 interaction appears to play a key role in the development of EAE lesions. Microglia, macrophages and perivascular mononuclear cells show some degrees of immune reactivity for CCR2 in chronic active plaques in several studies, but the expression of CCR2 is generally low in MS lesions. Nevertheless, the data from EAE and observations in MS suggest that the CCR2 – CCL2 interaction is important in the development of plaques. This view was recently proposed and will be discussed below.
CCR8, the receptor for CCL1, has been detected in vitro on TH2 and regulatory lymphocytes, macrophages and microglia. Using immunohistochemistry, Trebst et al [19] detected CCR8 on phagocytic macrophages and activated microglia in type II and type III demyelinating MS lesions. CCR8 expression correlated with the demyelinating activity, but was not restricted to the MS pathology. Phagocytic macrophages and activated microglia in stroke and progressive multifocal leukoencephalopathy also expressed CCR8. Thus, CCR8 seems to identify a subset of activated microglia in different CNS pathologies.
CCLs in the multiple sclerosis brain
Using methods of immunohistochemistry and in situ hybridization, McManus et al [46] investigated the expression of three monocyte chemoattractant proteins, MCP-1 (CCL2), MCP-2 (CCL8) and MCP-3 (CCL7) in correlation with the temporal evolution of plaques. All three proteins were detected in high amounts in the center, but sharply decreased at the edges of acute and chronic active lesions. Hypertrophic astrocytes showed the strongest expresion, while infiltrating mononuclear cells showed variable reactivity in plaques. MCP-3 (CCL7) was also detected in the extracellular matrix. Reactivity for these CC chemokine ligands outside of plaques was otherwise restricted to hypertrophic astrocytes. In situ hybridization confirmed the observation for CCL2 at mRNA level. There seemed to be an inverse correlation between the age of plaques and expression of these three CCL molecules, with only a scanty appearance of immunoreactive astrocytes in chronic silent lesions. These methods did not detect MCP chemokines in the brains of normal controls.
Additional studies demonstrated the expression of CCL3 and CCL4 in macrophages and microglia, and CCL3 also in astrocytes [47-49]. CCL5 was primarily detected in perivascular inflammatory cells and astrocytes [48-50].
While most of the above studies used the method of immunohistochemistry, we recently assessed the mRNA expression levels for CCL2, CCL3, CCL5, CCL7, CCL8, CCL13 and CCL15 relative to β-actin in corresponding normal appearing white matter (NAWM), normal appearing gray matter (NAGM) and chronic active plaque containing specimens from ten post mortem MS brains. These specimens were characterized by hematoxyllin & eosin, Luxol Fast Blue and immune staining specific for CD68 and β2-microglobulin [51]. In addition, the expression distribution for pro- and anti-apoptotic molecules in these specimens was also assessed by real-time PCR [51]. The selection of the above listed CC chemokines was based on two considerations. First, we detected MS associated SNP haplotypes in the genes of CCL2, CCL11-CCL8-CCL13, CCL15 and CCL3 [8]. Second, previous studies suggested the involvement of CCL2, CCL7, CCL8, CCL5 and CCL3 molecules in the development of plaques [39,46]. While neither our genetic nor our mRNA studies revealed positive findings for CCL5, the three MCP chemokines CCL2 (MCP-1), CCL7 (MCP-3) and CCL8 (MCP-2) showed altered regional expressions in MS brains. We detected an increased expression of CCL2 in plaques as compared to NAWMs, and an increased expression of CCL7 in both plaques and NAWMs as compared to NAGMs. In contrast, the expression level of CCL8 was decreased in plaques as compared to NAWM or NAGM specimens (Banisor and Kalman, unpublished observation). This analysis of CCL mRNA molecules in various regions of MS brain complements the data from previous immunohistochemical studies, and further confirms the involvement of CCL2 and CCL7 (and possibly of CCL8) in the development of pathology. In consensus with others, however, we also note an increased CCL7, CCL8 and CCL13 expression in the white matter as compared to the gray matter in 5 other neurological disease controls (1 viral and 1 post-infectious encephalitis, 2 Alzheimer disease and 1 Parkinson disease). No differences were observed for any of these molecules in the white and gray matters of normal controls. We postulate that the expression of CCL molecules may be detected in various inflammatory conditions of the CNS, however, the temporal and cell specific upregulation of certain CCL and CCR molecules is pathology specific. Therefore, further exploration of the expression kinetics of these molecules may facilitate a better understanding of MS pathogenesis.
CCL and CCR detected in blood and CSF
Relatively limited numbers of studies are available regarding chemokines and their receptors in the blood circulation and in the cerebrospinal fluid (CSF) in MS patients. The expression of CCR5 was found to be higher on circulating T lymphocytes from MS patients than on those from normal controls. These T cells showed an increased migration towards CCL3 and CCL5, suggesting a functional significance of the altered receptor expression [42,52]. The migratory population represented predominantly TH1 / TH0 cells, while the non-migratory population was enriched for TH2 cells. The aberrant migration of T cells towards CCL3 and CCL5 was related to the increased expression of the CCR5 receptor, and could be blocked by anti-CCR5 antibodies. A fluctuation of CCR5 expression by T cells was also suggested in correlation with relapses and remissions in a small group of patients [53].
Sorensen and Sellebjerg [54] assessed the CCR expression profile on peripheral T cells of patients with relapse, remission or secondary progressive disease, and detected a higher percentage of CCR2-expressing T cells in secondary progressive MS (SPMS) than in other patient groups. CCR2-positive T cells displayed TH2 profile producing IL5 and tumor necrosis factor-α. The CCR5 expression associated with TH1 profile was significantly lower in SPMS than in patients with relapsing-remitting MS (RRMS) during relapse. Thus, the authors conclude that patients with SPMS have a high expression of CCR2, a chemokine receptor associated with TH2 profile, whereas patients with RRMS preferentially display T cells with CCR5 expression and TH1 profile. More CCR5 positive T cells produced tumor necrosis factor-α in patients with RRMS than those in patients with SPMS. CCR2 is known to be predominantly expressed on monocytes. However, when expressed on T cells, CCR2 is associated with the TH2 subtype as CCL2 induces differentiation of T cells into TH2 phenotype [11]. While Sorensen and Sellebjerg [54] detected significant differences in the CCR5 and CCR2 expression profile between RRMS and SPMS, they noted no differences in CCR expression between RRMS and controls. The observation regarding the association of CCR5 with RRMS is consistent with a previous study revealing that patients with the defective CCR5 receptor (CCR5 Δ32 deletion) have prolonged relapse free periods, but the long term prognosis of MS did not seem to correlate with the CCR5 Δ32 genotype [55]. Besides establishing the CCR characteristics in RRMS and SPMS, this study also suggests that targeting the CCL2-CCR2 axis with specific CCR2 antagonist or a combination of CCR2 and CCR5 antagonists might be an option in SPMS, whereas CCR5 antagonists alone may be considered in RRMS.
However, CCR5 on peripheral MNCs was not uniformly found to be differentially expressed in MS subtypes [56]. MNCs from blood constitutively expressed CCL4 and CCL5, the ligands for CCR5, in all patient groups and controls. This study also failed to detect CCL2 and CCL3 by ribonuclease protection assay in peripheral blood MNCs. Further, the complexity of information regarding CCR5 is reflected by a recent study suggesting the association of the CCR5 Δ32 genotype with early death in MS [57].
There is relatively limited information available regarding CCR and CCL expression levels in the CSF. Some investigators showed that CCR5+ mononuclear cells of MS patients were enriched in the CSF, representing a significant proportion of monocytes and only a minority of T cells. However, neither cell population differed quantitatively from those of controls, suggesting that CSF leukocytes may not be fully reflective of CNS inflammation [39,55].
Giunti et al [58] detected CCR5, CCR7 and CXCR3 positive T cells in the CSF of patients with MS and other inflammatory neurological disease (IND) (meningitis, encephalitis, CIDP, neuroborreliosis). Coexpression of these receptors was noted on a subset of memory cells. The increased ratio of CXCR3 / CCR4 was suggested as a molecular correlate of disease activity by Nakajima et al.[59] TH1 clones established from the CSF of patients with IND and of controls similarly migrated in vitro towards CXCL10, CXCL12 and CCL5. CXCL10, CXCL12 and CCL19 were increased in the CSF of these patients [58].
Amongst CC chemokines, CCL3 and CCL5 were most consistently found to be elevated in the CSF of MS patients during relapses as compared to normal controls [59-61]. In contrast, decreased CCL2 was found in the CSF in all clinical forms of MS by Scarpini et al.[62] More consistently, however, low CCL2 levels were detected only during relapses by others [41,59,61,63,64]. The drop of CCL2 in the CSF was not found during relapses of neuromyelitis optica [65]. Mahad et al [64] also found that CCL2 in the CSF was decreased not only in patients with MS but also in patients with IND when compared to those of non-inflammatory CNS disease controls. In contrast, Bartosik-Psujek and Stelmasiak [61] observed an increase in both CCL2 and CCL5 in the CSF of patients with IND, and suggested that the drop of CCL2 during relapses is characteristic only of MS. Further, CCL2 concentration increased as time from the last relapse increased and following corticosteroid therapy [63,64]. With the exception of well defined changes in the CCL2, CCL3 and CCL5 levels in the CSF during relapses, most investigators observed no differences in various clinical forms of the disease [56,61,64,66].
Pashenkov et al [67] studied two secondary lymphoid organ chemokines, CCL19 (exodus-3, MIP-3β) and CCL21 (exodus-2, SCL) in CSF and sera of patients with MS, clinically isolated syndrome (CIS) presenting as optic neuritis (ON), isolated ON, IND and non-inflammatory neurological disease controls (NINC). CSF of the NINC group contained CCL19 but not CCL21, while both chemokines were elevated in the CSF of patients with MS, CIS-ON and IND. The authors postulate that CCL19 and CCL21 may control the retention of dendritic cells and the recruitment of naïve T cells and activated B cells, or a de novo formation of lymphoid structures in plaques. These cells are known to express CCR7, the receptor for CCL19 and CCL21. EAE studies also support the notion that CCL19 and CCL21 play important roles both at the BBB and in the CNS [3].
To correlate previous data on CCL concentrations in the CSF of MS patients, Kivisakk et al [68] measured mRNA for CCL2 / MCP-1 and CCL5 / RANTES in MNCs in the CSF and blood of patients with MS, acute meningitis and normal controls. While high numbers of MNCs expressing CCL2 and CCL5 were found in some patients, overall no differences were observed between MS and acute meningitis. This study would argue that there is no systemic dysregulation of CC chemokines contributing to MS pathogenesis.
In sum, the above data suggest that CCL1, CCL2, CCL3, CCL4, CCL5, CCL7 and CCL8 are expressed by residential glia and perivascular leukocytes in plaques. Expression of the corresponding CCR1, CCR2, CCR3, CCR5 and CCR8 receptors has been demonstrated on infiltrating leukocytes, but also on microglia, dendritic cells and astrocytes. While the expression kinetics of CCR1 and CCR5 may discriminate between histological type II and type III lesions of MS, CCR8 is similarly expressed in both lesions types (Table 1).
Table 1 CCR and CCL molecules in plaques, blood and CSF of MS patients. This Table summarizes in a cross-sectional manner major findings regarding CCR and CCL expression in brain, blood and CSF of multiple sclerosis patients. The dynamic nature of changes is detailed in the text. The interactions of CCL-CCR molecules on specific cell types are depicted in Figure 1. Type II and III lesions refer to the histological classification proposed by Lucchinetti et al [45]. References are indicated in brackets.
In chronic active plaque expressed on In blood expressed on In CSF expressed on
CCR1 Monocyte, macrophage, lymphocyte [39,41,42]
CCR2 Monocyte, macrophage, lymphocyte [39-42] TH2 in SPMS [54]
CCR3 Monocyte, macrophage, lymphocyte [39-42]
CCR4 Monocyte, macrophage, lymphocyte [39]
CCR5 Monocyte, macrophage, lymphocyte [39-42] TH1/TH0 in RRMS [42, 52-54] MNC [39,55,58]
CCR7 T, dendritic [58]
CCR8 Macrophage, microglia in type II and III lesions [19]
In early -> late stage type II lesion
CCR1+/CCR5+ -> CCR1-/CCR5+ Monocyte, macrophage [43]
CCR1-/CCR5- -> CCR1-/CCR5+ Microglia [43]
In acute, and to lesser degrees, in chronic active plaques expressed by In blood expressed by In CSF
CCL2 Astrocyte, microglia, MNC [46] low in relapse [59,61,63,64]
CCL3 Astrocyte, microglia, macrophage [47-49] increased in relapse [59-61]
CCL4 Microglia, macrophage [47-49] MNC [56]
CCL5 MNC, astrocyte [48-50] MNC [56] increased in relapse [59-61]
CCL7 Astrocyte, microglia, MNC [46]
CCL8 Astrocyte, microglia, MNC [46]
CCL19 present in NIND, increased in MS, CIS-ON, IND [67]
CCL21 increased in MS, CIS-ON, IND [67]
The increase of CCL3 and CCL5 in the CSF during a relapse correlates with the increase in the expression of their receptor, CCR5 on TH1 lymphocytes, which results in an enhanced migratory activity of these cells towards CCL3 and CCL5. The consistently observed decrease in CCL2 levels in the CSF during or even prior to a relapse generated alternative considerations. The first consideration suggests, that the decreased CCL2 level likely relate to a decreased TH2 lymphocyte activity, as CCL2 induces TH2 polarization. Vice versa, CCL2 expression is controlled by TH2 cytokines such as IL4. The concept of CCL2 – TH2 coregulation is supported by the observation that clinical improvement and normalization of the inflammatory CSF profile after corticosteroid treatment correlate with the normalization of CCL2 in the CSF. Thus, measurements of CCL2 in the CSF may also reflect the fluctuation of TH2 activity during the course of MS. The second consideration was proposed by Dr. Ransohoff (oral presentation at the ECTRIMS meeting 2004) [4,69]. This interpretation reconciles the complex observations from the EAE model suggesting a key role for CCR2 – CCL2 in the development of inflammatory lesions, and from MS suggesting a low expression of CCR2 and increased expression of CCL2 in active plaques, but a decreased CCL2 level in the CSF. Based on this model: 1) CCL2 – CCR2 play an important role in the development of inflammatory demyelinating lesions both in EAE and MS; 2) CCL2 expressed in the CNS attracts CCR2+ monocytes and T cells into the developing plaque; 3) while CCR2 binds and internalizes CCL2 molecules in large amounts, CCL2 will be consumed resulting in a reduced CCL2 level in the intercellular fluids and the CSF; 4) when CCR2 encounters its ligand, the CCR2 / CCL2 complex will be internalized and CCR2 will be downregulated on the surface of inflammatory cells in the lesion.
Conclusion
Studies on EAE and MS suggest that CC chemokine ligands (most prominently CCL2, CCL3, CCL5, CCL7, CCL8, but also CCL1, CCL4, CCL19 and CCL21) expressed by residential immune cells in the CNS or by endothelial cells at the BBB are major chemoattractants for hematogenic immune cells (primarily monocytes / macrophages [CCL2, CCL7, CCL8, CCL22] dendritic cells [CCL19, CCL20, CCL21, CCL22] and T lymphocytes [CCL1, CCL2, CCL3, CCL4, CCL5, CCL19, CCL21, CCL22]) via interactions with their G-protein-coupled receptors (CCR1-CCR10). These CCL – CCR interactions play a key role in the recruitment, activation and retention of immune competent cells in the CNS, with the CCL1 – CCR8, CCL2 – CCR2, CCL3 – CCR1 / CCR5, CCL5 – CCR1 / CCR5, CCL7 – CCR1 / CCR2 / CCR3, CCL8 – CCR3, CCL20 – CCR6, CCL19 / CCL21 – CCR7, CCL22 – CCR4 interactions being the best characterized among them (Figure 1). The EAE model suggests that CCL19 and CCL21 produced by endothelial cells induce G-protein-mediated signaling via their receptor CCR7. This signaling leads to an enhanced adhesion of the leukocyte α4-integrin (VLA-4) to the endothelial VCAM-1 and results in a facilitated transmigration of leukocytes via the BBB. CCL-CCR interactions also define the differentiation and chemotaxis of T cell subpopulations, and thus may control the dynamic changes in the local balance of TH1 (CCL3 – CCR1 / CCR5, CCL5 – CCR1 / CCR5) and TH2 (CCL1 – CCR8, CCL2 – CCR2, CCL22 – CCR4) cell populations in lesion. Different CCL – CCR expression kinetics may characterize the different (initial, height, self-limiting) phases and histological subtypes (type II or type III) of inflammatory demyelination. This differential involvement of chemokines and their receptors in various stages and forms of MS, and the arising information concerning the involvement of genetic variants of CCLs suggest that small CCR antagonists may represent a realistic strategy in controlling the inflammatory activity that may have to be adjusted to individual disease characteristics.
Figure 1 Interaction between CCL and CCR molecules at the blood-brain barrier. This figure depicts CCL-CCR interactions at the BBB (endothelial cells and astrocytic processes) interfacing a venule and the CNS. CCL molecules (most prominently CCL2, CCL3, CCL7 and CCL8, but also CCL1, CCL4, CCL19 and CCL21) are produced by residential microglia, astrocytes and endothelial cells throughout the course of lesion development, and by infiltrating MNCs (CCL5) during late phases of plaque formation, and attract functionally different subsets of monocytes / macrophages, dendritic cells and T lymphocytes from the circulation via the BBB into the CNS. The temporal and spatial regulation of molecular events, the association of distinct CCR molecules with different histological subtypes of demyelination and the involvement of different CCL-CCR interactions in T cell polarization are detailed in the text. Here we illustrate in a simplified and cross-sectional manner the main groups of interacting receptors on various hematogenous cells and ligands released by residential immune cells of the CNS or by components of the BBB. Group A of receptors and ligands expressed by and acting on monocytes / macrophages, respectively: CCR1 / CCR2 / CCR3-CCL7, CCR2-CCL2, CCR3-CCL8, CCR4-CCL22; Group B of receptors and ligands expressed by and acting on dendritic cells, respectively: CCR4-CCL22, CCR6-CCL20, CCR7-CCL19 / CCL21; Group C of receptors and ligands expressed by and acting on T lymphocyes, respectively: CCR1-CCL3 / CCL5, CCR2-CCL2, CCR4-CCL22, CCR5-CCL3 / CCL4 / CCL5, CCR7-CCL19 / CCL21, CCR8-CCL1.
List of abbreviations
BBB – blood brain barrier
CCL – CC chemokine ligand
CCR – CC chemokine receptor
CIS – clinically isolated syndrome
CNS – central nervous system
CR-EAE – chronic-relapsing EAE
CSF – cerebrospinal fluid
EAE – experimental allergic encephalomyelitis
EBV – Epstein-Barr virus
IL – interleukin
IND – inflammatory neurological disease
LPS – Lipopolysaccharide
MBP – myelin basic protein
MCP – monocyte chemotactic protein
MIP – macrophage inflammatory protein
MNC – mononuclear cells
MOG – myelin-oligodendrocyte glycoprotein
MS – multiple sclerosis
NAGM – normal appearing gray matter
NAWM – normal appearing white matter
NF1 – neurofibromatosis, type I
NINC – non-inflammatory neurological disease controls
NK cells – natural killer cells
NOS2A – nitric oxide synthase 2A
NPL – nonparametric linkage
OMG – oligodendrocyte-myelin glycoprotein
ON – optic neuritis
PCR – polymerase chain reaction
PLP – proteolipid lipoprotein
QTL – quantitative trait loci
RANTES – regulated upon activation normally T expressed and secreted cytokine
RRMS – relapsing-remitting MS
SNP – single nuclear polymorphism
SPMS – secondary-progressive MS
TH1 – T-helper 1
TH2 – T-helper 2
TNF – tumor necrosis factor
VCAM-1 – vascular cell adhesion molecule-1
VLA-4 – very late antigen-4
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
Ileana Banisor, research assistant, was involved in the acquisition and analyses of our research data mentioned in the paper. She prepared the figure. Thomas P. Leist, collaborator, critically reviewed and edited the manuscript. Bernadette Kalman, P.I., designed the research studies mentioned from her lab, supervised the work processes, interpreted the data and drafted this manuscript. She also generated funding supports.
Acknowledgements
Dr. B. Kalman and her team are supported by research grants from the National Multiple Sclerosis Society, Wadsworth Foundation, and Serono Inc. The Multiple Sclerosis Research and Treatment Center at the Roosevelt Hospital generally provided the space and opportunity to carry out all research activities related to this paper.
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Bartosik-Psujek H Stelmasiak Z The levels of chemokines CXCL8, CCL2 and CCL5 in multiple sclerosis patients are linked to the activity of the disease Eur J Neurol 2005 12 49 54 15613147 10.1111/j.1468-1331.2004.00951.x
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RetrovirologyRetrovirology1742-4690BioMed Central London 1742-4690-2-141574353410.1186/1742-4690-2-14CommentaryNew York City HIV superbug: fear or fear not? Smith Stephen M [email protected] Section of Infectious Diseases, Department of Medicine, Saint Michael's Medical Center, New Jersey, USA2 Department of Preventive Medicine and Community Health, The New Jersey Medical School, Newark New Jersey 07102, USA2005 2 3 2005 2 14 14 28 2 2005 2 3 2005 Copyright © 2005 Smith; licensee BioMed Central Ltd.2005Smith; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
On February 11, 2005, the New York City Department of Health and Mental Hygiene announced that a city resident had recently been infected with a multi-drug resistant form of HIV and rapidly progressed to AIDS. The Health Commissioner, Thomas R. Frieden, called for increased vigilance against this new strain. Is this situation an emerging crisis or simply an unusual case report of rapid HIV progression?
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On February 11, 2005, New York City (NYC) health officials announced the discovery of a "rare strain of multi-drug resistant HIV that rapidly progresses to AIDS." According to the NYC Department of Health and Mental Hygiene, a man in his mid-40s was diagnosed with HIV infection in December 2004. Shortly after his diagnosis, testing, at the Aaron Diamond AIDS Research Center in Manhattan, revealed that his virus was resistant to almost all anti-HIV therapeutics. Further, despite being infected for only 2–20 months, the man had developed AIDS. NYC Health Commissioner Thomas R. Frieden, MD, MPH, stated, "This case is a wake-up call. First, it's a wake-up call to men who have sex with men, particularly those who may use crystal methamphetamine...now, we've identified this strain of HIV that is difficult or impossible to treat and which appears to progress rapidly to AIDS." Dr. Frieden called on this community to help stop the spread of this and other drug resistant strains of HIV. He also called on NYC doctors and the public health system to improve HIV prevention counseling, to perform HIV drug resistance testing among treatment naïve, HIV+ persons, and to improve anti-HIV drug adherence.
At the 12th Conference on Retroviruses and Opportunistic Infections (CROI) in Boston, Drs. David Ho and Martin Markowitz of the Aaron Diamond AIDS Research Center in Manhattan presented clinical and laboratory data regarding the NYC resident[1]. He had tested negatively for HIV-1 antibodies several times before and in May 2003. His total lymphocyte counts during these time points were repeatedly normal. Investigators believe that the NYC resident may have been infected in October 2004, when he, while on crystal methamphetamine, engaged in unprotected, receptive and insertive anal sex with multiple partners. In early November 2004, the NYC resident developed a febrile illness, and then in December 2004, he tested positive for antibodies against HIV. His personal physician, concerned over the possibility of recent acute HIV-1 infection, referred the NYC resident to Dr. Martin Markowitz. At the time of diagnosis, his CD4+ T-cell count was 80 cells/mm3 and it has since fallen to less than 50. The NYC resident meets one criterion for the diagnosis of AIDS; his CD4+ T-cell count is less than 200 cells/mm3. His viral load has varied from ~100 K to 650 K/ml. The NYC resident's virus was tested and found to be resistant to all but two anti-HIV drugs, efavirenz (Sustiva®) and enfuvirtide (Fuzeon®; T20). This high degree of drug resistance existed before the NYC resident was treated with any anti-HIV compound.
Is this case a harbinger of a new epidemic with this superbug or is it just an isolated, forme fruste of HIV infection? No one knows the answer to this question yet, but we do have plenty of data to suggest that the latter is the case. In people naïve to drug therapy, bone fide antiviral resistance is uncommon. A recent USA based study of treatment-naïve patients found that the prevalence of mutations associated with drug resistance was 8.8%[2]. This means 8.8% of the subjects' viruses tested positive by genotyping for 1 or more mutations associated with drug resistance. Having a single mutation associated with resistance does not necessarily make a virus drug resistant. For many drugs, HIV must contain several mutations to become resistant. This fact is true for most protease inhibitors (PIs) and for several nucleoside analogue reverse transcriptase inhibitors (NRTIs). Therefore, the overall level of drug resistance is well less than 8.8% reported in this study. Indeed, in this study, no significant resistance to protease inhibitors was seen. A similar study found the overall prevalence of drug resistance mutations was 8.3%, as also determined by genotyping[3]. However, when viruses containing these mutations were analyzed by phenotyping, only 39% demonstrated decreased reduced drug susceptibility. In other words, less than 3.5% of all isolates had phenotypic resistance.
A commonly held view on why the level of drug resistance is low is that most mutations associated with drug resistance, also decrease viral fitness. Except the K103N reverse transcriptase mutation, which confers resistance to the three approved non-nucleoside inhibitors (NNRTIs), other mutations, associated with high level drug resistance, are thought to significantly decrease viral fitness. Theoretically, in the absence of drug pressure in a newly infected individual, wild-type virus is either selected for during transmission or the transmitted, resistant virus mutates back towards the fitter wild type. The current observation is that the vast majority of viruses in treatment-naïve patients are sensitive to almost all drugs.
In addition to CD4, the virus, isolated from the NYC resident, uses CXCR4 or CCR5 to enter cells[1]. Such viruses, termed dual-tropic, are rarely seen in newly infected individuals. Typically, an R5 virus, which utilize CCR5, is the transmitted type. After years of infection, in approximately 50% of individuals, the viruses' tropism changes from CCR5 to CXCR4[4]. This phenotypic change is associated with an accelerated disease course. People, who are homozygous for the CCR5, 32-bp deletion, do not express functional CCR5 and have a high relative resistance to infection with HIV. In those Δ32 homozygotes, who have become infected with HIV (8 individuals reported), the disease course appears to be more rapid[5]. Most of these individuals had CD4+ T-cell counts less than 300 cells/mm3 at the time of diagnosis. It is unclear why these individuals became infected, while the vast majority of Δ32 homozygotes remain uninfected. Possibly, these 8 individuals have some other aberration, which allows them to become infected with an X4 virus and, in turn, leads to an accelerated disease course. Perhaps, the NYC resident has a similar abnormality, which has lead to an increased rate of CD4+ T-cell depletion.
Also at CROI this year, Drs. Stephen Gange and Alvarez Muňoz from Johns Hopkins University Bloomberg School of Public Health presented models of rapid HIV progression probability, based on two, large prospective cohorts[6]. These studies, the MACS and theWIHS, have been on-going for the past 21 and 11 years respectively and have collected longitudinal data on 391 seroconverters. In the pre-HAART era, the median time to AIDS was 8.3 years. Using the cohorts' data, Drs. Gange and Muňoz estimated the probability of clinical AIDS developing within 6–24 months or a low CD4+ T-cell count existing at the first visit after diagnosis (within the first 9 months of infection). Their model predicts that 7 in 10,000 patients develop clinical AIDS within 6 months of infection. This number increases to 45 in 10,000 after 12 months. Similarly, 10 in 10,000 HIV infected individuals have a CD4+ T-cell count less than 200 cells/mm3 after 4.5–9 months of infection.
Several reports of rapidly progressing HIV infection have been published. The rapid disease course of the NYC resident is rare, but hardly unique. To date, no cluster of rapid progressors has been described. All rapid progressors have been unrelated, either genetically or virologically. While multi-drug resistant (MDR) viruses may be overall less fit compared with wild-type, drug sensitive strains, MDR HIV still causes steady CD4+ T-cell depletion. Therefore, it is highly probable that the NYC resident has a genetic predisposition, which led to rapid progression, rather than a new strain of HIV-1, which is simultaneously super-aggressive and multi-drug resistant.
Our experience at a large, inner city HIV clinic is in agreement with the above data. We do not see rapidly progressing, newly diagnosed individuals. We also do not see MDR HIV in our treatment-naïve patients. Review of our data does not reveal any evidence of MDR virus in persons, who have never been on therapy.
To determine whether this "superbug" has spread to others, the NYC Department of Health is appropriately and aggressively investigating the sexual contacts of the NYC resident. The reason for the NYC Department of Health press release at this early point in the investigation is unclear. In the absence of a documented cluster of patients, should the entire health system react? No, we should wait for more information. I do agree that genotypic resistance testing for treatment-naïve HIV+ patients is prudent, especially when the person is thought to have been infected within the past year. Of course, most patients do not know when they were infected, so we are testing each new patient.
Given the availability of free, rapid testing for HIV in New Jersey, we are strongly encouraging any one with current or previous high-risk behavior to get tested and determine his/her HIV status. The best way to fight this disease is with knowledge: knowledge on one's infection status, knowledge on how to avoid becoming infected, and knowledge on how not to infect some one else. HIV is not the common cold. It is transmitted through well-described behaviors, predominantly sex, especially receptive anal intercourse, and intravenous drug use with shared needles. These behaviors can be modified to reduce or eliminate the risk of contracting HIV. Two recent studies conclude that universal testing for HIV is a cost effective way to combat this infection in the USA [7-9]. Outreach prevention education and widespread testing are probably more effective public health strategies than sensational press releases. Dr. Frieden's call for increased vigilance against drug resistant HIV implies that regular, old-fashioned HIV infection is not horrific enough. Any one who has seen this disease up close knows that is not the case. While we have partially effective therapies and we better understand its pathogenesis, HIV infection in this country, not as life threatening as it once was, remains quite life altering.
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Markowitz M Mohri H Mehandru S Shet A Berry L Kalyanaraman R Kim A Chung C Jean-Pierre P Horowitz A La Mar M Wrin T Parkin N Poles M Petropoulos C Mullen M Boden D Ho DD A Case of Apparent Recent Infection with a Multi-Drug-Resistant and Dual-Tropic HIV-1 in Association with Rapid Progression to AIDS: February 24; Boston. 2005
Novak RM Chen L MacArthur RD Baxter JD Huppler Hullsiek K Peng G Xiang Y Henely C Schmetter B Uy J van den Berg-Wolf M Kozal M Prevalence of antiretroviral drug resistance mutations in chronically HIV-infected, treatment-naive patients: implications for routine resistance screening before initiation of antiretroviral therapy Clin Infect Dis 2005 40 468 474 15668873
Weinstock HS Zaidi I Heneine W Bennett D Garcia-Lerma JG Douglas JMJ LaLota M Dickinson G Schwarcz S Torian L Wendell D Paul S Goza GA Ruiz J Boyett B Kaplan JE The epidemiology of antiretroviral drug resistance among drug-naive HIV-1-infected persons in 10 US cities J Infect Dis 2004 189 2174 2180 15181563 10.1086/420789
Philpott SM HIV-1 coreceptor usage, transmission, and disease progression Curr HIV Res 2003 1 217 227 15043204
Sheppard HW Celum C Michael NL O'Brien S Dean M Carrington M Dondero D Buchbinder SP HIV-1 infection in individuals with the CCR5-Delta32/Delta32 genotype: acquisition of syncytium-inducing virus at seroconversion J Acquir Immune Defic Syndr 2002 29 307 313 11873082
Gange S Munoz A Variations in the Natural History of HIV Seroconverters in US Military Cohorts: February 24; Boston. 2005
Bozzette SA Routine screening for HIV infection--timely and cost-effective N Engl J Med 2005 352 620 621 15703428 10.1056/NEJMe048347
Paltiel AD Weinstein MC Kimmel AD Seage GR Losina E Zhang H Freedberg KA Walensky RP Expanded screening for HIV in the United States--an analysis of cost-effectiveness N Engl J Med 2005 352 586 595 15703423 10.1056/NEJMsa042088
Sanders GD Bayoumi AM Sundaram V Bilir SP Neukermans CP Rydzak CE Douglass LR Lazzeroni LC Holodniy M Owens DK Cost-effectiveness of screening for HIV in the era of highly active antiretroviral therapy N Engl J Med 2005 352 570 585 15703422 10.1056/NEJMsa042657
| 15743534 | PMC554760 | CC BY | 2021-01-04 16:36:40 | no | Retrovirology. 2005 Mar 2; 2:14 | utf-8 | Retrovirology | 2,005 | 10.1186/1742-4690-2-14 | oa_comm |
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Respir ResRespiratory Research1465-99211465-993XBioMed Central London 1465-9921-6-221574062910.1186/1465-9921-6-22ResearchAcute effects of cigarette smoking on inflammation in healthy intermittent smokers van der Vaart Hester [email protected] Dirkje S [email protected] Wim [email protected] Machteld N [email protected] Brigitte WM [email protected] H Marike [email protected] Judith M [email protected] Reus Dorothea M [email protected] Henk F [email protected] Hacken Nick HT [email protected] Department of Pulmonology, University Medical Center Groningen, Groningen, the Netherlands2 Department of Pathology, University Medical Center Groningen, Groningen, the Netherlands3 Department of Allergology University Medical Center Groningen, Groningen, the Netherlands4 Department of Epidemiology and Statistics, University Medical Center Groningen, Groningen, the Netherlands2005 1 3 2005 6 1 22 22 28 9 2004 1 3 2005 Copyright © 2005 van der Vaart et al; licensee BioMed Central Ltd.2005van der Vaart et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Chronic smoking is the main risk factor for chronic obstructive pulmonary disease. Knowledge on the response to the initial smoke exposures might enhance the understanding of changes due to chronic smoking, since repetitive acute smoke effects may cumulate and lead to irreversible lung damage.
Methods
We investigated acute effects of smoking on inflammation in 16 healthy intermittent smokers in an open randomised cross-over study. We compared effects of smoking of two cigarettes on inflammatory markers in exhaled air, induced sputum, blood and urine at 0, 1, 3, 6, 12, 24, 48, 96 and 192 hours and outcomes without smoking. All sputum and blood parameters were log transformed and analysed using a linear mixed effect model.
Results
Significant findings were: Smoking increased exhaled carbon monoxide between 0 and 1 hour, and induced a greater decrease in blood eosinophils and sputum lymphocytes between 0 and 3 hours compared to non-smoking. Compared to non-smoking, smoking induced a greater interleukin-8 release from stimulated blood cells between 0 and 3 hours, and a greater increase in sputum lymphocytes and neutrophils between 3 and 12 hours.
Conclusion
We conclude that besides an increase in inflammation, as known from chronic smoking, there is also a suppressive effect of smoking two cigarettes on particular inflammatory parameters.
SputumChronic Obstructive Pulmonary DiseaseInflammationTobaccoCarbon Monoxide
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Background
Chronic obstructive pulmonary disease (COPD) is one of the leading causes of morbidity and mortality world-wide, and its prevalence is still rising [1]. In order to develop strategies for its prevention and treatment, it is important to understand the underlying pathophysiologic mechanisms of this disease. Since chronic smoking is the main risk factor to develop COPD most studies in this field have been carried out in chronic (ex)smokers with or without COPD. It is also important to study the initial response to cigarette smoke to better understand the effects of chronic smoking, since repetitive acute smoke effects may cumulate and ultimately lead to irreversible lung damage associated with COPD. In addition, to appropriately evaluate the impact of chronic smoking, the "background" effects of acute smoking should be determined.
Until now, only a few studies have investigated acute effects of smoking in humans [2]. Unfortunately, these studies investigated only a small number of time points after smoking, hence little information is available on the time course and resolution of smoking induced changes. Furthermore, all studies assessed acute effects of smoking in chronic smokers who refrained from smoking for maximally 24 hours. It is unknown whether this is sufficiently long to exclude the influence of previous smoking on the acute smoke results. Finally, no study so far investigated acute smoke effects in sputum.
In the present study we investigated acute effects of smoking of two cigarettes by healthy intermittent smokers who refrained from smoking nine days before the study period. In this way, temporary effects on the airways due to chronic smoking will probably not affect the acute response to smoke. We assessed the time effects of cigarette smoking on both induction and resolution of the inflammatory response in exhaled air, induced sputum, blood and urine. We hypothesised that smoking of two cigarettes would induce an increase in inflammatory cells and markers within a limited time interval.
Methods
Design of the study
We performed a randomised, two-period cross-over, pilot study. Subjects were randomised into smoking two cigarettes or no smoking. Subjects refrained from smoking during nine days before each study period, verified by exhaled carbon monoxide (CO < 6 ppm) and urinary cotinine (< 25 ng/ml). The time interval between the two study periods varied between 9 to 20 days. Measurements of exhaled CO, exhaled Nitric Oxide (NO), blood sampling and Forced Expiratory Volume in 1 second (FEV1) were performed immediately before (baseline) and 1, 3, 6, 12, 24, 48, 96 and 192 hours after smoking and at the same time points in the no smoking period. Sputum was induced at 3, 6, 12, 24, 48, 96, 192 hours after smoking and no smoking. All subjects smoked two cigarettes from the same brand within 30 minutes and were encouraged to inhale deeply (Caballero unfiltered cigarettes, tar 12 mg, nicotine 1.0 mg, commercially obtained, no gifts). Adequacy of smoke inhalation was verified by the investigator. The working groups sputum induction from the ERS stated recently that sputum inductions should not be repeated within 48 hours to avoid carry over effects [3]. Taking this into account, we used a cross-over design (including no smoking) in this study to correct for this carry over effect. We have analysed the results of the control arm as a separate study in order to investigate the induction and resolution of the inflammatory response generated by repeated sputum inductions [4].
Subjects
Sixteen healthy intermittent smokers were recruited by advertisements in the local newspaper. Intermittent smoking was defined as smoking more than one cigarette a month, but not daily, during the last 6 months. We chose to investigate intermittent smokers because they are able to refrain from smoking for a certain time period (in contrast to most current smokers) and they are used to inhale smoke (in contrast to non-smokers). Included were subjects older than 40 years, with normal lung function (prebronchodilator FEV1/IVC [Inspiratory Vital Capacity] > 89% of predicted for women and > 88% of predicted for men [5] and a prebronchodilator FEV1 > 1 litre). Excluded were subjects with: 1) a history of asthma, allergic rhinitis, or allergic eczema; 2) atopy, confirmed by a positive skin prick test; 3) any current respiratory disease, symptoms of cough or sputum production; 4) a respiratory tract infection within the preceding 8 weeks or a nasal infection within the preceding 4 weeks; 5) treatment with glucocorticosteroids within the preceding 8 weeks; 6) use of aspirin, NSAIDs, paracetamol or antihistamines within the preceding 4 weeks. Subjects were asked to avoid places with high environmental tobacco smoke exposure during the study periods. The study was approved by the medical ethics committee of the University Medical Center Groningen, the Netherlands. Written informed consent was obtained from all subjects.
Pulmonary function, exhaled NO and CO
FEV1 and IVC were measured according to the guidelines of the European Respiratory Society [5], using a pneumotachograph (Jaeger, Wurzberg, Germany). Exhaled NO levels were determined according to the guidelines of the American Thoracic Society [6], exhaling with a flow of 100 ml/sec against a resistance between 5 and 20 cm H2O, using a chemiluminescence analyser (Ecophysics CLD 700 AL). Exhaled CO levels were measured using an infrared CO analyser (UNOR 6N, Maihak AG, Hamburg, Germany) [7].
Blood analyses
Blood differential cell counts were analysed automatically with a haematology flow cytometer (Coulter-STKS, Beckman Coulter, Miami, USA). Flow cytometric analysis was performed on blood cells using peridinin chlorophyll protein (PerCP) labelled anti-human leukocyte antigen (HLA)-DR, phycoerythrin (PE) labelled anti-CD11b, allophycocyanin (APC) labelled CD14 and fluorescein-isothiocyanate (FITC) labelled CD63 monoclonal antibodies (Becton Dickinson, Franklin Lakes, NJ USA). HLA-DR, CD63 and CD11b are activation markers for respectively monocytes and granulocytes. CD14 is used to discern between monocytes and granulocytes. Functional assays were performed on unstimulated and lipopolysacharide (LPS, 1 ng/ml, BioWhittaker, Walkerville, USA) stimulated blood cells, measuring tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-8 and IL-10 by ELISA (Sanquin, Amsterdam, the Netherlands).
Sputum induction and processing
Sputum was induced according to a modified standard technique [8], using 4.5% hypertonic saline. Whole sputum was processed within 120 minutes according to the modified method of Rutgers and colleagues [8]. The cell-free supernatant was collected and stored in aliquots at -80°C pending analysis of soluble mediators.
Sputum analyses
Flow cytometric analysis was performed on sputum cells using PerCP labelled anti-HLA-Dr, PE labelled anti-CD11b, APC labelled CD14 and FITC labelled CD63 monoclonal antibodies (Becton Dickinson, Franklin Lakes, NJ USA). Immunocytology was performed to quantify the percentage of inducible NO synthase (iNOS) positive macrophages. Cytospins were double stained with a monoclonal antibody against CD68 (IgG1 isotype, Dako, Glostrup, Danmark) as a marker for macrophages and rabbit polyclonal antiserum against iNOS (Transduction Laboratories, Lexington, KY, USA).
The following soluble mediators were measured in sputum supernatant. NO2-/NO3- was measured using the Griess reaction, eosinophilic cationic protein (ECP) using the fluorenzyme immunoassay UniCAP ECP (Pharmacia, Uppsala, Sweden). IL-8 and Leukotriene B4 (LTB4) were measured by a commercial ELISA (IL-8: Sanquin, Amsterdam, the Netherlands, LTB4: Amersham Biosciences, UK). Matrix metalloproteinase-9 (MMP-9) was measured by gelatine zymography [9], and tissue inhibitor of metalloproteinase-1 (TIMP-1) by ELISA (R&D, Abingdon, UK). Neutrophil elastase (NE) activity was measured by chromogenic substrate assay (N-methoxysuccinyl-ala-ala-pro-val-p-nitoanilide, Sigma, UK)] [10].
Urinary measurements
Before inhalation of smoke (or control), a urine portion was collected to measure urine cotinine. Cotinine was measured by gaschromatography-mass-spectrometry (Pharmacy Department, Groningen, the Netherlands). Furthermore, urine was collected over 24 hours in five consecutive fractions: 0–1 hour, 1–3 hours, 3–6 hours, 6–12 hours and 12–24 hours from all subjects to assess leukotriene E4 levels (ELISA, Amersham Biosciences, UK).
Statistical analyses
Since the start and duration of the acute effects of smoking of two cigarettes on our parameters were unknown, time series of all variables were plotted. Based on visual inspection of these plots the time intervals to be analysed were selected. The slopes of parameters were estimated using linear mixed effect models [11] by including the variables time (hours), smoking (yes or no) and their interaction. For the sputum parameters no baseline values were present, therefore time point 192 hours was used as baseline value. After log-transformation of all blood and sputum variables, the residuals of the models were normally distributed. All analyses were performed in S-plus 2000 (Insightful Corporation, Seattle, WA, USA). A p value <0.05 was considered statistically significant.
Results
Subjects
Clinical characteristics of the 16 subjects are listed in table 1. Fifteen subjects successfully refrained from smoking for nine days. One subject smoked one cigarette five days before the start of the study, but the urinary cotinine and exhaled CO levels were within the required range. The analyses are performed on data from all 16 subjects.
Table 1 Subject characteristics (healthy intermittent smokers).
Sex, male/female 12/4
Age, years 49 (39–71)
Smoked pack years 4 (0–40)
Smoked cigarettes per month 14 (1–60)
FEV1, % predicted 119 (68–144)
FEV1/ IVC, % 77.6 (68.1–87.0)
Values expressed as medians (ranges). FEV1: Forced Expiratory Volume in 1 second, IVC: Inspiratory Vital Capacity.
Exhaled NO and CO and FEV1
Exhaled CO increased significantly more with smoking than without between 0 and 1 hour and subsequently decreased significantly more between 1 and 12 hours (table 2, figure 1). Smoking had no significant effect on exhaled NO (data not shown) or FEV1 (table 2).
Figure 1 Time course of smoking of two cigarettes on exhaled carbon monoxide (CO). Black circles represent the values after smoking two cigarettes and grey circles represent the values of the control period.
Table 2 Linear mixed effect models: CO and FEV1
Independent variable Time interval B 95% CI P value
CO, ppm 0–1 hour 3.61 2.67–4.54 <0.0001
1–12 hours -0.29 -0.38 – -0.21 <0.0001
FEV1, L/sec 0–1 hour 0.06 -0.07–0.20 0.38
The time intervals of the above parameters were selected based on visual inspection of the plots. The slopes of the parameters were estimated using linear mixed effect models [11] by including the variables time (hours), smoking (yes or no) and their interaction. B: regression coefficient for the variable time (for further information see the method section). CO: carbon monoxide; FEV1: Forced Expiratory Volume in 1 second.
Blood
The number of blood eosinophils decreased more with smoking than without between 0 and 3 hours (table 3 and figure 2). Smoking had no significant effect on the number of other blood cells (table 3 and additional file 1, table 1). IL-8 release from LPS stimulated blood cells increased more with smoking than without between 0 and 3 hours (table 3). Smoking had no significant effect on TNF-α, IL-10 and IL-1β release compared with no smoking (additional file 1, table 2). There was no significant difference in the expression of CD11b, CD63 and HLA-DR on CD14 high and CD14 low cells between smoking and no smoking (data not shown).
Table 3 Linear mixed effect models: blood cells, IL-8 and TNF-α
Independent variable Time interval B 95% CI P value
Log (leucocytes, 109/L) 0–12 hours 0.00 -0.01–0.01 0.44
Log (neutrophils, 109/L) 0–12 hours 0.00 -0.01–0.02 0.58
Log (monocytes, 109/L) 0–1 hour 0.04 -0.10–0.19 0.55
Log (eosinophils, 109/L) 0– 3 hours -0.11 -0.18 – -0.03 0.01
Log (lymphocytes, 109/L) 0–12 hours -0.00 -0.01–0.01 0.65
Log (IL-8, pg/ml)* 0–3 hours 0.09 0.04–0.14 0.001
Log (TNF-α, pg/ml)** 0–3 hours 0.02 -0.08–0.12 0.75
The time intervals of the above parameters were selected based on visual inspection of the plots. The slopes of the parameters were estimated using linear mixed effect models [11] by including the variables time (hours), smoking (yes or no) and their interaction. B: regression coefficient for the variable time (for further information see the method section). * Release from whole blood cells after lipopolysacharide (LPS) stimulation. ** Spontaneous release from whole blood cells IL: interleukin, TNF-α: tumor necrosis factor-α
Figure 2 Time course of smoking of two cigarettes on blood eosinophils. Black circles represent the values after smoking two cigarettes and grey circles represent the values of the control period.
Sputum
The total number and percentage of sputum cells within the first 24 hours after smoking and no smoking are shown in table 4. The number of neutrophils increased significantly more with smoking than without between 3 and 12 hours (table 5, figure 3). The number of sputum lymphocytes decreased more with smoking than without between 0 and 3 hours (table 5, figure 4). Subsequently, however, the percentage and number of sputum lymphocytes increased more with smoking than without between 3 and 12 hours (table 5, figure 4). Smoking had no significant effect on the percentage and number of sputum eosinophils (table 5, figure 5) and macrophages (table 5). Smoking had also no significant effect on the levels of inflammatory mediators in sputum (additional file 1, table 3) and the expression of CD11b, CD63 and HLA-DR on CD14 high and CD14 low cells and the number of iNOS positive macrophages (data not shown).
Table 4 Inflammatory cells in sputum after smoking and no smoking.
Baseline (192 hours) 3 hours 6 hours 12 hours 24 hours
SMOKING
Sputum cells, 106/ml 1.8 (0.1–16.3) 2.0 (0.3–6.6) 2.4 (0.1–7.3) 2.4 (0.5–6.6) 2.6 (0.0–9.0)
Neutrophils, % 56.9 (22.0–97.3) 56.4 (4.0–96.0) 83.2 (13.7–97.3) 77.5 (32.2–98.3) 67.3 (39.0–84.3)
Macrophages, % 37.9 (2.5–74.5) 42.2 (3.7–84.8) 13.8 (2.5–68.5) 16.8 (1.7–61.7) 27.4 (14.8–57.7)
Eosinophils, % 0.1 (0.0–6.2) 0.5 (0.0–5.2) 0.0 (0.0–0.3) 1.1 (0.0–8.3) 1.0 (0.0–5.2)
Lymphocytes, % 1.1 (0.0–3.8) 0.4 (0.0–1.7) 1.0 (0.0–2.0) 1.2 (0.0–7.3) 0.4 (0.0–1.8)
NO SMOKING
Sputum cells, 106/ml 2.8 (0.8–23.8) 3.1 (0.1–20.4) 2.0 (0.7–7.9) 2.1 (0.4–6.2) 2.1 (0.6–9.5)
Neutrophils, % 50.9 (20.3–84.8) 58.9 (31.8–94.2) 73.2 (22.8–94.7) 83.2 (26.7–98.3) 64.5 (29.0–80.3)
Macrophages, % 46.9 (15.0–77.7) 38.5 (4.2–64.0) 20.8 (4.5–71.2) 10.3 (1.7–67.8) 28.7 (16.0–66.5)
Eosinophils, % 0.2 (0.0–3.2) 0.3 (0.0–1.2) 0.2 (0.0–4.2) 1.7 (0.0–15.5) 2.2 (0.5–12.5)
Lymphocytes, % 0.7 (0.0–4.0) 0.9 (0.0–2.8) 0.4 (0.0–3.5) 0.2 (0.0–3.7) 0.9 (0.0–1.5)
Values are expressed as medians (ranges).
Table 5 Linear mixed effect models of sputum cells
Independent variable Time interval B 95% CI P value
Log (total cells, 106/ml) 0–3 hours 0.075 -0.15–0.30 0.52
Log (neutrophils, % and 106/ml)
% 0–3 hours -0.17 -0.34 – -0.00 0.07
number 0–3 hours -0.22 -0.49–0.30 0.11
% 3–12 hours 0.03 -0.02–0.07 0.27
number 3–12 hours 0.13 0.04–0.22 0.007
Log (macrophages, % and 106/ml)
% 0–3 hours 0.12 -0.01–0.25 0.08
number 0–3 hours 0.13 -0.11–0.36 0.31
Log (eosinophils, % and 106/ml)
% 0–6 hours -0.22 -0.71–028 0.39
number 0–6 hours -0.12 -0.30–0.06 0.20
% 3–6 hours -0.84 -3.63–1.96 0.57
number 3–6 hours -0.31 -1.35–0.73 0.57
Log (lymphocytes, % and 106/ml)
% 0–3 hours -0.28 -0.59–0.02 0.08
number 0–3 hours -0.26 -0.42–-0.11 0.004
% 3–12 hours 0.19 0.06–0.31 0.006
number 3–12 hours 0.23 0.09–0.36 0.002
The time intervals of the above parameters were selected based on visual inspection of the plots. The slopes of the parameters were estimated using linear mixed effect models [11] by including the variables time (hours), smoking (yes or no) and their interaction. B: regression coefficient for the variable time (for further information see the method section).
Figure 3 Time course of smoking of two cigarettes on sputum neutrophils. Black circles represent the values after smoking two cigarettes and grey circles represent the values of the control period.
Figure 4 Time course of smoking of two cigarettes on sputum lymphocytes. Black circles represent the values after smoking two cigarettes and grey circles represent the values of the control period.
Figure 5 Time course of smoking of two cigarettes on sputum eosinophils. Black circles represent the values after smoking two cigarettes and grey circles represent the values of the control period.
Urine
Smoking had no significant effect on leukotriene E4 levels in urine compared to no smoking (data not shown).
Discussion
In order to better understand the effects of chronic smoking, it is important to study the initial (acute) response to cigarette smoke, since repetitive acute smoke effects may cumulate and ultimately lead to irreversible damage. We therefore investigated the acute effects of cigarette smoking on both induction and resolution of the inflammatory response in healthy intermittent smokers. This study shows that smoking of two cigarettes acutely suppresses blood eosinophils. Furthermore, smoking induces a biphasic response in sputum lymphocytes, after an initial smoke-related suppression, the cells increase more with smoking than without. Finally, smoking increases sputum neutrophils and the release of IL-8 from whole blood cells.
A remarkable finding in our study is that smoking of two cigarettes decreases eosinophils in blood. Three other studies have reported similar results: eosinophils decreased in blood from healthy female smokers within two hours after smoking 12 cigarettes [12], in lung tissue of rats within 6 hours after smoke exposure [13], and in lung lavage fluid of ovalbumin sensitised mice after 3 weeks smoke exposure [14]. A decrease in eosinophils may be due to a direct (apoptotic) effect by toxic substances in cigarette smoke [15], or to anti-inflammatory substances in cigarette smoke, like CO [16,17]. Smoking did not show a significant suppressive effect on sputum eosinophils in our study, although the figures show that sputum eosinophils are decreasing more from 3 hours onwards with smoking than without. The reason for this is probably a lack of study power, due to the lower number of successful measurements in sputum than in blood, or due to the low baseline levels of sputum eosinophils in our healthy non-atopic subjects. One has to realise that the decrease in eosinophils in blood in our study is significant but relatively small.
This study is the first to report that sputum can be used to study acute smoke effects. The number of sputum neutrophils increased between 3 and 12 hours after smoking. In line with this, we demonstrated a higher release of IL-8 by LPS stimulated blood cells after smoking, which may have contributed to increased neutrophil chemotaxis. The rise in neutrophils is in line with two studies on acute effects of smoking in humans, showing increased neutrophils in bronchoalveolar lavage fluid 1 hour after smoking [18] and increased neutrophil retention in the lung during smoke exposure [19]. The fast increase in neutrophils in sputum might result from detachment of neutrophils from the pulmonary vascular endothelium (the so-called marginated pool) [20] or from recruitment from the bone marrow [21,22].
Smoking also shortly suppressed the number of lymphocytes in sputum. Thereafter sputum lymphocytes increased more with smoking than without. The initial decrease might result from increased adherence of lymphocytes in the lung tissue due to the fast upregulation of adhesion molecules after smoking [23] or may also be caused by the suppressive CO as mentioned in the prior paragraph [16]. The subsequent increase in sputum lymphocytes may reflect the outwash of lymphocytes from the tissue into the sputum, which can be regarded as the waste bin of lung inflammatory cells.
Smoking did not affect all inflammatory markers we investigated. A few factors may contribute to this lack of response. First, the number of subjects and the number of cigarettes (n = 2) might have been too low. Second, we may have included a heterogeneous group of subjects regarding their response to cigarette smoke. We know that approximately 80% of all smokers never develop COPD. Therefore it is conceivable that a part of our healthy smokers does not respond to cigarette smoking. Third, we included subjects with a broad range in current and past smoking. Fourth, sputum may reflect only a part of the acute inflammatory changes of the airway wall [8]. It would be interesting to study the acute effects of smoking on lung tissue. Finally, CO in cigarette smoke may have dampened the inflammatory response, especially in the early phase. After continuous smoking the damaging and irritating effects may prevail, giving rise to more pronounced inflammation.
Studying the acute effects of smoking in intermittent healthy smokers has both advantages and disadvantages. We choose the presented model for a number of reasons. First, intermittent smokers can refrain from smoking for three weeks in contrast to most current smokers. Second, intermittent smokers have a normal lung function (in contrast to COPD), and likely (nearly) no structural airway changes, which may affect a normal response to cigarette smoke. Third, we assumed that detecting an acute inflammatory response to cigarette smoking after an abstinence period of 9 days would be easier than detecting a response on top of chronic smoke exposure. Finally, intermittent smokers are used to inhale cigarette smoke (in contrast to non-smokers). We realise that our model has the disadvantage that the results of our study cannot easily be extrapolated to the chronic effects of smoking or COPD development. Nevertheless, when comparing the airway inflammation of our subjects with that of smoking COPD patients, both show increased levels of neutrophils, lymphocytes and IL-8 in sputum. However, in COPD patients after quitting smoking lymphocytes and neutrophils do not normalise [24], in contrast to the short-lived acute effects of smoking in this study. This suggests that not smoke but structural changes in the airways are responsible for the ongoing inflammation in COPD. Despite above limitations we think that knowledge on both the acute and chronic effects of smoking will help to better understand the mechanisms of cigarette smoke induced inflammation, which may underlie the development of COPD.
Conclusion
We conclude that besides an increase in inflammation, as known from chronic smoking, there is also a suppressive effect of smoking of two cigarettes on particular inflammatory parameters. Although this seems beneficial, it may disturb physiologic responses, like repair processes, in which inflammatory cells play a role.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
HV: Participated in the design of the study, performed the study and drafted the manuscript.
DP: Conceived the study, participated in the design and co-ordination of the study and helped draft the manuscript.
WT: Participated in the design of the study and helped draft the manuscript.
MH: Participated in the design of the study, co-ordinated the FACS analyses and helped draft the manuscript.
BW: Performed some of the laboratory analyses, participated in the design of the study and helped draft the manuscript.
HB: Performed statistical analyses and helped draft the manuscript.
JV: Performed statistical analyses and helped draft the manuscript.
DR: Performed and co-ordinated most laboratory analyses and helped draft the manuscript.
HK: Co-ordinated laboratory analyses, participated in the design of the study and helped draft the manuscript.
NH: Conceived the study, participated in the design and co-ordination of the study and helped draft the manuscript.
Supplementary Material
Additional File 1
Table 1. Number of blood cells (109/L) after smoking and no smoking. Table 2. Release of IL-1β, IL-10, IL-8 and TNF-α from blood cells after smoking and no smoking. Table 3. Inflammatory mediators in sputum after smoking and no smoking.
Click here for file
Acknowledgements
The authors thank M.A. Star-Kroezen, A.J. van der Laan-Boers, E.M.D.H. Swierenga (Lung function department) for the many lung function measurements and sputum inductions they performed; I. Sloots, M. van der Toorn, H.A. Buivenga-Steketee, J.A. Noordhoek (laboratory of Pulmonology and Allergology) for all measurements in sputum and blood; M.D.W. Barentsen (laboratory of Pathology) for the counting of the iNOS positive macrophages on cytospins; Prof. Dr. R.A. Uges and dr. B. Greijdanus (department of Pharmacy) for the measurements of the urinary cotinine. This study was funded by AstraZeneca, Lund, Sweden. Parts of this study were presented in abstract form at the ATS 2003.
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| 15740629 | PMC554761 | CC BY | 2021-01-04 16:36:26 | no | Respir Res. 2005 Mar 1; 6(1):22 | utf-8 | Respir Res | 2,005 | 10.1186/1465-9921-6-22 | oa_comm |
==== Front
Mol CancerMolecular Cancer1476-4598BioMed Central London 1476-4598-4-101574062610.1186/1476-4598-4-10ResearchA cluster of genes located in 1p36 are down-regulated in neuroblastomas with poor prognosis, but not due to CpG island methylation Carén Helena [email protected]är Katarina [email protected] Susanne [email protected] Luke [email protected] Farida [email protected]öberg Rose-Marie [email protected] Cecilia [email protected] Tommy [email protected] Department of Clinical Genetics, Institute for the Health of Women and Children, Göteborg University, Sahlgrenska Univ. Hospital-East, SE-41685 Gothenburg, Sweden2 Section of Medical and Molecular Genetics, Department of Paediatrics and Child Health, University of Birmingham, The Medical School, Edgbaston, Birmingham B15 2TT, UK2005 1 3 2005 4 10 10 1 12 2004 1 3 2005 Copyright © 2005 Carén et al; licensee BioMed Central Ltd.2005Carén et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
A common feature of neuroblastoma tumours are partial deletions of the short arm of chromosome 1 (1p-deletions). This is indicative of a neuroblastoma tumour suppressor gene being located in the region. Several groups including our have been studying candidate neuroblastoma genes in the region, but no gene/genes have yet been found that fulfil the criteria for being a neuroblastoma tumour suppressor. Since frequent mutations have not been detected, we have now analyzed the expression and promoter CpG island methylation status of the genes UBE4B, KIF1B, PGD, APITD1, DFFA and PEX14 in the 1p36.22 region in order to find an explanation for a possible down-regulation of this region.
Results
The current study shows that gene transcripts in high stage neuroblastoma tumours are significantly down-regulated compared to those in low stage tumours in the 1p36.22 region. CpG island methylation does not seem to be the mechanism of down-regulation for most of the genes tested, since no methylation was detected in the fragments analyzed. One exception is the CpG island of APITD1. Methylation of this gene is also seen in blood from control individuals and is therefore not believed to participate in tumour development.
Conclusion
The genes UBE4B, KIF1B, PGD, APITD1, DFFA and PEX14 are down-regulated in high stage NB tumours, a feature that can not be explained by CpG island methylation.
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Background
Neuroblastoma (NB) is the most common paediatric solid tumour, responsible for 15% of cancer deaths of childhood. It is a tumour of the postganglionic sympathetic nervous system that develops from immature or dedifferentiated neural crest-derived cells [1]. The distal part of chromosome 1p shows loss of heterozygosity (LOH) in 20–40% of NB tumours and has therefore been alleged to contain one or more tumour suppressor genes. We and others have previously analyzed the chromosomal region 1p36.2-3 [2-12] and we have recently focused on the gene region involving the genes: UBE4B-KIF1B-PGD-APITD1-CORT-DFFA-PEX14. These genes have been analyzed for mutations and a few have been found in rare tumours [13-17].
In search of tumour suppressor genes, the focus has in the last years moved towards epigenetics and methylation of promoter regions. Methylation of cytosines in CpG-dinucleotides is a common modification in mammalian genomes. Methylated cytosines are more susceptible to deamination, which have lead to an erosion of the number of CpG-dinucleotides. The vast majority of CpGs resides within repetitive elements and is methylated. There are also stretches of DNA rich in CpG that are gene associated, i.e. CpG islands, which are normally unmethylated [18]. Methylation is generally associated with repression of transcription. Gene regulation by methylation includes tissue-specific regulation during development and processes as X-chromosome inactivation and genomic imprinting, reviewed by Herman and co-workers. [19]. Cancer is associated with a genome-wide hypomethylation and a more gene-specific hypermethylation. Hypermethylation of CpG islands has been shown to be a common mechanism for the inactivation of tumour suppressor genes and is found in a wide range of tumour types [20-23]. According to the Knudson two-hit hypothesis, two successive mutations are required to inactivate a tumour suppressor gene and turn a normal cell into a malignant one. Inactivation could be due to deletions or mutations. Epigenetic events, such as hypermethylation of promoter-associated CpG islands have come in focus during the last decade as a route to inactivation [19].
The most common way to analyze methylation status is based on bisulphite modification of DNA [24]. In this reaction, unmethylated cytosines are deaminated to uracil, while methylated cytosines remain unconverted. The region of interest can be amplified using non-discriminating primers, amplifying both methylated and unmethylated templates in one reaction, or with methylation-specific PCR (MSP) in which methylated and unmethylated templates are amplified in separate reactions [25].
Some genes have been analyzed in NB tumours focusing on methylation status. For example, CASP8, on 2q33, was one of the first genes found to be methylated in neuroblastoma, with a frequency of about 40 % in primary NB tumours [26]. On 3p21.3, RASSF1A and BLU have been shown to be frequently methylated [27-29]. In a study by van Noesel and co-workers, 34 genes in 12 different chromosomal regions were analyzed in neuroblastoma cell lines [30]. A total of six genes that were methylated in at least three of the 22 cell lines were found. CASP8, as already known, was one of these genes, also FLIP at 2q33 was methylated as well as four genes in the chromosome region 8p21; DR4, DR5, DCR1 and DCR2. Genes on 1p36 were also included in this study, none of which were found to be methylated. No genes in the NB tumour suppressor candidate region on 1p36.22 were included. Alaminos and co-workers have in a study screened 45 genes in NB cell lines [31]. 34 genes were found to be methylated in at least one of the ten cell lines. The analysis also demonstrated that the percentage of methylated genes was higher in cell lines with MYCN-amplification than in those without. UBE4B, located in 1p36.22, was included in this study and found to be unmethylated.
The genes in the 1p36 consensus deleted region are all, except for CORT, associated with a CpG island in their respective promoter regions. The fact that this region is deleted in a subset of NB tumours and that frequent mutations are not found, makes it a possible candidate region for hypermethylation of genes. In the current study, we wanted to analyze the expression pattern of these genes in the 1p36.22 region using TaqMan technology and the methylation status of promoter regions associated with CpG islands, in a panel of NB cell lines and primary NB tumours.
Results
Expression analysis
GUSB was selected as an endogenous control for real-time PCR quantification, and further used as an internal reference for normalization. Real time-PCR studies of all the genes in the region on cDNA-samples showed a significant reduction of mRNA levels in high stage NB tumours compared to low stage tumour mRNA levels, ranging from 49% for PEX14 to 79% for APITD1 (Table 4) [16].
Table 4 Average relative gene expression (gene/GUSB).
Gene Average relative expression in NB cell lines Average relative expression in low stage NB Average relative expression in high stage NB % less expression in high stage NB compared to low stage NB
UBE4B 1.1 1.2 0.33 73%
KIF1B 0.51 3.6 0.99 72%
PGD 0.95 1.1 0.38 65%
APITD1 1.3 0.35 0.075 79%
DFFA 1.5 1.2 0.51 57%
PEX14 1.1 1.9 0.96 49%
Data analysed by TaqMan, and grouped in neuroblastoma cell lines, low stage primary tumours and high stage NB primary tumours.
The average expression in NB cell lines compared to primary tumours varied between the different genes in the region. Cell lines showed a higher expression of APITD1 and DFFA compared to primary tumours. Lower expression in cell lines was seen in KIF1B and equal levels of cell lines and stage 2 primary tumours in UBE4B, PGD and PEX14 (Table 4; Fig. 1). The 1p-deleted cell line SK-N-AS generally showed lower expression of the genes in the region compared to the other NB cell lines tested (Fig. 1).
Figure 1 Expression studies using the TaqMan-technique for detecting mRNA in NB cell lines and primary tumours. The results are grouped into three groups indicated at the top; also the 1p-deletion status for each sample is indicated at the top. The genes tested are indicated to the left. The Y-axis indicates relative expression level compared to the housekeeping gene GUSB (gene-mRNA concentration / GUSB-mRNA concentration). The identity of each sample is indicated at the bottom.
Methylation analysis
The analyzed fragments of the genes UBE4B, KIF1B, PGD, DFFA and PEX14 were not methylated in the panel of NB primary tumours and cell lines. For APITD1, the region -393 to -222 relative to initiation codon showed methylation. This region was therefore amplified and cloned with a T/A cloning kit in order to give a value of the percentage of clones being methylated (Fig. 2). The region contains 16 CpG sites and these sites show various degree of methylation, in different clones as well as in different samples. Methylation of cytosine in front of guanine is concentrated to the beginning of this "methylated region"; the three first cytosine bases showing the highest degree of methylated clones (Fig 3). No methylation was seen up-stream of -393 in the fragments analyzed. At some CpG sites in the region, methylation was never detected. Only one sample had no clones with methylation for any of the sites; the stage 2 tumour 20S9. Blood from control individuals also showed various degree of methylation at these sites. The cell lines did not show methylation down-stream of CpG -212 in the analyzed fragments, clones from tumours were not methylated down-stream of CpG -311. Healthy controls showed a smaller region of methylation, no methylated clones were detected down-stream of CpG -280.
Figure 2 Methylation status of APITD1. Black boxes indicate methylation, white boxes no methylation, position relative to start codon is indicated at the top.
Figure 3 Organization of the 5' region of APITD1. The CpG island, predicted promoter and analyzed region including the region of partial methylation are indicated.
Discussion
Deletion of parts of chromosome 1p is a common feature of neuroblastoma tumours. We have previously narrowed down the shortest region of overlap of deletions (SRO) to 25 cM in our tumour material [6,7]. By including a germ cell tumour with 1p deletion we could confine the SRO to 5 cM [11]. This SRO was further confirmed when a cell line with a homozygous deletion of 500 kb was found within this region [32]. We have previously screened the 500 kb region for mutations and only rare have been detected. The aim of this study was to investigate the expression of the genes in the region and to correlate this with the methylation status. TaqMan expression data showed a clear down-regulation in gene expression of genes in the region tested when comparing high stage NB tumours (stage 3 and 4) and low stage tumours (stage 2). This down-regulation was seen regardless if the high stage tumour was 1p-deleted or not (Fig. 1). This finding further supports that this region might be involved in NB development and progression. We can speculate about some central mechanism for the expression of the genes in the entire region that is affected in tumours with poor prognosis, either due to the deletion of one allele and/or by some other mechanism. An attractive explanation for this would be methylation of CpG islands that can lead to inactivation of the respective gene. This does not seem to be the case though, since none of the genes, APITD1 excepted, showed methylation. Furthermore, DNA from healthy controls is also methylated in the same region of the APITD1 gene, making methylation a less likely explanation for the difference in APITD1 gene expression between advanced and favourable primary NB tumours.
CpG methylation is not the only possible explanation for the down-regulation in gene expression shown in high stage tumours. Analysis of chromatin modifications could be the next step in a further analysis of this region, in search of a mechanism that could lead to down-regulation of the genes. Deficiencies in transcription factors and upstream elements could also account for the decrease in gene transcripts. Another possibility is that the variations are due to differences in the treatment of the tumours before and after surgery. Radiation and chemotherapy could probably have a vast affect on the expression patterns of a substantial amount of genes in the tumour cell. It is however unlikely that all the genes in the analyzed region would be affected by the treatment alone, rather this might explain the differences in the level of down-regulation between different genes.
The difference in expression between NB cell lines and primary NB could be explained by the difference in cell environment between cells in culture and in vivo. In primary tumours, there are always some non-NB cells in the RNA preparations that might affect the gene expression results. On the other hand, genetic events happen to the cells when they get immortalized in cell culture, some genes tend to be down-regulated and others up-regulated. We can also speculate that some of these genes, for example APITD1, might even be so crucial that only cells with expression of this gene are capable of surviving in culture.
Conclusion
In conclusion, in our aim to find a mechanism that could inactivate the genes in the candidate gene region we analyzed the expression status and the methylation profile of six genes. The techniques used were TaqMan real-time-RT-PCR technology and bisulphite DNA sequencing. Of the genes analyzed all were down-regulated in high staged NB tumours as compared to low stage tumours. Promoter methylation was not detected in the genes analyzed, except for the CpG island of APITD1. This methylation does not seem to be tumour specific, since methylation was also detected in healthy blood controls. Hence, the six genes UBE4B, KIF1B, PGD, APITD1, DFFA and PEX14 are down-regulated in high stage NB tumours, a feature that can not be explained by methylation, rather by a mechanism still remaining to be discovered.
Methods
Cell lines and patients
A panel of 10 tumours from primary NBs (4 Stage 2, all from patients with no evidence of decease at last follow-up, and 6 Stage 3 or 4 tumours, all from patients with adverse outcome) and 7 NB cell lines (IMR-32, SK-N-AS, SK-N-BE (2), SK-N-SH, SK-N-DZ, SK-N-F1 and SH-SY-5Y) were analyzed (Table 1).
Table 1 Clinical data for the primary tumours used in this study.
Patients/cell lines NB stage 1p-del Ploidi Outcome
18F8 2A neg NED
20S9 2 neg NED
23S4 2 neg 3n NED
25S9 2 neg NED
4F1 4 neg DOD
10S2A 4 pos DOD
13S0 4 pos DOD
13S1 3 pos DOD
15S3 4 neg/pos DOD
17S2 4 neg DOD
Column 3: 1p-del, 1p-deletion; neg, negative; pos, positive; neg/pos; ambiguous results based on microsatellite marker analysis (according to Martinsson et al. [6]) and FISH. Column 5: NED, no evidence of disease; DOD, dead of disease.
Expression analysis
cDNA preparation
Total RNA was extracted from frozen (-70°C) NB tumour tissue using RNeasy RNA extraction kit (Qiagen, Hilden, Germany). 2.4 μg total-RNA of each sample was reversed transcribed to cDNA using Superscript II (Amersham, Buckinghamshire, UK) and random hexamer primers, all according to supplier's protocol. All cDNAs were quality tested by amplification of the housekeeping genes UNPH and GAPDH.
Real time PCR -Endogenous control
To select the most appropriate endogenous control for the real-time PCR quantification analysis, we tested eight different primary NB samples of different stages for their expression levels of ten commonly used housekeeping genes with TaqMan Human Endogenous Control Plate, (Applied Biosystems, Foster City, CA). Analysis was performed according to supplier's protocol. GUSB (β-glucuronidase) and B2M (β2-microglobulin) showed least variations in ΔCT levels, and were expressed at constant levels in all samples regardless of NB-stage. GUSB was selected, and further used as an internal reference for normalization in the real-time PCR quantification analysis (Abel et al., submitted).
Real time PCR- TaqMan
TaqMan primers and probes were derived from the commercially available "TaqMan® Assays-on-Demand™ Gene Expression Products" (URL:). Real-time PCR was performed in 384-well plates using ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems). Amplification reactions (10 μl) were carried out in duplicate with 0.1 μl template cDNA according to manufacturers protocol (Applied Biosystems). In each assay, a standard curve with six cDNA dilutions was recorded and two non-template controls were included.
Quantification was performed by the standard-curve method. The mean CT-value for duplicates were calculated, and the gene concentration (or gene copy numbers) of test samples was interpolated based on standard curves. All samples were normalized by dividing the concentration of the test gene with the concentration of the housekeeping gene β-glucuronidase (GUSB) in the same cDNA sample.
The logarithms of the expression levels were compared with Student's two-sided t-test on each group of tumours; low stage and high stage tumours.
Methylation analysis
Bisulphite modification
DNA was phenol extracted with the use of phase lock gel (Eppendorf AG, Hamburg, Germany) according to standard procedure and was, with some minor changes, modified according to previously published papers [24,33]. Briefly, 1 μg of genomic DNA was digested with restriction endonucleases that cut close but outside the region of interest. The DNA was then denaturated in 0.3 M freshly prepared NaOH at 40°C for 15 minutes. Sodium metabisulphite (Sigma-Aldrich CO, St Louis, MO) and urea, at a final concentration of 1.73 M and 5.36 M respectively, were added in order to sulphonate the unmethylated cytosines, along with hydroquinone (0.5 mM). Conversion was carried out at 55°C for 16 hours, with a temperature rise to 95°C for 30 seconds every third hour. DNA was purified with Wizard DNA clean up system (Promega Corporation, Madison, WI) according to the manufactures instructions and desulphonated in 0.3 M NaOH at 37°C for 15 minutes. DNA was then precipitated in ethanol, resuspended in distilled H2O and stored at -20°C.
Promoter analysis and DNA amplification
The putative promoter regions of the genes were predicted using Genomatix Promoter Inspector software (URL: ) and CpG islands with MethPrimer software (URL: ; Table 2) [34]. These regions, or parts of them, were amplified with one primer pair, or if needed, with semi-nested primers (Table 3). The methylation status was analyzed using bisulphite sequencing. Conditions for PCR amplification were 1× PCR Gold Buffer (Applied Biosystems), 0.5 mM dNTPs, 2.0–3.0 mM MgCl2, 0.4 μM of forward and reverse primers respectively and 1 unit of AmpliTaq Gold, in a total volume of 50 μl. Reactions were denatured at 95°C for 10 min followed by 5 cycles of 95°C for 1 min, 49–55°C for 2 min, 72°C for 3 min and 30 cycles of 95°C for 30 sec, 49–55°C for 2 min, 72°C for 1 min 30 sec and ending with 10 min extension at 72°C. The PCR products were immediately sequenced or cloned into a sequencing vector using the TOPO T/A cloning kit (Life Technologies Invitrogen, Carlsbad, CA), where after 10–30 clones were picked. PCR products were purified with ExoSAP-IT™ (USB Corporation, Cleveland, Ohio) and sequencing was carried out using forward or reverse primer with ABI Prism BigDye™ cycle sequencing Ready Reaction Kit (Applied Biosystems). The samples were analyzed in an ABI 3100 or an ABI 3730 Genetic Analyzer.
Table 2 Putative promoter regions and CpG island predictions.
Gene Promoter region CpG island Amplified region
UBE4B -1212 to -492 -1157 to -150 -959 to -494
KIF1B -22392 to -21141 -22444 to -20935 -22517 to -21874
PGD -185 to +7 -680 to +728 -157 to +144
APITD1a -250 to +30 -389 to +643 -418 to +321
DFFA +65 to +264 -164 to +256 -266 to +142
PEX14 - -2806 to -2362 -2752 to -2481
a For APITD1, the methylation status was analyzed for three different fragments. Column 2: Putative promoter regions according to Genomatix Promoter Inspector software. No promoter region was detected in PEX14. Column 3: CpG island prediction by MethPrimer (for CpG island criteria, see materials and methods). Column 4: Region amplified with the primers used in this study.
Table 3 PCR primers for amplification; all primers are designed for the sense strand.
Gene Primer Sequence Length of fragment (bp) Accession number
UBE4B FP 5'-TTGTTAGTTTATTTGGTTTAGGTT-3' 466 NM_006048
RP 5'-TAACAAAACCCAACACTATAAAAAAAACCCCT-3'
KIF FP 5'-TTTTTAAGGGTATTTTTTAGAAGGG-3' 644 NM_015074
RP 5'-ACTATAACCAATCACAACACAAAACTC-3'
PGD FP-A 5'-GTGAGTTGTTATGGTTATAGTTG-3' 301 NM_002631
FP-B 5'-ATGGTGTGGTTTTATGGTTTTATTT-3'
RP 5'-CAAAATCACAAAACCCCAAATAA-3'
APITD1 1FP-A 5'-GATTTTGTAAGATATATTTGAGGTAT-3' 231 chr1_29_927.b
1FP-B 5'-ATGGAGTTTTTGATAATGTGTATTG-3'
1RP 5'-AACCCCCTACTCAACTTACTCTAC-3'
2FP-A 5'-ATTAGGTTTTGGGGTGTAGTAGTGAT-3' 199
2FP-B 5'-GTAGAGTAAGTTGAGTAGGGGGTTG-3'
2RP 5'-ACCCTAAACAAAAACAAAAAAAC-3'
3FP-A 5'-GTAGAGTAAGTTGAGTAGGGGGTTG-3' 350
3FP-B 5'-TTGTTTTTGTTTAGGGTCGGTT-3'
3RP 5'-CAAAACCAAAAAATAACCTCTC-3'
DFFA FP 5'-AAGTTAAAAATAATTTTTAGGTTGAAT-3' 407 NM_004401
RP 5'-ACCAACCCTTACTCCTCAAATCT-3'
PEX14 FP 5'-TGATTAGTTAGGTTTTAGAAAGATGG-3' 333 NM_004565
RP 5'-CAAATAAAACCAAAAATACTAACAAAC-3'
Column 2: FP, forward primer; RP, reverse primer; FP-A and FP-B, primers used for semi-nested PCR together with the same reverse primer. Column 5: UCSC Genome Browser August 2001 draft sequence was used as reference sequence (URL: ).
Authors' contributions
HC participated in the design of the study, carried out the methylation analysis and drafted the manuscript. SF, LH and FL participated in parts of the methylation study. KE and CK carried out the TaqMan runs. KE also contributed to drafting the manuscript. RMS participated in the cloning. TM coordinated the study. All authors read and approved the final manuscript.
Acknowledgements
This work was supported by grants from the Swedish Cancer Society, the Children's Cancer Foundation, the King Gustav V Jubilee Clinic Cancer Research Foundation, the Assar Gabrielsson Foundation, the Wilhelm and Martina Lundgren Research Foundation and the Sahlgrenska University Foundation. We would like to thank the Swegene Gothenburg Genomics resource unit for access to the ABI 7900HT Sequence Detection System and the ABI 3730 Genetic Analyzer.
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| 15740626 | PMC554762 | CC BY | 2021-01-04 16:36:36 | no | Mol Cancer. 2005 Mar 1; 4:10 | utf-8 | Mol Cancer | 2,005 | 10.1186/1476-4598-4-10 | oa_comm |
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