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BMC Plant BiolBMC Plant Biology1471-2229BioMed Central London 1471-2229-4-201559600810.1186/1471-2229-4-20Research ArticleMolecular characterization and functional expression of flavonol 6-hydroxylase Anzellotti Dominique [email protected] Ragaï K [email protected] Molecular Membrane Dynamics Laboratory, Department of Anatomy and Cell Biology, McGill University, Montreal, H3A 2B2, Canada2 Plant Biochemistry Laboratory and Center for Structural and Functional Genomics, Department of Biology, Concordia University, Montreal, H4B 1R6, Canada2004 13 12 2004 4 20 20 26 7 2004 13 12 2004 Copyright © 2004 Anzellotti and Ibrahim; 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
Flavonoids, one of the major groups of secondary metabolites, play important roles in the physiology, ecology and defence of plants. Their wide range of activities is the result of their structural diversity that encompasses a variety of functional group substitutions including hydroxylations. The aromatic hydroxylation at position 6 of flavonols is of particular interest, since it is catalyzed by a 2-oxoglutarate-dependent dioxygenase (ODD), rather than a cytochrome P450-dependent monooxygenase. ODDs catalyze a variety of enzymatic reactions implicated in secondary metabolite biosynthesis.
Results
A cDNA fragment encoding an ODD involved in the 6-hydroxylation of partially methylated flavonols, flavonol 6-hydroxylase (F6H), was isolated and characterized from Chrysosplenium americanum using internal peptide sequence information obtained from the native plant protein. This novel clone was functionally expressed in both prokaryotic and eukaryotic expression systems and exhibited ODD activity. The cofactor and cosubstrate requirements of the recombinant proteins are typical for ODDs, and the recombinant enzymes utilize 3,7,4'-trimethylquercetin as the preferred substrate. The genomic region encoding this enzyme possesses two introns at conserved locations for this class of enzymes and is present as a single copy in the C. americanum genome.
Conclusions
Recombinant F6H has been functionally expressed and characterized at the molecular level. The results demonstrate that its cofactor dependence, physicochemical characteristics and substrate preference compare well with the native enzyme. The N-terminal region of this protein is believed to play a significant role in catalysis and may explain the difference in the position specificity of the 6-hydroxylation reaction.
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Background
Flavonoid compounds constitute one of the major groups of secondary metabolites and play important roles in plant development, reproduction and defence. This diverse spectrum of activities results from their structural diversity and a variety of functional group substitutions [1]. Chrysosplenium americanum (Saxifragaceae), a semi-aquatic weed, accumulates a variety of tetra- and penta methylated flavonol glucosides substituted at various positions of the flavonol ring [2]. Their biosynthesis from the parent aglycone, quercetin, is catalyzed by a number of stepwise, substrate-specific, position-oriented O-methyltransferases and two distinct O-glucosyltransferases in a highly ordained sequence (Figure 1) [2]. During the course of their biosynthesis, the partially methylated intermediate, 3,7,4'-trimethylquercetin (TMQ) is hydroxylated at position 6 to 3,7,4'-trimethylquercetagetin (TMQg) by a 2-oxoglutarate-dependent dioxygenase (ODD), flavonol 6-hydroxylase [3].
Plant ODDs (EC 1.14.11.-) constitute a class of non-heme, iron-containing cytosolic enzymes that utilize an oxoacid as a cosubstrate and a reducing agent for the reactive iron moiety, typically ascorbate. This widespread class of enzymes has been implicated in a variety of plant metabolic pathways, including the biosynthesis of some amino acids, hormones, signalling molecules and a variety of secondary metabolites [4]. Hydroxylation at position 6 of partially methylated flavonols is of particular interest, since it is catalyzed by a 2-oxoglutarate-dependent dioxygenase rather than a cytochrome P450-dependent monooxygenase.
Using the peptide sequence information obtained from the purified plant protein [3], degenerate primers were designed for the screening a C. americanum cDNA library and the isolation of a F6H clone. The identity of this clone was confirmed after identifying internal peptide sequences in the translated amino acid sequence of the isolated cDNA clone, as well as by functional expression of F6H in both prokaryotic and eukaryotic systems. The molecular characterization of this gene, with respect to its genomic organization and phylogenetic relationship to other ODDs involved in plant secondary metabolism, contributes to the growing pool of information on this class of enzymes.
Results and discussion
Isolation and cloning of F6H
Several peptides were obtained from tryptic digestion of the native protein, one in particular, Micro1 – DNGWILLHIGDSNGHR, exhibited significant similarity to other known flavonoid ODDs such as the flavanone 3-hydroxylase (F3H) [5], flavonol synthase (FLS) [6] and anthocyanidin synthase (ANS) [7]. Two other peptide sequences, Micro2 – KIVEACEDWG and Micro3 – TLMAGLACKLLGVL, exhibited limited homology to this class of enzymes. Of the different approaches used, two methods allowed the isolation of putatively positive cDNA fragments from a C. americanum bacteriophage expression library [8]. The PCR-based strategy utilized several primer combinations and resulted in the isolation of a fragment termed, F6H·1, that exhibited a significant degree of homology to flavonoid ODD genes. In addition, a portion of the deduced amino acid sequence of F6H·1 matched another fragment obtained from microsequencing. Subsequently, the PCR-based screening approach resulted in the amplification of a 1,245 bp-long cDNA fragment, termed cF6H, that contained the 3'-end of this putative cDNA clone, including an in-frame stop codon (TAA), a putative polyadenylation signal (ATATAA) and a short polyA tail (Accession # AY605048), although it lacked the translation start site. The same cDNA library was also screened with an oligonucleotide probe derived from the F6H·1 sequence. Putatively positive clones were isolated and characterized, but none yielded a full-length F6H cDNA clone, although several clones were isolated with homology to related sequences, particularly F3H and aminocyclopropanecarboxylate oxidase (ACCO). Further attempts to isolate a complete F6H ORF were unsuccessful, including the use of inverse PCR and the GenomeWalker technique (ClonTech).
The truncated DNA fragment, cF6H, translates into a 376 amino acid-long sequence with a predicted molecular mass of 40.9 kDa and a pI of 5.1. The fact that the native protein exhibits a molecular mass of 43 to 45 kDa following gel filtration or SDS-PAGE analysis respectively [3], suggests that the cDNA fragment lacks ~10 to 15 N-terminal amino acid residues. However, the conserved regions and catalytically important residues located in the C-terminal of ODDs [9] are present in the translated cF6H sequence. The amino acid residues involved in iron (His222, His285, Asp224) and α-KG-binding (Arg295, Ser297), as well as other conserved residues are present within the cF6H sequence (Figure 2). The near-full length cF6H, although truncated at the N-terminal, was confirmed to be an authentic fragment of the gene encoding the F6H, since the internal peptide sequences obtained from the native protein were present in the deduced amino acid sequence of the cDNA clone (Figure 2). Sequence analyses of ODD genes including cF6H demonstrate that the conserved regions and catalytically important amino acids are located primarily within the C-terminal half of these proteins, whereas the less conserved N-terminal region may be involved in substrate binding or maintaining the protein's tertiary conformation. Given the inherent variability of the N-terminal, that in certain cases it does not contribute significantly to ODD enzyme activity as was the case with the desacetoxyvindoline 4-hydroxylase of Catharanthus roseus [10], and that all possibilities for the isolation of the remaining fragment were exhausted, the cF6H fragment was cloned into prokaryotic and eukaryotic expression vectors to assess its enzyme activity.
Expression of recombinant F6H
The induction and solubility of the bacterial expressed F6H (rF6Hb) fusion protein were assessed over a 4-h period and recombinant F6H enzyme activity was assayed. The level of expression of the fusion protein was monitored by Western bolt analysis of cell lysates using anti-(His)6 antibodies. The antibody reacted with a 45 kDa protein band in cell lysates carrying the pTrc-His-cF6H construct after induction (Figure 3). This corresponds to the correct mass of the recombinant protein since the (His)6-tag and extraneous amino acids resulting from the cloning process account for an additional ~3.5 kDa in the fusion construct. The Ni-NTA-purified recombinant protein (Figure 3) exhibited a F6H activity of 0.103 pkat/mg following size exclusion chromatography (Table 1). The level of enzyme activity did not vary significantly regardless of the time or temperature of induction.
In order to test for any inhibitory effect resulting from the (His)6-tag, the fusion protein was cleaved by incubation with enterokinase (EK). The enzyme activity of the cleaved protein was significantly reduced compared to the Ni-NTA-purified rF6Hb (Table 1). The loss of activity was attributed to destabilization of the protein resulting from dialysis to remove inhibitors of the EK reaction, followed by incubation at room temperature with EK. Control experiments indicated that the (His)6-tag did not stimulate overall recombinant enzyme activity, since the intact rF6Hb treated in parallel with the preparation undergoing cleavage exhibited a similar reduction in enzyme activity.
The translated cF6H sequence possesses two potential glycosylation sites at Ser139 and Ser305 that may contribute to catalytic activity, perhaps by stabilizing a favorable tertiary conformation or through the modulation of interactions with other proteins. The effects of such post-translational modifications and protein folding were taken into account by expression of the recombinant protein in the eukaryotic expression system Pichia pastoris. This system is relatively easy to manipulate and exhibits many of the advantages of eukaryotic expression, including protein processing, folding and modification. Recombinant protein expression was both intracellular (rF6Hy) and secreted (rF6Hys). The chosen culture media (BMGY/BMMY) contained protein stabilizing factors, such as peptone and yeast extract, shown to reduce proteolysis, particularly of secreted proteins [11]. Protein expression, monitored by Western blot analysis using anti-(His)6 antibodies, was maximal at 3 to 4 days post-induction in both intracellular and secreted systems (Figure 4), and the protein eluted at a volume corresponding to ~40 kDa from a Superose 12 column. The Ni-affinity-purified preparations exhibited a specific activity of 0.21 and 0.11 pkat/mg for the intracellular (rF6Hy) and the secreted (rF6Hys) constructs, respectively. Their specific activities could not be further improved by size exclusion chromatography instead of or in addition to, Ni-NTA purification (Table 1).
In both prokaryote and eukaryote expression systems rF6H was soluble, exhibited similar physicochemical properties to the native protein and was no more susceptible to proteolytic degradation. Interestingly, as with the native plant protein [3], the recombinant F6H was functional as a monomer, but was also shown by gel filtration to associate in vitro (Figure 3C). In the case of the native F6H, this may result from the disruption during isolation of any weak interactions with other proteins, particularly those involved in polymethylated flavonol biosynthesis. A hydrophobic prediction plot of the translated cF6H (data not shown) indicates the presence of a nonpolar region at the very end of the protein sequence, which may participate in dimerization or aggregation processes.
The substrate specificity of recombinant F6H was tested using the Ni-NTA-purified enzyme with different flavonol substrates (Table 2). The results indicate that, although the overall specific activity of both recombinant proteins is reduced as compared to the native protein, 3,7,4'-TMQ is the preferred substrate for both rF6Hb and rF6Hy, which accepted neither quercetin nor 3-methylquercetin to any significant extent. On the other hand, 3,7-dimethylquercetin was accepted at 56% and 63% of the control activity by rF6Hb and rF6Hy, respectively.
Given the fact that neither of the recombinant proteins utilized naringenin as a substrate, it seems unlikely that F6H catalyzes a side reaction of F3H or a bifunctional activity as F3H-FLS, as has been demonstrated with the Citrus unshiu bifunctional dioxygenase [12]. Therefore, this enzyme may putatively be classified as a narrow-specificity ODD [12]. Taken together, these results suggest that Chrysosplenium F6H exhibits a specificity for partially methylated flavonoids involved in polymethylated flavonol biosynthesis characteristic of the species.
The cofactor requirements for the bacterially expressed recombinant protein were similar to those of the native plant protein [3]. Ferrous ions had the greatest effect on enzyme activity and enzyme reactivation in affinity-purified samples, as demonstrated by abolition of enzyme activity upon incubation with 5 mM EDTA. A preliminary assessment of the apparent Km values of rF6Hb for the flavonol substrate (63 μM) suggests that the binding affinity has been reduced 17-fold compared to the native protein (0.27 μM) [3]. In contrast, that for α-KG was only slightly affected (78 μM instead of 60 μM), indicating that the binding site for the cosubstrate has not been substantially altered. Nevertheless, a detailed kinetic analysis of the recombinant protein was not conducted given the truncation of the N-terminal and reduced enzyme activity.
The hydroxylation of an aromatic carbon may be the result of substrate positioning in relation to the oxidizing moiety within the active site. Mehn and colleagues [13] in attempting to elucidate the role of the cosubstrate in oxygen activation by ODDs through the use of synthetic iron complexes have also demonstrated the hydroxylation of phenolic substrates. In addition, the nature of the substituents on the phenolic moiety, dramatically affects the reaction rate. These results suggest that the structure of the substrate and its positioning within the active site play a crucial role in aromatic hydroxylations by ODDs as opposed to more fundamental differences in reaction mechanisms. This may explain the importance of the N-terminal to F6H enzyme activity, particularly if it is involved in substrate binding either by directly contributing to the flavonol binding site, or by maintaining the appropriate conformation for substrate recognition. This hypothesis is reinforced by the fact that rF6H exhibited a reduced specific activity in comparison to the native F6H, regardless of the expression system, degree of purification or the location of the polyhistidine tag.
3.2 Molecular characterization
In order to isolate the genomic region coding for F6H, genomic C. americanum DNA was used as a template for amplification reactions with primers designed to the outermost regions of the cF6H sequence. This produced a fragment of ~3.1 kbp (gF6H) that contained two potential intronic sequences (Figure 5). The first intron, 421 bp long is centrally located at position 684 of cF6H, whereas the second intron is significantly longer, 964 bp and is located towards the 3'-end of the gene at position 943.
Southern analysis of genomic DNA, probed with the cF6H partial ORF, gave rise to single bands in BamHI- and EcoRI-digested genomic DNA (Figure 6). The internal sequence of gF6H does not contain any recognition sites for the above enzymes; however, one recognition site is located within the known sequence for KpnI, NcoI, XhoI. Two major bands were detected in these lanes at estimated sizes ranging from ~4.3 to 10.0 kbp. These results indicate that F6H is present as a single copy gene in C. americanum, particularly since potential tandem F6H repeats would have resulted in the observation of fragments larger than 6.2 kbp, which were not present in lane 1. Under conditions of reduced stringency, certain lanes exhibited more than the two expected bands (data not shown), indicating the existence of related sequences within the genome.
The ORFs of the clones isolated using different approaches were identical in sequence, indicating that they represent the same protein. This is in agreement with the results obtained from Southern analysis suggesting that F6H is present as a single copy in the C. americanum genome. The deduced amino acid sequence of cF6H exhibits homology to other ODDs, particularly at the C-terminal, and this conservation can be extended to the organization of the gene as a whole. The fact that gF6H possesses two introns at conserved locations for ODDs [14], suggests that ODD sequences arose through divergence from a common ancestor. The single copy nature of this enzyme also suggest a regulatory role in polymethylated flavonol biosynthesis and may be significant, in relation to a second hydroxylation that occurs at the 2'-position of the TMQ intermediate (Figure 1). A comparable reaction was recently reported to be catalyzed by a cytochrome P450-dependent monooxygenase involving the 2'-hydroxylation of isoflavones in Medicago truncatula [15]. It has been proposed that the cytosolic enzymes involved in the sequential methylation of Chrysosplenium flavonoids are organized on the surface of a multienzyme aggregate, thus allowing for a more efficient regulation of the pathway as a whole [16]. It is likely that the F6H is a component of this enzyme complement, as it introduces a hydroxyl group at position 6 of 3,7,4'-trimethylquercetin for subsequent O-methylation at this position by a flavonol 6-O-methyltransferase (F6OMT). Immunolocalization of both enzymes, F6H and F6OMT, should provide further evidence in support of this view.
cF6H from C. americanum exhibits highest similarity, at the amino acid level, to various F3H homologs. Phylogenetically, it is evident that homologous genes encoding biochemically related proteins in single pathways are clustered into related subgroups (Figure 7), perhaps as a result of gene duplication and divergence. Genes encoding proteins in the same pathway of a given species, although not within the same subgroup, exhibit expectedly higher identity than unrelated genes. cF6H clusters with the F3H group of genes, although at a position distal to those homologs from other species, thus suggesting an evolutionary relationship with this particular subgroup of biosynthetically related enzymes. It is evident from Figure 7 that flavonol ODDs cluster into 2 distinct clades; the first consisting of enzymes with a narrow substrate specificity including F3H, FSI and F6H, whereas the second is comprised of FLS and ANS, both possessing broad substrate specificity as has been previously described [12,17,18]. It seems that based on biochemical and phylogenetic considerations, Chrysosplenium F6H exhibits high substrate and position specificity.
At the phylogenetic level, ODDs comprise a superfamily with numerous subgroups whose subsets are defined by shared motifs in the encoded proteins, such as those involved in flavonoid biosynthesis. These motifs often comprise the active site of the enzyme and/or the binding domains for substrates and cofactors. In addition, the degree of similarity between genes encoding dioxygenases with different substrate preferences suggests a common reaction mechanism. The molecular characterization of F6H and related ODDs could identify potential commonalities in structure or reaction mechanisms, as well as reveal motifs or residues that may determine reaction type and/or substrate preference. When aligned with related sequences, cF6H exhibits a high degree of conservation of the motifs required for iron and cosubstrate binding, whereas other regions show little or no identity and are presumed, most likely, to contribute to differences in substrate specificity and/or reaction type. This applies particularly to the variable N-terminal and a region where a gap insertion within an ODD sequence motif is required for proper alignment with other flavonoid biosynthetic enzymes. Further evidence of such distinctness is the fact that, although F6H clusters with the F3H group of flavonoid dioxygenases (Figure 7), it appears to have evolved paraphyletically from a common ancestor with respect to this group of genes.
Conclusions
A novel ODD cDNA involved in the 6-hydroxylation of partially methylated flavonols was isolated and characterized from C. americanum. The substrate specificity observed with the recombinant F6H compares well with the native enzyme [3]; displaying a specificity for position 6 of partially methylated flavonols, with 3,7,4'-trimethylquercetin being the preferred substrate. The fact that it hydroxylates position 6 of the aromatic ring A of a partially methylated flavonol contrasts with other aromatic hydroxylations which are commonly catalyzed by cytochrome P450 monooxygenases, such as the flavonol 8-hydroxylase of Tagetes patula [19] and the flavonoid 6-hydroxylase of soybean involved in isoflavone biosynthesis [20]. Given that both the native and recombinant F6H exhibit a preference for relatively hydrophobic substrates further distinguishes this protein from other dioxygenases characterized to date, that are known to accept relatively polar flavonoid aglycones [21-24]. Further evidence of such distinctness is the fact that, although cF6H clusters with the F3H group of flavonoid dioxygenases (Figure 7), it appears to have evolved in a paraphyletic fashion with respect to this group of genes. General flavonoid biosynthesis may have arisen from a multifunctional dioxygenase, since F3H and F6H seem to share a common ancestor, but significant divergence has since occurred resulting in the evolution of this novel activity.
A clone with full enzyme activity, which can be isolated based on the cF6H sequence provided fresh tissue is available, can be used to assess the versatility of this enzyme with respect to substrate preference through site-directed mutagenesis, as well as to assess its potential applications in metabolic engineering.
Methods
Materials
Chrysosplenium americanum (Schwein x Hooker; Saxifragaceae), was collected from St. Anicet, Québec, and was maintained in the greenhouse under conditions simulating its natural habitat regarding light intensity, temperature and humidity.
Flavonol substrates were from our laboratory collection. All other chemicals were of analytical reagent grade. Buffers: Assay buffer, 50 mM Tris-HCl (pH 7.3), 10 mM DTT, 150 mM NaCl, 10% glycerol (v/v); Wash solution I, 2 × SSC, 0.1% SDS (w/v); Wash solution II, 0.1 × SSC, 0.1% SDS (w/v); Blocking solution, 5% SDS (w/v), 125 mM NaCl, 25 mM sodium phosphate (pH 7.2); Blot washing solution, 0.5% SDS (w/v), 12.5 mM NaCl, 2.5 mM sodium phosphate (pH 7.2). FPLC buffers were filtered, degassed and stored at 4°C.
Isolation of an F6H clone
The ligand-affinity purified F6H protein [3] was subjected to digestion and sequencing at the Harvard Microchemistry Facility using microcapillary, reverse-phase HPLC nano-electrospray tandem mass spectrometry (MS/MS) on a Finnigan LCQ quadrupole ion trap mass spectrometer. The MS/MS spectra obtained were correlated with known sequences using the Sequest algorithm developed at the University of Washington [25,26].
A C. americanum cDNA Lambda UniZap XR library was screened by PCR as described in [27] with 5 μL of the cDNA library (representing 1 × 106 pfu) per reaction. The following degenerate oligonucleotide primers were derived from two internal peptide sequences obtained from the native protein, Micro1 and Micro2;
Micro1For-5'-GCTGGATCCTCCTTCATATA-3';
Micro1Rev-5'-(GC)GATAC(AT)GG(TC)AA(TC)(CG)TTAG(ATGG(CT)TATAC-3';
Micro2For-5'-AATTGTTGAAGCATGTGAAG-3';
Micro2Rev-5'-(TC)GGGGTTAG(AG)AG(TC)GT(AC)CG(AG)AA-3';
T3 + 6 - 5'-GCT CGA AAT TAA CCC TCA CTA AAG GG-3'
T7 + 6 - 5'-GAA TGG TAA TAC GAC TCA CTA TAG GGC G-3'
Primer combinations: i) Micro1Rev and Micro2For; ii) Micro1For and T3; iii) Micro1Rev and T7; iv) Micro2For and T3; v) Micro2Rev and T7. PCR products were subjected to DNA sequencing for characterization after cloning into the pGEM-T vector (Promega). DNA sequencing was carried out using the dideoxy-mediated chain termination method [28]. Fragment F6H·1 was amplified using a mixed primer set, T7 and Micro1Rev, and contained the peptide sequence to which the primer was designed, as well as certain conserved ODD motifs and a second peptide sequence. In order to isolate full-length putative F6H clones, new sets of primers were designed specifically to the ends of the F6H·1 sequence and the screening procedure was repeated with vector primers under stringent conditions. Alternatively, the bacteriophage cDNA library was screened as described in [29], using the F6H·1 fragment (602 bp) as an oligonucleotide probe. The near-full-length cDNA fragment isolated that putatively encoded the F6H was termed cF6H. Given the lack of fresh C. americanum tissue and its disappearance from its natural habitat, for use in 5'-rapid amplification of cDNA ends (RACE) experiments or primer extension, genomic DNA-based techniques such as inverse PCR and GenomeWalker (ClonTech) were employed in attempts to isolate the remaining 5'-end of the putative F6H sequence. Nevertheless, neither of these approached proved successful.
To isolate the genomic region coding for the cF6H, C. americanum genomic DNA (0.1 to 1.0 μg) was employed as a template for PCR using primers designed to the ends of the cF6H sequence. Putatively positive fragments were subsequently cloned into the pGEM-T vector for DNA sequencing.
To determine the copy number of F6H, genomic DNA (15 μg) was digested overnight and subjected to Southern analysis according to [30]. The probe, cF6H, was labeled using the BioPrime Kit (Amersham) designed for the preparation of biotinylated nucleotide probes for blotting with the IRDye800-conjugated Streptavidin (Rockland Immunochemicals) as the detection system. The membrane was pre-hybridized using UltraHyb-OS (Ambion) at 42°C for 2–3 h. Hybridization was carried out with denatured, biotinylated probe solubilized in a fresh aliquot of UltraHyb-OS overnight at 42°C. The membrane was washed according to manufacturer's instructions using wash solutions I and II. The membrane was blocked with blocking solution for one hour at room temperature, prior to probing with IRDye800-labelled streptavidin (1:10,000) in blocking solution for 1 h at room temperature. The membrane was washed with blot washing solution three times, 15 min each, prior to detection.
Cloning and expression strategies
The cDNA fragment encoding the near-full length F6H, cF6H was cloned into an E. coli expression system (pTrc-His; Invitrogen) The fragment was amplified by PCR using gene-specific primers, containing BamHI and HindIII recognition sites, respectively. The DNA insert is positioned downstream and in-frame with a sequence encoding the (His)6-tag and an enterokinase cleavage recognition site. The pTrc-His-F6H construct was transformed into Top 10 cells by heat shock, and selected on ampicillin (50 μg/mL) containing media. Recombinant protein production in E. coli strain Top 10 was induced by the addition of 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 4 h at 37°C. After sonication of the cell pellet and centrifugation, the supernatant was assayed for F6H enzyme activity or purified on Ni-NTA resin (Qiagen). The affinity-purified protein was eluted in presence of 250 mM imidazole and subjected to buffer exchange on a PD-10 column against the assay buffer. Enzyme assays and analysis of reaction products were carried out as previously described [3].
Heterologous expression in the Pichia pastoris expression system (EasySelect pPicZ-His; Invitrogen) can be either intracellular (pPicZ) or secreted (pPicZα), depending on the presence of an in-frame signal peptide. The cF6H fragment was amplified by PCR using gene-specific primers, containing KpnI and SacII recognition sites, respectively. For the pPicZ construct, the initiation ATG was part of the yeast consensus sequence (AATAATGTCT) included in the 5' gene-specific cloning primer. For the pPicZα construct, the insert was cloned in-frame with the N-terminal signal sequence and C-terminal His-tag. The pPicZ-cF6H and pPicZα-cF6H constructs were transformed into Pichia cells by electroporation and putative multi-copy recombinants were selected on Zeocin-containing media according to manufacturer's instructions (Invitrogen).
Recombinant F6H production was induced by the addition of 0.5% methanol (in the absence of glucose) every 24 h for 3 to 4 days. A buffered culture medium (BMGY/BMMY; containing 100 mM potassium phosphate, pH 8.0) was used for cell growth and protein induction in order to enhance protein stability and limit enzyme inactivation. In the case of intracellular recombinant protein production, cells were lysed using glass beads and extracts were prepared according to manufacturer's instructions (Invitrogen). Secreted proteins were collected and concentrated by ammonium sulfate precipitation (35 to 70% saturation), and used for activity assays after desalting or for subsequent affinity purification.
Recombinant proteins obtained from both prokaryotic and eukaryotic expression systems were subjected to SDS-PAGE [31] and Western blot [32] analysis using chemiluminescent (HRP; Amersham) or IRDye800 (Li-Cor) detection.
Phylogenetic analyses
Fifteen ODD genes involved in secondary metabolism where biochemical information was available, belonging to different plant species, were selected from the PubMed and GenBank searches. The amino acid sequences were aligned using CLUSTAL-W [33] and PHYLIP output format. The data was transferred into MacClade 4.03 [34] for visual inspection and manual editing prior to analysis by a phylogenetic tree building and analysis program, PAUP 4.0, beta 4 [35]. The optimality criterion employed for the distance method was Neighbor-Joining [36]. The aligned amino acid sequences were analyzed using a heuristic search algorithm with 1000 random addition sequences. Bootstrapping analysis was carried with heuristic search based on 1000 replicates and the distance measure was set to be equal to mean character difference.
List of abbreviations
cF6H F6H cDNA
F6H flavonol 6-hydroxylase
gF6H genomic F6H
ODD 2-oxoglutarate-dependent dioxygenase
α-KG α-ketoglutarate
ORF open reading frame
rF6H recombinant F6H
rF6Hb recombinant F6H expressed in bacteria (E. coli)
rF6Hy recombinant F6H expressed in yeast (P. pastoris) – intracellular
rF6Hys recombinant F6H expressed in yeast (P. pastoris) – secreted
TMQ 3,7,4'-trimethylquercetin
TMQg 3,7,4'-trimethylquercetagetin
Authors' contributions
DA carried out experiments in the project, participated in the design and coordination of the project, as well as the writing of the manuscript. RKI conceived of the project and contributed to the writing of the manuscript. All authors read and approved the final manuscript.
Acknowledgments
This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada and the Fonds de recherche sur la nature et les technologies to R.K.I, and postgraduate scholarships to D.A.
Figures and Tables
Figure 1 Proposed pathway for polymethylated flavonol biosynthesis in C. americanum
Figure 2 Translated cF6H sequence. Illustrates the conserved residues involved in iron binding (dashed circles), cosubstrate binding (underlined), other strictly conserved residues (encircled) and location of internal peptide sequences (boxed).
Figure 3 rF6Hb expression as detected by immunoblotting using anti-His antibody (A) Time course induction of rF6Hb expression. Soluble fraction represents total cell lysate. (B) Purification of rF6Hb on Ni-NTA; Lanes: 1, flow-through (10 μg); 2, wash (5 μg); 3, Bound 1 (1 mL eluate, 2 μg); 4, Bound 2 (2 mL eluate, 2 μg). (C) Purification of rF6Hb eluted from Superose 12 (10 μg/fraction).
Figure 4 Expression of rF6Hy/rF6Hys as detected by immunoblotting using anti-His antibody (A) Time course induction of rF6Hy. (B) Purification of rF6Hy on Ni-NTA. Lanes: 1, Bound 2 (2 mL eluate; 5 μg); 2, Bound 1 (1 ml eluate; 5 μg); 3, wash (10 μg); 4, flow-through (10 μg). (C) Time course induction of rF6Hys. (D) Purification of rF6Hys on Ni-NTA. Lanes: 1, Bound 3 (3 mL eluate; 2 μg); 2, Bound 2 (2 mL eluate; 2 μg); 3, Bound 1 (1 mL eluate; 5 μg); 4, wash (10 μg); 5, flow-through (10 μg).
Figure 5 Genomic organization of gF6H (Adapted from [14])
Figure 6 Southern blot of C. americanum genomic DNA Lanes: 1, BamHI, 2, EcoRI, 3, KpnI, 4, NcoI, 5, XhoI, 6, Undigested vector containing probe sequence
Figure 7 Phylogenetic analysis of cF6H Accession numbers for above genes: Potato FLS (X92178); Petunia FLS (Z22543); Parsley FLS (AY230249); Arabidopsis FLS (U84259); Arabidopsis ANS (U70478); Apple ANS (X71360); Carrot ANS (AF184273); Potato F3H (AY102035); Petunia F3H (X60512); Apple F3H (X69664); Parsley Flavone synthase (AY230247); Parsley F3H (AY230248); Hyoscyamus hyoscyamine 6β-hydroxylase (M62719); Catharanthus desacteoxyvindoline 4-hydroxylase (U71604).
Table 1 Specific activity of recombinant F6H proteinsa
Step Specific Activity (pkat/mg)
rF6Hb
Cell Lysate 0
Ni-NTA 0.091
Ni-NTA (30°C) 0.087
Ni-NTA + Superose 12 0.103
Superose 12 0.075
Cleavage of (His)6-tag after Ni-NTA 0.044
rF6Hy (intracellular)
Cell Lysate 0
Ni-NTA 0.17
Ni-NTA + Superdex 75 0.21
Superdex 75 0.087
rF6Hys (secreted)
(NH4)2SO4 (35–70%) 0
(NH4)2SO4 + Ni-NTA 0.11
(NH4)2SO4 + Ni-NTA + Superdex 75 0.08
Superdex 75 0.04
a All assays were carried in the presence of assay buffer, 5 μM 3,7,4'-trimethylquercetin and ODD cofactors using the F6H enzyme assay as described in [3].
Table 2 Substrate specificity of the recombinant F6H Both rF6Hb and rF6Hy were purified on Ni-NTA resin then Superose 12, and fractions were assayed with the indicated substrates, using the direct enzyme assay.
Substratea Relative enzyme activity (%)
rF6Hb b rF6Hy c
3,7,4'-Trimethylquercetin 100 100
3,7-Dimethylquercetin 56 63
3,7,3',4'-Tetramethylquercetin 11 21
3-Methylquercetin 0 4
a No F6H activity was detected with kaempferol, quercetin, myricetin, naringenin, eriodyctyol, apigenin or luteolin when used as substrate, regardless of concentration. Rhamnetin (7-O-methylquercetin, 17% at 50 μM), tamarixetin (4'-O-methylquercetin, 13 % at 50 μM) and isorhamnetin (3'-O-methylquercetin, 4 % at 50 μM)
b Estimated as 0.10 pkat/mg for 100% activity and 5 μM substrate.
c Estimated as 0.19 pkat/mg for 100% activity and 5 μM substrate.
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| 15596008 | PMC544895 | CC BY | 2021-01-04 16:03:51 | no | BMC Plant Biol. 2004 Dec 13; 4:20 | utf-8 | BMC Plant Biol | 2,004 | 10.1186/1471-2229-4-20 | oa_comm |
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BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-5-961561055710.1186/1471-2164-5-96Research ArticleFunnyBase: a systems level functional annotation of Fundulus ESTs for the analysis of gene expression Paschall Justin E [email protected] Marjorie F [email protected] Jeffrey D [email protected] Jennifer L [email protected] J Andrew [email protected] Gerald J [email protected] Kevin J [email protected] Douglas L [email protected] Division of Molecular Biology and Biochemistry, 5100 Rockhill Rd., University of Missouri-Kansas City 64110, USA2 Department of Environmental & Molecular Toxicology, North Carolina State University; Raleigh, NC 27695-7633 USA3 Division of Marine Biology and Fisheries, NIEHS Marine and Freshwater Biomedical Sciences Center, Rosenstiel School of Marine & Atmospheric Science, University of Miami, Miami, FL 33149, USA2004 20 12 2004 5 96 96 17 8 2004 20 12 2004 Copyright © 2004 Paschall et al; licensee BioMed Central Ltd.2004Paschall 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
While studies of non-model organisms are critical for many research areas, such as evolution, development, and environmental biology, they present particular challenges for both experimental and computational genomic level research. Resources such as mass-produced microarrays and the computational tools linking these data to functional annotation at the system and pathway level are rarely available for non-model species. This type of "systems-level" analysis is critical to the understanding of patterns of gene expression that underlie biological processes.
Results
We describe a bioinformatics pipeline known as FunnyBase that has been used to store, annotate, and analyze 40,363 expressed sequence tags (ESTs) from the heart and liver of the fish, Fundulus heteroclitus. Primary annotations based on sequence similarity are linked to networks of systematic annotation in Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) and can be queried and computationally utilized in downstream analyses. Steps are taken to ensure that the annotation is self-consistent and that the structure of GO is used to identify higher level functions that may not be annotated directly. An integrated framework for cDNA library production, sequencing, quality control, expression data generation, and systems-level analysis is presented and utilized. In a case study, a set of genes, that had statistically significant regression between gene expression levels and environmental temperature along the Atlantic Coast, shows a statistically significant (P < 0.001) enrichment in genes associated with amine metabolism.
Conclusion
The methods described have application for functional genomics studies, particularly among non-model organisms. The web interface for FunnyBase can be accessed at . Data and source code are available by request at [email protected].
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Background
Investigating patterns of gene expression using mouse and human microarrays has produced insights into cancer [1,2], cardiac diseases [3-6], and metabolic disorders [7-12]. These and many other functional genomics studies rely on full genomic sequence to establish well-annotated databases. Yet, microarrays based on EST collections are increasingly being used for diverse species, from honey bees to fish [13-20] and including simple diploblastic organisms [21]. These studies within a diversity of organisms provide insights not provided by 'model' species (species that are genetically well defined or with annotated genomes [22]). For example, 'non-model' organisms have provided insight into the natural variation in gene expression [23], social castes among bees [24,25], hypoxia [26], and physiological responses to variation in the thermal environment [27,28]. To investigate adaptive variation in gene expression we use the teleost Fundulus heteroclitus (killifish) [23,29].
The killifish Fundulus heteroclitus are distributed along the eastern coast of North America which has one of the steepest thermal clines in the world: northern populations have environmental temperatures more than 12°C below southern populations across 12 degrees of latitude. Migration among populations is sufficient to minimize random genetic drift [30] but not frequent enough to extinguish local adaptation [31,32]. Populations are large (>10,000) and affected by historical, demographic and selective constraints, providing a framework for the partitioning of variation in gene expression within and among populations. Additionally, the well-established phylogenetic relationship among Fundulus species can be used to discern adaptive changes [23,33,34]. These characteristics make F. heteroclitus an ideal species to investigate adaptive variation in gene expression.
Microarrays from diverse EST collections offer opportunities to address many biological problems, but to effectively use this information often requires a locally generated bioinformatics approach. Tools like the TIGR Gene index [35] and Unigene [36] provide significant information on many species, yet these databases do not meet the needs of functional genomics projects for many non-model species. Currently, TIGR and NCBI provide gene indices for 28 and 23 animal species, respectively. Yet, there are 63 animal species with more than 10,000 ESTs [37]. The number of species with ESTs >10,000 has continued to grow, and there was approximately a 20% increase in the preceding three months. While annotation from these resources can be accessed through web-based homology searches, for many laboratory collections of ESTs it is difficult to use existing tools to achieve a systems-level view of gene functions and relationships. Rather than simply browsing functional information over the web for a different group's project, laboratories that produce novel EST collections and microarrays require customized databases providing access to integrated functional annotation as expression data are being analyzed.
We have developed FunnyBase to meet these functional genomics needs. FunnyBase provides functional information for >40,000 ESTs from the teleost fish Fundulus heteroclitus, provides the means to quickly process, evaluate, and store annotation based on similarity searches of public resources, and integrates these data with species-specific clustering and microarray analysis. Perhaps ironically, the greatest challenge for functional annotation based on similarity searches is an overabundance of data. There are a number of databases to chose from, and often the single best hit from a given database search is not the most informative. FunnyBase implements a strategy to make maximum use of systems-level functional information from Gene Ontology (GO) [38] assignments and membership in metabolic pathways as defined by the Kyoto Encyclopedia of Genes and Genomes (KEGG) [39]. Specifically, several sequence databases are queried and results integrated to maximize the number of annotated sequences. Alignments and scores for all homology based associations are tracked, allowing further evaluation and statistical studies.
Microarray data using genes annotated in FunnyBase can be systematically analyzed in the context of biological functions. We present a case study to illustrate how assessment of systems-level annotation can identify statistically significant functional differences among sets of genes.
Results and discussion
FunnyBase (Fig. 1) is divided into 3 modules: Sequence Pipeline, Hierarchical Annotation, and Microarray Production and Analysis. The Sequence Pipeline takes sequences and quality output files from the sequencer, applies vector screening, quality trimming, clone tracking, and clustering (described below) to produce a set of unique sequences that are deposited in the 'Sequence Data' and 'Cluster Data' tables. Notice "unique sequences" are a combination of singletons (single unique ESTs) and clusters of overlapping sequences.
Figure 1 FunnyBase annotation scheme. The integration of the three FunnyBase modules: sequence pipeline, hierarchical annotations and microarray production and analysis. Database tables are shown in cylinders, arrows are data flow, and dashed lines indicate the integration of data from multiple sources.
The Hierarchical Annotation module uses the consensus sequences from the clusters or singletons, and integrates primary annotation such as gene name and description with associated pathways and systems-level functional annotation. This may include gene function (e.g., enzyme catalyst), metabolic or signal pathway (e.g., oxidative phosphorylation), or biological function (e.g., protein translation). Sequence data from the first module and functional annotation from the second are matched using database similarity searches (BLASTX and BLASTN). E-values, bit scores and local alignments are stored in the 'Similarity Data' table for all significant matches. One of the strengths of FunnyBase is the use of different sequence databases (SwissProt [40], NCBI UniGene [36], and NCBI non-redundant NR [41]) to provide separate annotations. Although these databases are not completely independent, the three separate annotations provide verification of gene names.
The third module, Microarray Production & Analysis provides a list of unique genes to be printed and integrates expression data from microarray experiments with the Hierarchical Annotation module. This provides functional annotation for expression data. FunnyBase annotation is accessible through the web or through local SQL queries and data-mining scripts.
EST isolation and sequencing
The overall strategy used to isolate and sequence thousands of Fundulus cDNAs was (1) generate a high quality unidirectional cDNA library, (2) normalize the library, (3) randomly pick colonies and amplify by PCR the cDNA within the vector, (4) sequence and identify PCR products, and (5) after approximately every 1,000 clones, subtract these from the normalized library and repeat steps 3–5. Details for all protocols are provided at and were used in the Comparative Functional Genomic course at Mount Desert Island, ME 2000.
We sequenced 46,433 ESTs and 40,043 of these are available in the dbEST database at NCBI (dbEST identification numbers: 23,480,307 to 23,515,306; 23,520,047 to 23,525,409 and 24,320,128 to 24,320,184) as of June 26, 2004. Sequences in FunnyBase are identified by a number series: unique sequence number, array number, plate number and well identifier (example: 23434_125_001_H04). The remaining 6,966 un-submitted sequences failed to meet one of the sequence quality parameters. Two criteria are used for defining "good" sequences: 1) >100 bp of sequence with Phred score >20 or 2) form an overlapping cluster with other sequences. Of the 19,937 sequences processed with the current version of FunnyBase, 17,893 (90%) passed one of these quality parameters. In earlier iterations of FunnyBase, visual inspection and later a sequencer-specific quality measure equivalent to a Phred score of 15 were used as filters resulting in 3,603 of the first 5,760 sequences (63%) and 13,922 of the next 15,168 sequences (92%) meeting quality standards, respectively. Re-sequences account for 5,668 sequences, and 4,625 of these were submitted.
Controls
One of the most important steps for producing microarrays from cDNA libraries is being able to associate the bacteria containing the cDNAs of interest with the EST annotation. High-throughput procedures are highly prone to tracking errors including: loading plates into an automatic sequencer in the wrong order, orienting symmetric plates in the wrong direction, or mislabeling of plates. The ability to identify these types of mistakes requires controls for identifying plates and plate orientation. The FunnyBase system has a number of integrated quality control steps. First, a Ctenophore cDNA (NCBI: accession number: CN992733) that is unlike anything else in GenBank is used as a control. Controls are placed in 96-well plates in wells corresponding to the plate number and two orientation wells (A5 and F9). Sequences from 96-well plates are automatically scanned for these controls so that the identity and orientation are confirmed and a report is generated for manual review. Secondly, all clones used for microarray production are re-sequenced. This is necessary because individual cDNAs are cherry picked, re-grown and re-amplified, and each of these steps has the potential to introduce or magnify an error. For example, for a 6,000 gene array, a 5% error rate would result in 300 incorrect clones. Re-sequenced array plates are compared using pair-wise BLAST [42] against previous sequencing results so that the identity of printed microarray spots are verified.
EST clustering
EST projects generate a number of redundant sequences due to the random selection of cDNAs from tissue libraries (Table 1). Clustering redundant sequences is a critical first step of analysis in order to identify genes to target for subtraction. The program CAP3 by Xiaong Huang [43] was used to cluster EST sequences with a 30 bp overlap and 75 percent similarity.
Table 1 The Ten Most Frequently Annotated ESTs. Clusters with the greatest number of annotated ESTs, the sequence id, number of ESTs that cluster together, and the e-value (probability of similarity) are listed.
ID Number of ESTs Evalue Description
1616 977 0 Vitellogenin I precursor
2262 734 2e-88 Cytochrome c oxidase polypeptide II
1348 507 1e-102 Alpha-1-antitrypsin homolog precursor
1026 481 5e-61 Zona pellucida sperm-binding protein 3 precursor
1727 401 0 Serotransferrin precursor
555 397 0 Cytochrome c oxidase polypeptide I
640 369 8e-61 Zona pellucida sperm-binding protein B precursor
1549 351 2e-77 Apolipoprotein A-I precursor
570 331 1e-111 Cytochrome c oxidase polypeptide III
1178 304 2e-45 ATP synthase a chain
FunnyBase contains a total of 40,043 EST sequences from F. heteroclitus heart and liver. Clustering with CAP3 yields 3,776 clusters that contain 30,688 ESTs (77%). The remaining 8,991 ESTs (23%) are singletons. By storing the results of clustering with annotation, FunnyBase easily identifies these genes and aids in the selection of genes to be used for library subtraction with the goal of picking less common transcripts.
The 10 annotated clusters with the most sequences are listed in Table 1. In microarray experiments these genes tend to be highly expressed and the fluorescent signal tends to saturate the photomultiplier tube. These genes also serve to verify microarray printing because the predicted spots for these genes have the strongest signal.
The distribution of the number of clusters with two or more ESTs is depicted in Figure 2. Although most clusters (2,581 or 68%) in FunnyBase have two or three-to-four sequences (Fig. 2), a small number of highly expressed genes form clusters with a large number of ESTs. For example, there are three clusters that contain 512 to 1,024 ESTs and ten clusters that contain 256 to 512 ESTs (bottom two bars for killifish in Fig. 2). This distribution is similar to other teleost fish EST collections (Fig 2). Notice, as more ESTs are added, clusters tend to get larger (more ESTs per cluster) rather than new small clusters growing in frequency. Of the 3,779 killifish clusters, 14% have more than eight ESTs, yet of the 14,714 Medaka clusters, 48% have eight or more ESTs. These distributions suggest that adding more EST sequences has diminishing returns.
Figure 2 Frequency of cluster size class in teleosts. The frequency of the number of ESTs in each cluster is shown for Fundulus heteroclitus (Killifish: total number of clusters 3,779), Oryzias latipes (Medaka: total number of clusters 7,401), Oncorhynchus mykiss (Rainbow Trout: total number of clusters 11,405) and Danio rerio (Zebrafish: total number of clusters 14,714). For example, the first bar indicates that approximately 1,000 clusters contain 2 ESTs in all four teleost fish. Clusters for other species (not F. heteroclitus) are based on NCBI UniGene.
One of the objectives of EST projects is to isolate most, if not all, genes expressed in a tissue or organism. The increasing size of larger clusters with more sequencing efforts indicates that strategies to increase the probability of isolating new genes need to be employed. We used two strategies. First, we normalized the library to reduce the differences among expressed genes to less than 10-fold among rare and abundant mRNAs [44,45]. Using this technique we were able to reduce redundancy in annotated genes from 33% in the non-normalized library to 11% after normalization. Second, we targeted specific sequences for subtraction: annotated cDNAs with high frequencies were targeted in order to focus effort on picking new, rare sequences. Through subtraction we were able to increase the rate of discovery of new annotatable sequences from 24% to 36%. However, analysis of these results indicate that a set of highly expressed sequences, some of which were not subtracted because they were not in the set of annotated genes, still make up much of the EST library and should be the focus of future subtractions.
Gene annotation
Of the 12,776 unique ESTs (3,776 clusters and 8,991 singletons), 3,877 (30%) were annotated. The distribution of e-values for these annotations is shown in Figure 3. Most (84%) of these ESTs have e-values less than 10-5. Among the clusters, 2,265 of 3, 779 (60%) were annotated as compared to 333 of 1,131 (30%) of confirmed high quality singletons. The lower percent of annotated singletons suggests that these are either rare fish-specific genes, or represent otherwise divergent, likely non-coding (5' or 3' UTR), regions.
Figure 3 Distribution of e-values for annotations. Gene annotation is based on sequence similarity. The e-values, which describe the probability of random sequence similarity, are shown as the negative log value (e.g., 10-5 = 5).
Annotations for ESTs are based on similarity using BLASTX or BLASTN [42] to sequences in one of six-public databases: Swiss-Prot, Human UniGene, Danio rerio UniGene, Oncorhynchus mykiss Unigene, Oryzias latipes Unigene, or GenBank NR. FunnyBase includes locally parsed copies of these databases in a relational format. Thus, all searches are done locally and annotation features beyond the FASTA description can be queried. Consensus sequences from the Fundulus EST clusters as well as high quality single unique sequences (singletons) are used as query sequences for BLAST searches against these public databases. The use of consensus sequences, when available, allows sequences that do not contain regions of significant similarity with known protein (e.g., 5' or 3' noncoding regions) to be annotated if they are members of an annotated cluster. All hits with e-value less than 0.001 and their associated alignments are stored in the database and tracked with any associated functional annotation. Users can specify a custom level of significance when assessing the validity of homology based annotation. This record, which goes beyond storing a certain number of 'best hits', is critical because in many cases additional results may have a negligibly lower alignment score, but provide much more useful functional data.
The use of multiple databases increases the total number of annotated ESTs (Fig. 4) as compared with any one source and provides opportunity to compare annotation between all three sources for 1,841 (47%) sequences. GenBank NR provided the most number of annotations, but these tend to be less informative (see systematic functional annotations below). Human Unigene provided an additional 311 (8% of total) annotations. SwissProt provided an additional 32 annotations (1% of total) with 743 fewer annotated sequences than the NR. However, SwissProt is uniformly well annotated as compared to NR where informative functional annotation can easily be buried by numerous uninformative hits at similar e-values. Besides increasing the number of annotations, comparing the annotations from multiple databases ensures that mistakes in the curation can be detected and information such as alternative gene names can be compiled from multiple sources.
Figure 4 Venn diagram of annotations from three different databases. Number of unique ESTs annotated by three different databases: GenBank non-redundant (NR, total annotated = 3,534), Swiss-Prot (total annotated = 2,829), and human Unigene (total annotated = 2,390).
Systematic functional annotation: KEGG and Gene Ontology
In conjunction with performing similarity searches by BLAST, FunnyBase includes locally parsed representations of public databases such as SWISS-PROT in a relational database format. These databases provide additional information that cross-references other public resources such as GO, KEGG or OMIM [46] that are not available in the single FASTA description line returned by BLAST search.
KEGG [39] is a unique tool that represents metabolic and signal-transduction pathways both visually and computationally. FunnyBase links annotated genes to enzymes in KEGG pathways based on enzyme commission (EC) numbers. These pathway associations are stored and queries can readily identify genes from a given pathway that show specific patterns of expression. For visual inspection of the pathway, the web interface links directly to the graphical KEGG pathways in which a gene occurs.
Of the 3,877 annotated ESTs in FunnyBase, 588 (14%) participate in one or more pathways defined by KEGG. These 588 ESTs represent 105 different pathways. Table 2 provides a breakdown of the number of ESTs in FunnyBase for the 10 pathways associated with the largest number of distinct sequences (contigs or singletons). The extent that a given pathway is represented in FunnyBase can be used to identify metabolic differences among tissues [47] or in different species.
Table 2 Number of distinct sequences in the Top 10 most common KEGG pathways. The KEGG pathway name and number of distinct sequences (clusters or singletons) from FunnyBase are presented. There are more distinct sequences than enzymes in a pathway because many enzymes have several protein subunits and many proteins have several different loci encoding the same subunit (e.g., NADH dehydrogenase, a protein complex of oxidative phosphorylation, has 26 protein subunits and 42 loci for these subunits).
Sequence count for TOP 10 pathways
Glycolysis/Gluconeogenesis 89
Oxidative phosphorylation 86
Fatty acid metabolism 70
Pyruvate metabolism 69
Tryptophan metabolism 66
Butanoate metabolism 48
Glycerolipid metabolism 47
Valine, leucine and isoleucine degradation 46
Glycine, serine and threonine metabolism 44
Propanoate metabolism 44
The Gene Ontology project (GO) has produced a structured vocabulary in the form of an acyclic directed graph that biologists can use to annotate genes in a systematic manner [38]. FunnyBase includes two non-trivial steps to make the best possible use of GO terms. First, many GO annotations are lost if only the single 'best hit' from a homology search is considered because GO annotation is applied most often to a few model species such as human that may not appear as the single 'best hit' in a list of BLAST results. FunnyBase identifies the gene name associated with the 'best hit' BLAST result and then uses all GO annotation associated with hits from the complete BLAST results that have the same gene name as the 'best hit' and an e-value of e < 10-12. The goal of this approach is to identify annotation associated with a single 'best hit' gene based on results that may come from multiple species (orthologous genes) and therefore may have varying degrees of sequence similarity due to phylogenetic distance, but to avoid the problem of selecting an inconsistent set of GO terms arising from gene families that share regions of sequence similarity but may have different functions.
Secondly, GO annotation in public databases tends to annotate sequences with only the most specific GO term available, for example RNA polymerase II transcription factor activity, enhancer binding (GO:0003705) rather than the more general parent term transcription regulator activity (GO:0030528). However, in functional genomic analysis, significant patterns of expression may exist at the more general level of functional description. FunnyBase takes advantage of the connected parent-child relationship of GO terms provided by using the relational database version of GO available for download at to identify such relationships. These data are used to extract the tree of more general GO terms related to those provided by public databases. A FunnyBase script then re-annotates genes with this more complete set of GO terms.
Of the 3,877 annotated genes, 1,912 (54%) are assigned one or more GO terms with a total 6,728 GO assignments being made directly based on information in public databases such as SwissProt. Using parent-child GO term relationship backtracking, an additional 34,112 GO term assignments were made, resulting in a final count of GO assignments of 36,024 excluding the most general terms that divide GO into three categories. Thus, on average, 19 GO terms are assigned to each of 1,912 annotated genes.
Gene scaffolding: clustering of clusters
Humans have approximately 30,000 expressed genes, yet there are over 1,000,000 human UniGenes (NCBI). Clearly, these clusters of cDNAs greatly overestimate the number of unique genes. Similarly, FunnyBase has multiple clusters for the same gene: 15 apolipoprotein I, 10 cytochrome oxidase I, and 53 vitellogenin clusters. To provide a more precise estimate of the number of unique genes, consensus sequences were queried against the 27,695 sequences from the Human RefSeq [48] database, then grouped by identical gene symbol. Of the 2,376 Fundulus clusters that were similar to a sequence in Human RefSeq (e-value < 10-10), 1,818 (76%) had distinct gene annotations. This method of clustering clusters by similarity to well-annotated reference sequences provides a method to more accurately define the number of unique genes represented by an EST set.
Case study: using functional annotation for microarray analysis
As a case study in how functional annotation in FunnyBase can be integrated with microarray data in a rigorous manner, we used a data set based on a microarray of metabolic genes printed from ESTs annotated in FunnyBase [47]. Statistical analysis of this set of 363 metabolic genes identified a set of 62 genes that showed statistically significant regression between gene expression levels and temperature along the Atlantic coast. That is, among individuals collected from different locations along the thermocline and then acclimated to common physiological conditions for at least nine months before analysis, 17% of the metabolic genes had a linear relationship between the amount of mRNA and the environmental temperature these animals evolved in. Our hypothesis was that this set of 59 genes represents a functionally different set than those genes that do not show regression with temperature. To test this hypothesis we examined the frequency of genes annotated with a given GO term in the statistically significant gene set versus the non-significant genes. Figure 5 shows the relevant proportions in each set for GO terms that are represented by 5 or more ESTs in the significant set. For example, the GO term Amine Metabolism (GO:0009308) is assigned to 14% of the 62 statistically significant genes but only 3% of the non-significant genes (those that do not show significant regression with temperature). A Fisher-exact test indicates these frequencies (14% vs. 3%) represent different underlying distributions (p < 0.001). Specifically, genes involved in amine metabolism are overrepresented in the set of genes that show regression with temperature as compared with the remaining sequences. This significant increase is found for two other non-mutually exclusive GO terms: amino acid and derivative metabolism, and amino acid metabolism (p < 0.05). Other GO terms show a reverse trend although none were statistically significant. For example, ion transport (p = 0.08) and cell growth (p = 0.16) had few genes with a clinal variation in expression. These data suggest that the functions of genes influence whether they are affected by ecologically interesting patterns of expression (Fig 5).
Figure 5 Distribution of significant and non-significant genes relative to GO terms. The relationship between gene expression from "common gardened" fish and the environment they evolved in was statistically analyzed and grouped by GO terms. Black bars represent genes whose levels of expression has a significant regression with the environmental thermal cline among populations (p < 0.05). Hatched bars represent the set of genes with no significant relationship to the thermal environment.
Web interface
The web interface provides public access to the FunnyBase system and dataset. Searches can query by keyword in annotation, gene name, GO term, metabolic pathway, clone or plate id, and BLAST homology search. All data including raw sequences, cluster memberships, cluster alignments, and alignments with homologous sequences are provided for the user to examine the source of annotations. Links associated with each annotation are made to external resources such as GO's AMIGO browser, KEGG pathways, SwissProt, and NCBI records.
Other Fundulus sequences
FunnyBase was constructed to annotate sequences for the analysis of gene expression. It provides identification and annotation for genes in the Crawford laboratory with a primary goal of identifying clones useful for the construction of microarrays. As such, other Fundulus sequences in Genbank are not included. However, FunnyBase forms the basis of the TIGR Killifish gene index that includes publicly available F. heteroclitus sequences.
Conclusions
Customized species specific EST databases are available for many species [17,18,21,49-56]. FunnyBase provides an integrated method to annotate ESTs with the most biologically relevant set of associations and provides several innovations for the production of ESTs for microarrays. Control sequences are identified in each 96-well plate so that mislabelled or inverted plates are automatically detected. Annotations are based upon several different public databases. The multiple annotations provide greater assurance about gene description and greater frequency of annotation than any one database. The most functionally informative innovation of FunnyBase is the process of culling through numerous primary similarity search results in order to identify links to systematic functional databases in GO and KEGG. These provide a discrete set of terms that can be analyzed statistically and that are organized into networks that represent biological knowledge of higher-level functional and pathway associations. The range of databases queried by similarity search and the tracking of homology beyond a single 'best' hit maximizes the opportunity to obtain this annotation. A richer set of GO terms is achieved by using all hits with e-values less than 10-11 that represent the same gene as the 'best hit'. Additional GO terms that represent more general functions than those found in public annotation are derived through the parent-child relationship of the Gene Ontology. EC numbers provide links, via KEGG, to metabolic pathways and these stored terms can be used to investigate the relationship between gene expression in specific metabolic pathways including cardiac metabolism [57]. To provide a more accurate accounting of the number of unique genes, consensus sequence from clusters of ESTs were queried against the Human RefSeq database and those sequences sharing the same gene symbol are grouped based on this scaffolding information. These approaches use publicly available bioinformatics tools (BLAST, CAP3, Phred, Cross-Match, Perl, and the MySQL database management system). The application of theses tools in an appropriate framework as outlined in FunnyBase can be used to create a systems level functional genomics annotation system useful for EST databases to study biological processes among a rich diversity of organisms.
Methods
Organism
The animal protocols used in the present study have been approved by the University of Miami Institute Animal Care and Use Committee. The teleost fish Fundulus heteroclitus used for ESTs were collected from two sites: Scorton Creek in Sandwich, MA, and Stone Harbor, NJ. These populations are in the central portion of the thermal cline and have relatively high levels of heterozygosity [32]. These fish were subjected to the following environmental regime before tissues were harvested for mRNA extraction: kept in controlled temperature and aeration conditions, and acclimated to common conditions (20°C, 15 ppt salinity) in re-circulating aquaria for at least nine months before experiments. Following this common acclimation a subset of fish were subjected to one of several stresses: 4°C, 34°C, hypoxia, or a complex mix of hydrocarbons.
cDNA library
To effectively isolate and sequence thousands of cDNAs for the production of microarrays, a unidirectional cDNA library with few non-recombinants was required. We created four cDNA libraries: heart libraries from non-stressed and stressed fish and liver libraries from non-stressed and stressed fish. The non-stressed F. heteroclitus cardiac and liver libraries were provided by Drs. S. Karchner and M. Hahn, WHOI [58] and were constructed using the UniZap λ cDNA Gigpack Gold cloning kit (Stratagene, La Jolla, CA, USA). The cardiac library was produced from 27 fish hearts (both sexes) sampled from Scorton Creek in Sandwich, MA. The cDNAs in these libraries are oriented such that the 5'end of each cDNA is ligated to EcoR1 and 3' poly A is ligated to XhoI. These libraries had less than 1% non-recombinants, i.e. 2 of 300 random clones from a non-normalized library had no inserts. The stressed libraries included 4 fish subjected to the four stressors (above) and 4 non-stressed individuals. Unidirectional heart and liver libraries were constructed such that the 5'end of each cDNA is ligated to EcoR1 and 3' poly A is ligated to XhoI of the plasmid vector pSmart (Lucigen, Middleton, WI, USA). The pSmart-cDNA vector was designed for EST work. The vector expresses kanamycin-resistance and has a terminator on both sides of the cDNA insertion site preventing expression of cDNA. These two attributes (non-expression and Kan-resistance) increase the stability of different genes in the library versus cDNA libraries in Amp libraries with Lac promoters (Crawford, unpublished). These libraries had less than 1% non-recombinants.
Normalization of cDNA libraries reduces the differences among expressed genes to less than 10-fold among rare and abundant mRNAs [44,45]. Normalized libraries were produced by isolating cDNAs from approximately 1012 plasmids. The cDNAs were isolated using PCR amplification with vector specific primers immediately 5' and 3' to the insertion site (EcoRI and XhoI sites). These PCR products (PCR-cDNAs) were denatured and hybridized to single stranded plasmids from the cardiac cDNA library. Taking advantage of Cot values, the most abundant cDNAs were annealed to the more abundant PCR products and were removed selectively by hydroxyapatite-column chromatography. The single-stranded plasmids in the flow-through were converted to double strands using the Sequenase DNA polymerase (Amersham, Piscataway, NJ, USA). DH10s E. coli (BRL) were transformed with these double-stranded plasmids by electroporation. The number of recovered plasmids and the resulting complexity of the normalized library depended on the duration of hybridization or Cot values. Two normalized libraries were made using either a 12 or 24 hour hybridization. The library from the 12-hour hybridization yielded 250,000 plasmids. The library from the 24 hour hybridization yielded 3,000 plasmids and had a greater representation of rare mRNAs and greater frequency of non-recombinants.
Isolation and sequencing of cDNAs
Characterization of cDNAs (growth of individual bacterial colonies containing plasmids, PCRs, purification of PCR products, sequencing reactions) used 96 well plates and octopipettes. To characterize cDNAs, 96 individual bacterial colonies from the normalized library were randomly chosen, and each was grown in 1.25 ml of Superbroth in 2 ml-96 well plates. After 18 hours of growth, two 250 ul bacterial glycerol stocks were made and stored in 96 well plates at -80°C. One microliter of these bacterial growths was used for PCR reactions using forward and reverse plasmid specific primers: (PucF = CGCCAGGGTTTTCCCAGTCACG, PucR = GAGCGGATAACAATTTCACACAGGAAA). PCR reactions had 0.2 mM dNTPs, 10 pmoles of each primer, 1 unit of Promega Taq (0.2 ul), and reaction buffer with detergents and DMSO (final concentrations: 50 mM Tris HCl, pH 9.2 (25°C), 16 mM (NH4)2SO4, 2.25 mM MgCl2, 2% (v/v) DMSO, 0.1% (v/v) Tween 20). Two-step thermal cycle conditions were used (94°C for 10 seconds; then 32 cycles of 94°C for 30 seconds followed by 70°C for 5 minutes; then 72°C for 15 minutes). PCR products were purified manually in 96 well format using Sephadex G-50 in a deep well plate with a 0.2 microfilter (Millipore, Billerica, USA) or robotically using AmPure (Agencourt, Beverly, MA, USA) and EvolP3 96 pipetting liquid handling system (PerkinElmer Life Sciences Inc., Boston, MA, USA).
PCR products were sequenced from the 5' end (relative to the mRNA) on an ABI 373 or ABI 3730 sequencer using ABI "Big Dye" reaction mix. We typically used 1/16 the amount of reaction mix, yielding 300 to 400 unambiguous bases. Sequences were purified using biotin primers and streptavidin coated magnetic beads (for the ABI 373) or Agencort CleanSeq (for the ABI 3730).
Validation
We used three procedures to verify that the correct sequence was associated with each cDNA. 1) Each 96-well plate had three wells with a "marker cDNA" (Ctenophore cDNA #5, a random cDNA with no similarity to any sequence in GenBank). Two wells (#40 and #67) always contained the marker cDNA, and thus any misloading or mislabeling of sequencing lanes was identifiable. The third marker cDNA was placed in a well that corresponds to the plate number (e.g., plate 2 had the marker in well 2). 2) After the production of 12 plates, one row (8 wells) from each plate was re-sequenced. Thus, 8/96 or ~8% of all sequences and their locations were confirmed. 3) cDNAs used for microarrays were re-sequenced. These measures are important to ensure that the correct and known cDNAs are printed.
Subtraction
The complexity of the normalized library was reduced by subtracting the characterized cDNAs previously isolated from the normalized library. Subtraction greatly reduced the probability of isolating the same cDNA and thus improved the efficiency of screening the library for unique clones. Subtraction used a 100-fold molar excess of biotin-labeled antisense cDNAs produced by PCR using all the characterized cDNAs as substrates and vector-specific primers in which the 3' primer was labeled with biotin. These PCR products were hybridized to the cDNA libraries in the presence of oligo-dA and vector-specific oligos (that prevented non-specific hybridization to oligo-dT or vector sequences). After a 24 hour hybridization, genes in the library that bound to these biotin-labeled PCR products were removed with the use of magnetized, streptavidin coated beads. DH10s E. coli were transformed with the subtracted library by electroporation.
Hardware and software
Computational work was done on an Apple G5 dual 2 GHz processor system with 4 GB of RAM. Data are stored in a MySQL database, perl scripts were used extensively for parsing and loading data, and PHP was used on an APACHE web server to construct the user interface. Additional programs available from their authors are mentioned within context. Software and databases are described in Table 3.
Table 3 Software and Databases. Publicly available software and databases used for FunnyBase. The version and/or download date are listed.
Resource Version and/or Download Date
Software
Stand-alone BLAST 2.2.8
Cross match 0.990329
CAP3 January, 2004
Public Sequence Similarity Databases
Swiss-Prot
NR 44
Human RefSeq June, 2004
Human Unigene June, 2004
Zebrafish Unigene June, 2004
Medaka Unigene June, 2004
Rainbow Trout Unigene June, 2004
June, 2004
Functional Annotation
Gene Ontology 2004-06-04
KEGG June, 2004
Microarrays
Microarrays were printed using a select 384 cDNAs from F. heteroclitus cardiac library encoding essential proteins for cellular metabolism isolated from over 40,000 expressed sequences . These 384 cDNAs were amplified with amine-linked primers and printed on 3-D Link Activated slides (Surmodics Inc., Eden Prairie, MN, USA) using GeneMachine OminGrider, and blocked following slide manufacturer protocols. The suite of 384 amplified cDNAs was printed as a group in four spatially separated replicates. Four hybridization zones of these four replicate arrays were printed per slide, with each zone set separated by a hydrophobic barrier. Samples were hybridized twice; once with Cy3 and once with Cy5 resulting in overall technical replication of 8-fold per sample.
Sample preparation and hybridization
RNA was extracted from tissue homogenate in a chaotropic buffer using phenol/cholorform/isoamyl alcohol and RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). RNA for hybridization was prepared by amplification using a modified Eberwine protocol [59] using the Ambion Amino Allyl MessageAmp aRNA Kit. Cy3 and Cy5 were hybridized to slides and incubated 12–18 hours at 42°C. Following hybridization, slides were scanned using the Packard Bioscience ScanArray Express microarray scanner (PerkinElmer Life Sciences Inc., Boston, MA, USA) and images processed using ImaGene (Biodiscovery Inc., Marina del Rey, CA, USA).
Authors' contributions
JP designed, scripted and implemented FunnyBase and provided statistical analyses of database. MFO initiated, designed protocols and provided sequences for F. heteroclitus' EST project. JDV optimized robotic interfaces for sequencing and sequenced ESTs. JLR and KJK sequenced ESTs. GJW collaborated on bioinformatics and database development. JAW provided microarray data and analyses. DLC initiated F. heteroclitus' EST project and developed the database and annotation schemes for FunnyBase. All authors read and approved the final manuscript.
Acknowledgements
Initial support for the F. heteroclitus EST project was provided by NSF BioInformatics post-doctoral fellowship 0074520 to MFO and NSF/IBN grant 9986602 to DLC. We would like to thank Kristin Horgan of M.J. research for arranging the loan of Tetrad-thermal cycler for the Compartive Functional Genomic Course. Additionally, we would like to thank AP-Biotech and specifically Dr. Robert Feldman for use of MegaBace used to re-sequence cDNAs.
Current EST isolation, sequencing and bioinformatics is supported by NSF/OCE grant 0221879 and NIH/NHLBI R01 HL65470 to DLC and NIH/NIEHS ES011588 to MFO.
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| 15610557 | PMC544896 | CC BY | 2021-01-04 16:39:23 | no | BMC Genomics. 2004 Dec 20; 5:96 | utf-8 | BMC Genomics | 2,004 | 10.1186/1471-2164-5-96 | oa_comm |
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BMC Med EthicsBMC Medical Ethics1472-6939BioMed Central London 1472-6939-5-910.1186/1472-6939-5-9Research ArticleVariations and voids: the regulation of human cloning around the world Pattinson Shaun D [email protected] Timothy [email protected] Sheffield Institute of Biotechnological Law and Ethics (SIBLE), University of Sheffield, UK2 Health Law Institute, Faculty of Law and Faculty of Medicine and Dentistry, University of Alberta, Canada2004 13 12 2004 5 9 9 12 10 2004 13 12 2004 Copyright © 2004 Pattinson and Caulfield; licensee BioMed Central Ltd.2004Pattinson and Caulfield; 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
No two countries have adopted identical regulatory measures on cloning. Understanding the complexity of these regulatory variations is essential. It highlights the challenges associated with the regulation of a controversial and rapidly evolving area of science and sheds light on a regulatory framework that can accommodate this reality.
Methods
Using the most reliable information available, we have performed a survey of the regulatory position of thirty countries around the world regarding the creation and use of cloned embryos (see Table 1). We have relied on original and translated legislation, as well as published sources and personal communications. We have examined the regulation of both reproductive cloning (RC) and non-reproductive cloning (NRC).
Results
While most of the countries studied have enacted national legislation, the absence of legislation in seven of these countries should not be equated with the absence of regulation. Senator Morin was not correct in stating that the majority of recent legislation bans both RC and NRC. Recent regulatory moves are united only with regard to the banning of RC. While NRC is not permitted in seventeen of the countries examined, it could be permitted in up to thirteen countries.
Conclusions
There is little consensus on the various approaches to cloning laws and policies, and the regulatory position in many countries remains uncertain.
==== Body
Background
"The immense majority of countries who have passed legislation recently do ban both reproductive and therapeutic cloning" (Senator Morin, The Standing Senate Committee On Social Affairs, Science And Technology, Ottawa, Canada, Wednesday 18 February, 2004).
In February 1997 an article was published in Nature announcing the birth of what was to become the most famous sheep in history [1]. That sheep, known as Dolly, was the product of asexual reproduction. As the world's media unhesitatingly announced, she was a clone. The prospect of a human clone led to immediate calls for regulatory controls on the technology. There were, however, divisions, particularly when it became apparent that the potential uses of the technique were not limited to reproduction. Other potential uses came one step closer when, in the following year, it was announced that embryonic stem cells had been successfully extracted from non-cloned human embryos [2]. Now, a year after the death of Dolly [3], it is appropriate to review the current regulatory position on the creation and use of cloned embryos around the world [4,5] – particularly considering the public debate that has surrounded the recent cloning experiments in Korea, the granting of the first "research cloning" license in the UK, and the past and impending UN cloning debates.
This paper is concerned with the creation of functional embryos, whether by nuclear transfer or embryo splitting. As this suggests, we will use 'embryo' to refer to any human entity considered theoretically capable of implantation and development in the womb. Many regulatory positions distinguish between the creation of a cloned embryo for reproductive purposes and for other purposes. For our purposes, the former will be called reproductive cloning (RC for short) and the latter non-reproductive cloning (NRC for short). This paper will examine the regulatory position of the thirty countries for which we have been able to obtain reliable information (see Table 1). Where possible, we have relied on copies of the original legislation or of English translations of that legislation. In some situations we have also found it necessary to rely on other published sources [6] and personal communications.
Table 1 Summary of regulation*
Country National legislation and effect, or approach in the absence of national legislation
Australia Prohibition of Human Cloning Act 2002: Prohibits both RC and NRC
Austria Act No.275 of 1st July 1992: Implicitly prohibits both RC and NRC
Belgium Law of 11th May 2003: Prohibits RC but permits NRC
Canada Assisted Human Reproduction Act 2004: Prohibits both RC and NRC
China None Ministerial regulations prohibit RC and allow NRC
Denmark Law No.460 of 1997: Prohibits both RC and NRC
Finland Medical Research Act No.488 of 1999: Prohibits RC, but NRC might not be included within this prohibition
France Law of July 2004: Prohibits both RC and NRC
Germany Embryo Protection Act 1990: Prohibits both RC and NRC
Greece Law 3089 of 23rd December 2002: Prohibits RC, but does not cover NRC
Iceland Law No. 55 of 29th of May 1996: Prohibits both RC and NRC
India None Guidelines reject RC but might allow NRC
Ireland None Constitutional provision might prohibit both NRC and RC
Israel Prohibition of Genetic Intervention (Human Cloning and Genetic Manipulation of Reproductive Cells) Law 1999: Imposes a moratorium on RC, silent on NRC
Italy Law of 2003: Prohibits both RC and NRC
Korea Bioethics Law 2003: Prohibits RC, silent on NRC
Luxembourg None
Mexico General Health Law of 7 May 1997: Prohibits both RC and NRC
Netherlands Embryo Act 2002: Prohibits RC and imposes a moratorium on NRC
New Zealand Medicines (Restricted Biotechnical Procedures) Amendment Act 2002: Prohibits RC, silent on NRC
Norway Law No. 100 of 5 December 2003: Prohibits both RC and NRC
Peru General Law No. 26842 of 9 July 1997: Prohibits both RC and NRC
Portugal None Ratified Convention (discussed below)
Russia Law on the Temporary Prohibition of Human Cloning 2002: Imposes a Moratorium on RC
Spain Law 35 of November 1988: Prohibits both RC and NRC
Sweden Law No. 115 of 14th March 1991: Implicitly prohibits both RC and NRC (with possible gaps)
Switzerland Federal Law of 18 December 1998: Prohibits both RC and NRC
Thailand None
UK Human Fertilisation and Embryology Act 1990: Permits NRC under licence Human Reproductive Cloning Act 2001: Prohibits RC
United States None Some states have legislation prohibiting RC or both RC and NRC
* The International Digest of Health Legislation provides translations of the legislation of Austria (1993, vol 44); Denmark (1997, vol 48); Israel (2000, vol 51), Norway (2003, vol 54), Peru (1998, vol 49), and Switzerland (1999, vol 50, and 2003, vol 54). The Bulletin of Medical Ethics provides translations of the legislation of Finland (Feb 2000, p7) and Germany (Dec 1990, p9). In addition, we have used English translations of the legislation of Greece , the Netherlands , and Sweden (Ministry of Health and Social Affairs, Swedish Act concerning Use of Gene Technology on Human Beings and Experiments with Fertilised Ova, 1991).
Understanding the complexity of this "regulatory patch work" is essential [7]. It provides a sense of the vast differences between nations, the issues on which there are different views, and the existing regulatory uncertainties. In addition, it highlights both the challenges associated with the regulation of a controversial and rapidly evolving area of science and the need for a regulatory framework that can accommodate this reality. Finally, it demonstrates that policy makers cannot rely on the existence of a single regulatory trend to inform policy development. Only on the banning of RC (reproductive cloning) do the world's legislatures and policy-makers display anything approximating a single mind. Senator Morin, who is a member of Canadian Senate, was not right to claim that a majority of recent legislation bans both RC and NRC (non-reproductive cloning). Even looking beyond recent legislation, only a narrow majority of the thirty countries studied actually prohibit NRC. What is more, a large minority of countries have yet to enact national legislation.
This paper is divided into sections. The next, section II, will examine the degree of regulatory variation in the countries studied. Section III will ask whether the existence of legislation answers all regulatory questions. Using examples drawn from those countries with legislation, we seek to show how broad interpretative strategies are sometimes required to avoid unintended lacunae. Section IV uses examples to demonstrate the evolving nature of regulatory positions and increasing reliance on legislation. Section V examines the impact of international initiatives in European (ie the European Convention on Human Rights and Biomedicine) and the United Nations. Section VI will explain why the regulatory outcome usuallysays little about the ethical approach adopted by a particular jurisdiction. Section VII is the conclusion.
Variation between countries
As many commentators have noted, there is great variation in regulatory approaches even within countries that have decided to create relevant laws and policies [4,5,7,8]. No two countries have adopted identical regulatory measures on cloning, though the effect of those adopted in some countries is very similar. There is only one area of regulatory agreement – no jurisdiction has, yet, adopted legislation or guidelines permitting RC. As a result, there are essentially only two regulatory approaches to RC: prohibition or regulatory silence. Regulatory silence usually means that RC is technically legal in the jurisdiction in question, though if it were attempted, there would likely be a rapid regulatory response.
The majority of the countries studied have now enacted national legislation (see Table 1). Only seven have yet to do so. The absence of national legislation in these seven countries should not, however, be taken to amount to an absence of regulation or, in the case of the US, an absence of state legislation. Legislation is just one of many possible regulatory responses. Ireland provides an illustrative example. The Eighth Amendment to the Irish Constitution (which forms Article 40.3.3) states that,
"The State acknowledges the right to life of the unborn and, with due regard to the equal right to life of the mother, guarantees in its laws to respect, and, as far as practicable, by its laws to defend and vindicate that right."
While this provision does not mention cloning, it has been taken to protect in vitro embryos and thereby prohibit NRC. In addition, doctors must comply with the guidance of the Medical Council, as this body has the power to remove their licence to practise in Ireland. The Medical Council's guidelines declare that " [t]he creation of new forms of life for experimental purposes or the deliberate and intentional destruction of human life already formed is professional misconduct" [9]. Also, it limits the manipulation of sperm or eggs to the "improvement of health" and adds that "if the intention is...the creation of embryos for experimental purposes, it would be professional misconduct" [9]. Thus, the absence of legislation in Ireland does not render all things permissible.
Variation and non-reproductive cloning (NRC)
NRC represents the source of considerable regulatory variation. While NRC is not permitted in seventeen of the countries studied, it could be permitted in up to thirteen other countries. Regulatory uncertainties make it impossible to be sure in some of these counties. Also, some countries (such as the US) have many jurisdictions, each capable of adopting a different regulatory position. Given the superficial similarity of many of these countries and jurisdictions, it is hard to explain such stark variation on cultural differences alone [8,10].
Only Belgium and the UK have deliberately enacted or extended legislation for the purpose of permitting the creation of cloned embryos for research [6,11]. The UK licensing authority has, in fact, granted its first licence to conduct NRC [12]. Similarly permissive approaches, albeit non-legislative, have been adopted by China (which issued Ministerial Regulations in August 2003 to allow cloning research for therapeutic purposes) [6,13] and Korea (where the government is in the process of approving limited research on limited somatic nuclear transfer research) [6].
In contrast, Finland, Greece, Israel, Russia and Sweden appear to allow NRC only because their legislation has potential gaps [14]. The Greece legislation is the most striking, because it is the most recently enacted. The Greek Law 3089/2002 explicitly prohibits 'human reproduction' by any cloning method, but no mention is made of NRC. This must have been deliberate, because the provision allowing embryo research does so by allowing research (with consent) on "fertilized ova" that are surplus following assisted reproductive treatment. Indeed, the legislation's Explanatory Memorandum declares that
"only reproductive cloning is prohibited. It could be thus construed that therapeutic cloning is permitted....This position has also been supported in the Report of the National Bioethics Committee regarding the use of stem cells in biomedical research and clinical practice (21.10.2001), submitted to the Prime Minister on 11.1.2002."
This is nonetheless controversial in Greece, which has ratified both the European Convention on Human Rights and Biomedicine, and its Additional Protocol on cloning (discussed below).
The US, India, and Portugal are anomalous. In the US, what little national legislation there is only concerns the use of federal funds [4] and some States (such as California and New Jersey) have even adopted permissive legislation [15]. The Indian Council of Medical Research has declared that "research on cloning with intent to produce an identical human being, as of today, is prohibited", but has not declared NRC to be so prohibited [16]. However, an Indian Government policy document "opens the door to therapeutic cloning considered on a case-by-case basis by the National Bioethics Committee" [6]. Portugal has no national legislation, but has ratified the European Convention and its Additional Protocol (see below).
Legislative gaps and uncertainties
There are, of course, many nations that have long standing laws that are relevant to cloning technologies. In many of these nations, however, the laws were designed prior to Dolly and the recent advances in stem cell research. As such, how these laws might apply to cloning is sometimes unclear. Also, these laws are not a result of a public and political dialogue about the complex scientific and ethical issues that are associated with cloning and stem cell technologies. [10] In addition, there are a number of countries where recent legislative intervention has failed to answer all legal questions relating to human cloning.
The legislation of some countries clearly encompasses somatic cell nuclear transfer (SCNT). The Spanish Law 35 of November 1998 is an example of a pre-Dolly legislation of this type. This Act not only renders it an offence to create identical human beings where it is aimed at race selection, it also renders it an offence to create "human beings by cloning in any of the variants or any other procedure capable of originating several identical human beings." The Canadian Assisted Human Reproduction Act 2004 is an example of post-Dolly legislation of this type. Under this Act, the creation and implantation of a "human clone" are prohibited. "Human clone" is defined under s. 3 to mean "an embryo that, as a result of the manipulation of human reproductive material or an in vitro embryo, contains a diploid set of chromosomes obtained from a single – living or deceased – human being, foetus or embryo". This clearly captures SCNT.
In a number of contrasting countries, SCNT is only captured by a broad, non-literal interpretation of the relevant provisions. The Swedish Law No. 115 of 14 March 1991 is an example of pre-Dolly legislation of this type. This Act only regulates experiments performed on "fertilised ova" or gametes "before fertilisation". The Finnish Medical Research Act 1999 is a rare example of post-Dolly legislation of this type. This Act has many provisions with respect to research on embryos (including a prohibition on the creation of embryos for research) and it prohibits all research conducted with the aim of cloning a human being. However, s. 2 of the Act defines an embryo as "a living group of cells resulting from fertilisation not implanted in a woman's body". Thus, the Dolly technique only appears to be covered insofar as its use involves "research with the aim of cloning human beings" (Also, s.1 of the Constitution secures the inviolability of human dignity, but there is no authoritative interpretation on whether (and how) this provision could apply to cloning). If this aim is only correctly attributed to RC, then NRC might not be covered at all.
The dangers of non-literal interpretation of pre-Dolly provisions should not be exaggerated. Although there are a number of countries that have such legislation (notably, Austria and Germany and, until very recently, France) [5], in reality the courts are likely to adopt a broad, purposive approach to interpretation. A very broad approach was, for example, taken when the domestic courts addressed the UK's Human Fertilisation and Embryology Act 1990 [17]. The Act explicitly prohibited only one form of cloning (the creation of a clone by replacing the nucleus of an embryo) leaving the licensing authority to regulate activities such as the creation, storage, and use of in vitro embryos. More precisely, the Act imposes a licensing requirement on the creation of an in vitro embryo (ss.3(1)(a) and 1(2)); storage or use of in vitro embryos (ss.3(1)(b) and 1(2)); storage of gametes (s.4(1)(a)); and use of gametes, unless 'services are provided for the woman and man together' (s.4(1)(b)). Yet, under s. 1(1) of the Act, "embryo" is defined as "a live human embryo where fertilisation is complete", including "an egg in the process of fertilisation". This raised the question of whether SCNT fell outside the Act altogether. Nonetheless, the House of Lords recently held that SCNT produces a functional embryo that falls within the ambit of this Act (in effect, holding that the Act's definition of embryo is non-exhaustive and restricted in purpose) [18].
Evolving nature of the laws
Not only is there a great deal of variation between nations and much uncertainty as to the scope of existing laws, many of the existing laws and policies are in a state of flux. Indeed, some countries have built in review provisions.
The Dutch legislation, the Embryo Act 2002, presents an example. This Act prohibits procedures undertaken for the purpose of creating genetically identical human individuals and prohibits the creation of embryos for research. Yet, s. 33 of the Act allows for the future repeal of the prohibition on the creation of embryos for research. Likewise, the recently enacted Canadian legislation states that a Parliamentary review of the law is required within three years of proclamation.
Some countries, such as Israel, New Zealand and Russia, have even adopted time-limited legislation. The Israel legislation of 1999, for example, states that, for a period of 5 years, no intervention will be carried out on human cells for the purpose of human cloning or to bring about the creation of a person by the use of reproductive cells that have undergone permanent intentional genetic modification [19]. What is more, the Act states that the Minister of Health may (upon satisfaction of a number of conditions) permit the creation of a human being through the use of genetically modified cells. Non-legislative bans are often time-limited or chosen because of the ease with which they can be reconsidered.
Other countries are in the process of considering a revision to their existing law. Ireland has set up a Commission on Assisted Human Reproduction in 2000 to explore this topic [20] and the Irish government has officially stated its opposition to cloning [21]. There are voices calling for revision of the Germany legislation [22]. The recently passed Italian legislation might have to be reconsidered because a referendum on a disputed law can be forced if 500,000 signatures are obtained and it has been reported that over a million people have signed a petition calling for a referendum [23]. The Swedish legislation might well be amended in the near future, to close the gaps mentioned in the last section. If the Government Bill 2003/04:148 on stem cell research is enacted, it will come into force on the 1st of January 2005. This Bill seeks to extend the existing legislation to make it clear that RC using the somatic nuclear transfer is encompassed and to explicitly allow somatic nuclear transfer as a way of creating embryos for non-reproductive purposes. Thus, Sweden is likely to join Belgium and the UK in permitting NRC by legislation.
International initiatives
i) European Convention
The European Convention on Human Rights and Biomedicine has now been signed by 31 of the 45 member States of the Council of Europe, of which 15 have also ratified the Convention [24]. It has not been signed by any of the non-member participants (which include Australia, Canada, the Holy See, Mexico, and the US). While this Convention does not specifically address human cloning, a number of its provisions have implications for cloning.
Article 18(2) of the Convention prohibits the "creation of human embryos for research purposes". The phrase "human embryos" is not defined by the Convention and subsequent negotiations of the working party on the protection of the human embryo and fetus appear to have failed to reach agreement on this and other issues. This provision only prohibits NRC if it captures the creation of all functional human embryos for research. While the Convention makes provision for referrals of questions of interpretation to the European Court of Human Rights (Article 29), referral is unlikely because the Convention arguably leaves such decisions to the discretion of individual States. The Strasbourg court itself allows individual States a wide discretion (known as the 'margin of appreciation') in controversial policy areas. The court has, for example, adopted this approach when considering whether the fetus is included in the provision of the European Convention on Human Rights and Fundamental Freedoms that grants 'everyone' a right to life (see the latest case: Vo v France (no. 53924/00)). Moreover, before signing or ratifying the Convention on Human Rights and Biomedicine, any State could make a reservation to this provision insofar as it is inconsistent with their pre-existing law (Article 36). This is what we would expect the UK to do if it eventually signs the Convention.
Whether RC is implicitly prohibited by the Convention is more controversial. Those who hold that cloning violates human dignity will no doubt point to Article 1, which requires parties to the Convention to "protect the dignity and identity of all human beings". This seems tenuous. There is, however, an Additional Protocol on the Prohibition of Cloning Human Beings [25]. Article 1 of the Additional Protocol declares that,
Any intervention seeking to create a human being genetically identical to another human being, whether living or dead, is prohibited.
Since "genetically identical" is defined, under Article 1(2), as "sharing with another the same nuclear gene set", use of the Dolly technique on humans is included within this prohibition. This provision clearly captures RC. What is more controversial is whether it covers NRC. To foreclose this possibility, when the Dutch government signed the Protocol it added an interpretative statement declaring that it "interprets the term "human beings" as referring exclusively to a human individual, ie a human being who has been born". This interpretative statement is arguably unnecessary, because, in the absence of a definition of human being in the Convention itself, States are free to interpret this provision in accordance with their own national policy.
The impact of these international instruments is particularly important with regard to the three countries that have ratified both: Portugal (which has no legislation), Greece (whose legislation only explicitly prohibits RC), and Spain (which has comprehensive legislation in this area). In Greece, the Explanatory Memorandum to the legislation declares that ' [i]t could be...that therapeutic cloning is permitted exactly as in Article 1 paragraph 1 of the Additional Protocol on Cloning'. We understand that conservative opinion is of the view that this interpretation is in conflict with Article 18(2) of the Convention itself. However, neither the Convention nor the Protocol on Cloning provide any sanctions for violation.
ii) United Nations
The United Nations' struggle to agree on a cloning treaty exemplifies both the variation of approaches and the challenges associated with seeking consensus in a morally contested area [26]. In December 2001, the UN General Assembly established an Ad Hoc Committee to consider "the elaboration of an international convention against the reproductive cloning of human beings" [27]. Since that time, a number of treaty proposals have been considered. A proposal by France and Germany, for example, recommended a narrow ban on RC only, leaving NRC for future debate [28]. A second proposal supported by Spain and the US, argued for a comprehensive ban on cloning, including NRC [29]. The most recent proposal, which was put forward by Costa Rica, would require states to establish criminal offences for all human cloning, including NRC [30].
There has, however, been little consensus on how to proceed. Though all countries agree that RC should be banned, there is deep division regarding NRC. Neither the Ad Hoc cloning committee nor the UN's Legal Committee could reach a consensus on which proposal to support and bring before the General Assembly. In November 2003 the Legal Committee voted (80-79) to recommend a two-year deferral on a General Assembly decision – a compromise that was put forward and supported by most of the members of the Organization of the Islamic Conference. This decision was largely seen as a victory for those countries supporting a more permissive approach to cloning policy [31]. Indeed, some viewed the two year delay as an ideal opportunity for the scientific community to promote the value of NRC [32]. However, in response to pressure from those countries seeking a comprehensive ban, the General Assembly came to yet another compromise. In January 2004 the General Assembly overturned the Legal Committee's recommendation and supported a one year delay on the debate over the cloning treaty. This October, the General Assembly re-opened the debate, again with no apparent compromise from either camp [33].
The fact that the deep division at the UN is primarily about NRC reflects the lack of any consistent approach to cloning policy. For example, one would expect an emerging trend toward the banning of NRC, as suggested in the quote by Senator Morin, to be reflected in the building of consensus or, at least, a degree of flexibility at the UN General Assembly.
Ethical considerations
Few areas of regulation are as evidentially driven by ethical views as the regulation of cloning and cloning research. This is not the place for in-depth analysis of the underlying debate. Elsewhere we have both argued that existing regulatory attempts to prohibit RC have rarely been underpinned by thoughtful exposition of underlying ethical principles [5,10,34]. Policy statements frequently rely on claims that are tautologous, under specified, poorly considered, or a combination of these things. Our claim here is more modest. In this section we seek to show why attempts to understand the ethical basis of the existing law cannot focus solely on the existing regulatory outcomes. And, of course, the regulatory outcome does not, necessarily, represent a jurisdictional consensus on the central ethical issues.
Lawyers rarely look to regulatory outcome to understand ethical debates. Unfortunately, in the area of cloning many commentators do that very thing. As our starting quotation demonstrates, politicians and commentators are all too ready to find support for their ethical views in regulatory positions adopted elsewhere. There are, however, varying levels of ethical agreement. Agreement on the appropriate regulatory position does not imply agreement on the underlying ethical principles.
Consider the relationship between ethical positions on the moral status (or dignity) of the cloned embryo and NRC. The cloned embryo could be considered to have full, no, or limited moral status [18,35]. The full status position would grant the embryo the same level of moral duties as you or I. The no status position would grant the embryo no more status or dignity than your hair or nails. The limited status position would grant the embryo a fixed or gradualist level of intrinsic moral value between these two extremes. The full status position will require the prohibition of NRC (destructive use of embryos is considered murder) and the no status position will usually require NRC to be permitted (unless such an approach will interfere with the moral interests of those who do matter). The limited status position is, however, potentially compatible with either regulatory position, depending on the particular status given to the early embryo and the weight given to potential benefits of NRC. It follows that the fact that the regulatory position permits or prohibits NRC does not do even tell us what status the embryo is considered to have. This is further complicated by the fact that supporters of the no or limited status position might be prepared to accept more restrictions than are strictly required by their position to protect a more important moral goal. Only supporters of the full status position cannot coherently make such pragmatic compromises [18].
Similarly, the existing positions on RC could be supported by radically different ethical views. Prohibitions could be supported by those who hold that RC is absolutely wrong (eg always violates human dignity) and by those who hold that cloning at present would be wrong. Even time-limited prohibition does not enable us to discern whether it is underpinned by, for example, the view that RC is wrong because of current safety issues or the view that RC is not wrong but the most effective way to get there is by initially prohibiting it.
In sum, the majority of regulatory outcomes could be coherently explained by reference to one or more underlying ethical positions. Thus, similar or even identical regulatory outcomes imply less by way of ethical agreement than some may be inclined to believe.
Conclusion
Cloning laws and policies are far from uniform across the globe and the legal position in some countries remains uncertain. This will give little comfort to scientists and policy makers hoping to gain clear direction from the international position. For the time being at least, policymakers must accept the reality of international "dissensus" and scientists wishing to undertaken research on NRC are best advised to consider conducting their research in only a handful of countries. Even where there is agreement as to the regulatory outcome, policy-makers should not confuse this with agreement on underlying ethical principles. Like many topics concerning the developing genetic and reproductive technologies, cloning remains controversial.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contribution
Both authors contributed to the original concept for the paper, the writing and revision of the manuscript and the analysis of the law.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
Professor Caulfield is a Canada Research Chair in Health Law and Policy and would like to thank the Alberta Heritage Foundation for Medical Research, Genome Prairie and the Stem Cell Network for research support. We would like to thank those who provided information on the laws of other jurisdictions, in particular, Tina Garanis-Papadatos (Greece), Salla Lötjönen (Finland), Deirdre Madden (Ireland), André Pereira (Portugal), and Elisabeth Rynning (Sweden). Finally, we would like to thank the reviewers for their useful comments.
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| 15596013 | PMC544897 | CC BY | 2021-01-04 16:31:54 | no | BMC Med Ethics. 2004 Dec 13; 5:9 | utf-8 | BMC Med Ethics | 2,004 | 10.1186/1472-6939-5-9 | oa_comm |
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BMC Med Inform Decis MakBMC Medical Informatics and Decision Making1472-6947BioMed Central London 1472-6947-4-191553325710.1186/1472-6947-4-19DebateCase-based medical informatics Pantazi Stefan V [email protected] José F [email protected] Jochen R [email protected] School of Health Information Science, University of Victoria, BC, Canada2 Department of Health Studies and Gerontology, University of Waterloo, Ont., Canada2004 8 11 2004 4 19 19 17 10 2003 8 11 2004 Copyright © 2004 Pantazi 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 "applied" nature distinguishes applied sciences from theoretical sciences. To emphasize this distinction, we begin with a general, meta-level overview of the scientific endeavor. We introduce the knowledge spectrum and four interconnected modalities of knowledge. In addition to the traditional differentiation between implicit and explicit knowledge we outline the concepts of general and individual knowledge. We connect general knowledge with the "frame problem," a fundamental issue of artificial intelligence, and individual knowledge with another important paradigm of artificial intelligence, case-based reasoning, a method of individual knowledge processing that aims at solving new problems based on the solutions to similar past problems.
We outline the fundamental differences between Medical Informatics and theoretical sciences and propose that Medical Informatics research should advance individual knowledge processing (case-based reasoning) and that natural language processing research is an important step towards this goal that may have ethical implications for patient-centered health medicine.
Discussion
We focus on fundamental aspects of decision-making, which connect human expertise with individual knowledge processing. We continue with a knowledge spectrum perspective on biomedical knowledge and conclude that case-based reasoning is the paradigm that can advance towards personalized healthcare and that can enable the education of patients and providers.
We center the discussion on formal methods of knowledge representation around the frame problem. We propose a context-dependent view on the notion of "meaning" and advocate the need for case-based reasoning research and natural language processing. In the context of memory based knowledge processing, pattern recognition, comparison and analogy-making, we conclude that while humans seem to naturally support the case-based reasoning paradigm (memory of past experiences of problem-solving and powerful case matching mechanisms), technical solutions are challenging.
Finally, we discuss the major challenges for a technical solution: case record comprehensiveness, organization of information on similarity principles, development of pattern recognition and solving ethical issues.
Summary
Medical Informatics is an applied science that should be committed to advancing patient-centered medicine through individual knowledge processing. Case-based reasoning is the technical solution that enables a continuous individual knowledge processing and could be applied providing that challenges and ethical issues arising are addressed appropriately.
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Background
A meta-level view of science
Our aim is to place Medical Informatics in the context of other sciences and to bring coherence in its formal education [1]. This will necessarily place the discussion at a meta-level view of science, which traditionally was the concern of philosophers. From such a general perspective, science could be defined as "the business of eliciting theories from observations in a certain context, with the hope that those theories will help to understand, predict and solve problems." Also revolving around the "business of creating theories," R. Solomonoff's ideas [2], summarized in [3], contribute to the basis of Algorithmic Information Theory (AIT) [4], a relatively new area of research initiated by A. Kolmogorov, R. Solomonoff and G. Chaitin, and regarded as the unification of Computer Science and Information Theory. According to Solomonoff's view, a scientist's theories are compressions of her observations (i.e., her experimental data). These compressions are used to explain, communicate and manage observations efficiently and, if valid, to help solving problems, understanding and predicting. Intuitively, the higher the compression achieved by the theory, the more "elegant" that theory and the higher its chances of acceptance. This very general perspective of the scientific endeavor also makes science to appear twofold: it comprises the creation of theories (i.e., theory elicitation) as well as their subsequent use in understanding, predicting and solving problems (i.e., theory application). Therefore, science seems to be driven by two opposite forces: that of creating theories, and that of applying those theories to practical applications.
The four-dimensional space-time continuum we live in (i.e., our universe) forms the reality (i.e., the context) of all scientific observations. The compression of the immense complexity and dynamicity of this reality in concise "theories of everything" was already demonstrated by Zuse [5,6] and recently Schmidhuber [7]. These results of theoretical computer science demonstrate the power of human theory elicitation and provide important answers to old questions of science and philosophy. However, their unfeasibility when applied to practical problems, which would be equal to building computing devices capable of running precise simulations of our reality, also widens the gap between theoretical research and practical sciences. For the time being, humanity still needs to divide science and define human knowledge as a collection of individual theories elicited from scientific observations. The immense number of theories that comprise the collective human knowledge about every possible subject, as well as its extraordinary dynamics, have forced us to divide it into what we commonly refer to as knowledge domains, thereby reducing the contexts of our observations to smaller space-time continuums. The attempts to process with computers the knowledge in a domain have taught us that we need to recognize the reality of the "knowledge acquisition bottleneck" [8] and to not underestimate the importance of common-sense knowledge (see [9] and [10-13]). The particularities with regard to the context retention, acquisition, representation, transferability and applicability of domain knowledge, causes us to distinguish between different modalities of domain knowledge, and place them on what we refer to as the knowledge spectrum.
The knowledge spectrum
The knowledge spectrum (Figure 1) spans from a complex reality (the source of experimental data and information gathered from observations and measurements) to high-level abstractions (e.g., theories, hypotheses, beliefs, concepts, formulae etc). Therefore, it comprises increasingly lean modalities of knowledge and knowledge representations media and the relative boundaries and relationships between them. Two forces manifest on the knowledge spectrum: that of creating abstractions and that of instantiating abstractions for practical applications. The former is the theory elicitation and is synonymous to processes of context reduction and knowledge decomposition. The latter, theory application, equates to context increase and knowledge composition processes. The engines behind the two knowledge spectrum forces are the knowledge processors, natural or artificial entities able to create abstractions from data and to instantiate abstractions in order to fit reality.
Knowledge is traditionally categorized into implicit and explicit (Table 1) and ranges from rich representations grounded in a reality, to highly abstracted, symbolic representations of that reality. The classical distinction between data, meta-data, information, knowledge and meta-knowledge is simplified by our subscription to the unified view of Algorithmic Information Theory (AIT) [4] which recasts all knowledge modalities and their processing into a general framework requiring a Universal Turing Machine, its programs and data represented as finite binary sequences. From this perspective a precise distinction between these modalities becomes unimportant.
Implicit knowledge (U, from unobvious, unapparent) is the rich, experiential, sensorial kind of knowledge that a knowledge processor acquires when immersed into an environment (i.e., grounded in an environment), or presented with detailed representations of that environment (e.g., images, models, recordings, simulations). It is very well applicable to specific instances of problems and relies on processing mechanisms such as feature selection, pattern recognition and associative memory.
Explicit knowledge (E) is the abstract, symbolic type of knowledge present explicitly in documentations of knowledge such as textbooks or guidelines. It requires a representation language and the capability of a knowledge processor to construe the meaning of concepts of that language. It is applicable to both specific and generic problems and relies on explicit reasoning mechanisms.
The distinction between implicit and explicit knowledge are useful to characterize the nature of human expertise, but become problematic when one wants to describe fundamental differences between theoretical and applied sciences: many applied sciences, especially knowledge intensive ones, in addition to general theories of problem solving, also make use of explicit knowledge in order to describe, with various degrees of precision, particular instances of problem solving and theory application. This represents the rationale for further dividing the knowledge spectrum into general and individual knowledge (Table 2).
General knowledge
General knowledge (G) is the explicit, abstract, propositional type of knowledge (e.g., guidelines), well applicable to context-independent, generic problems. However, it is more difficult to use in specific contexts because of the gap between the general knowledge itself and a particular application context. This knowledge gap translates into uncertainty when a general knowledge fact is instantiated to a specific situation. For example, knowing generally that a certain drug may give allergic reactions but being uncertain whether a particular patient may or may not develop any, is an example of what we consider the uncertainty associated with general knowledge. The creation of general knowledge (i.e., abstraction, generalization, context reduction, theory elicitation) is a relevance-driven process done by "stripping away irrelevancies" [9]. This causes general knowledge to have a lower complexity and be more manageable: "generalization is saying less and less about more and more" [9].
Formal representations of explicit knowledge have been common in early artificial intelligence (AI) applications in the context of expert system development. They operated under the "closed world assumption" and were meant to make the representation of knowledge manageable, reproducible and clear. However this assumption also rendered the expert systems "brittle" or completely unusable when applied to real world problems [14]. The completeness necessary for automatic reasoning using explicit reasoning mechanisms can be illustrated with the following formal definition of the concept of "a brick" in a limited, hypothetical world, containing only simple geometric objects such as bricks and pyramids (Figure 2) (adapted from [15]): "being a brick implies three things:
1. first, that the brick is on something that is not a pyramid;
2. second, that there is nothing that the brick is on and that is on the brick as well; and
3. third, that there is nothing that is not a brick and the same thing as the brick."
This definition could have the predicate calculus representation:
This representation shows that an intelligent agent who has no implicit knowledge of the hypothetical physical world and no capacity of generalization or analogy making, must be explicitly provided with all knowledge necessary to reason about "bricks" in that limited reality. Such approaches are known to suffer from a fundamental shortcoming, the "frame problem."
The frame problem
Daniel Dennett was the first philosopher of science who clearly articulated the "frame problem" and promoted it as one of the central problems of artificial intelligence [16] (also see [17]). Janlert [18] identifies the frame problem with "the problem of representing change." In [14] the frame problem is defined as "the problem of representing and reasoning about the side effects and implicit changes in a world description." In order to articulate and circumvent the abstract nature of its definition, Dennett has invented a little story involving three generations of increasingly sophisticated robots. These fictitious robots are products of early artificial intelligence (AI) technology that use automated reasoning based on formal representations similar to the brick example. These particular robots are specifically designed to solve a problem consisting of the retrieval of their life-essential batteries from a room, under the threat of a ticking bomb set to go off soon. Although increasingly sophisticated in their reasoning, all three successive versions of the robot fail:
• The first robot fails by missing a highly relevant side effect of pulling the wagon with the batteries out of the room: the ticking bomb sitting on the same wagon was also retrieved, together with the batteries.
• The second robot did not finish its extensive, irrelevant side-effect reasoning procedures before the bomb goes off. As Dennett ironically puts it, the robot "had just finished deducing that pulling the wagon out of the room would not change the color of the room's walls and was embarking on a proof of the further implication that pulling the wagon out would cause its wheels to turn more revolutions than there were wheels on the wagon – when the bomb exploded."
• The third robot failed because it was "busily (i.e., explicitly) ignoring some thousands of implications it has determined to be irrelevant" and its batteries were therefore lost in the inevitable explosion.
The frame problem can therefore be recast as a problem of relevance [17](see preface), which is compounded by time constraints. It demonstrates that relevance judgment mechanisms based on general knowledge are time consuming and cause the failure to solve time-constrained decision problems. It is a problem only because in the real world we do have time constraints.
Individual knowledge
Individual knowledge (I) or instance specific knowledge, on the other hand, is a knowledge modality very well applicable to real problems, because it identifies uniquely and matches precisely an application context. The knowledge gap and uncertainty are reduced but still exist because of our changing reality (time dimension) which may render individual knowledge about a patient collected in the past (e.g., value of blood pressure from a month ago), less applicable in the present or future. Because it preserves context (i.e., it is more grounded), individual knowledge has a higher complexity than general knowledge and hence is more difficult to manage (i.e., has high memory requirements). For example, knowing the drugs and the precise description (e.g., numeric, textual, visual) of the allergic reactions that they caused in a certain person, as well as many other particular knowledge facts about individual, is what we call individual knowledge. The uncertainty and knowledge gap related to the application of such knowledge to future instances of decision making involving that individual are reduced: individual knowledge is supposed to fit very well the application context where it was originally captured.
Case-based reasoning
Individual knowledge captured from a very specific context (e.g., diagnosing a particular patient with a particular disease) can be extrapolated to similar contexts. The higher the similarity between contexts, the smaller the knowledge gap and instantiation uncertainty and the higher the chances for a successful solution to a new problem. For this reason, individual knowledge processing has become increasingly important for artificial intelligence applications and is defined as the approach to solving new problems based on the solutions of similar past problems [14,19-21]. It has several flavors (e.g., exemplar-based, instance-based, memory-based, analogy-based) [21] which we will refer to in this paper interchangeably, through the generic term of "case-based reasoning" (CBR).
There are four steps (the four "RE") that a case-based reasoner must perform [14,20,21]:
1. RETRIEVE: the retrieval from memory of the cases which are appropriate for the problem at hand; this task involves processes of analogy-making or case pattern matching;
2. REUSE: the decomposition of the retrieved cases in order to make them applicable to the problem at hand;
3. REVISE: the compositional adaptation and application of the knowledge encoded in the retrieved cases to the new problem; and
4. RETAIN: the addition of the current problem together with its resolution to the case base, for future use.
CBR entails that an expert system has a rich collection of past problem-solving cases stored together with their resolutions. CBR also hinges on a proper management of the case base and on appropriate mechanisms for the matching, retrieval and adaptation of the knowledge stored in the cases relevant to a new problem. Ideally, the individual knowledge in a case-base will progress asymptotically towards an exhaustive knowledge base, which represents the "holy grail" of knowledge engineers. From a learning systems point of view, similarly to artificial neural networks [22,23] and inductive inference systems [24] that learn from training examples, a CBR system acquires new knowledge, stores it in a case base and makes use of it in new problem solving situations.
The absolute positions and shapes of boundaries between the four knowledge modalities, although admittedly not as precise as drawn on the knowledge spectrum in Figure 1, are not of importance for this discussion. However, the relative relationships between knowledge modalities are, and can be represented formally as a Venn diagram (Figure 3), which implies that:
• Individual knowledge has a higher complexity than the explicit knowledge elicited from the same context. This is equivalent to stating that, for example, the picture of a person encodes more knowledge than the textual description of that person's appearance.
• Implicit knowledge is a subset of the individual knowledge.
• General knowledge is a subset of the explicit knowledge.
• The set of individual knowledge represented explicitly formed by the intersection of individual knowledge with explicit knowledge is a nonempty set. This is equivalent to stating that it is possible, for example, for an explicit textual description to identify a context uniquely (e.g., the complete name and address of a person at a specified moment in time).
A meta-level view of Medical Informatics
The meta-level overview of sciences and the definitions and properties of the knowledge spectrum and knowledge modalities enable us to draw some fundamental differences between theoretical sciences and applied sciences such as Medicine [25] and Medical Informatics. From this perspective, theoretical sciences (e.g., theoretical computer science):
• Make use of observations which are highly abstract symbolisms and create far more limited contexts of application of their theories, when compared to the complexity of the human body or of any social or biological system,
• Have as a primary purpose the creation of general knowledge comprising valid, powerful theories which explain precisely and completely the observations, and therefore,
• Include a relatively limited number of precise theories which are evaluated primarily by their power of explaining experimental observations, elegance, generality, and
• Are less concerned with the acquisition of the individual knowledge required by the practical implementation and by the application of results to real world problems.
Applied sciences such as Medicine and Medical Informatics, on the other hand:
• Gather extensively data and observations (individual knowledge) from very complex systems [9,26] (e.g., human body), which are characterized by high individual variation and randomness;
• Have as a primary purpose not only distilling data and observations into general knowledge, but are also concerned with the implementation details and with the application of theories to individual problem solving (e.g., diagnosis and treatment of real patients),
• May lack the incentive to refine existing theories which are objectively wrong as long as practical success is achieved [25],
• Contain very few simple, "elegant" theories (general knowledge) that can solve individual problems completely or explain and predict accurately [27] because of the complexity of the human body and its individual variation and, therefore,
• May pursue the application of a multitude of mutually contradictory, poorly grounded, general theories (e.g., the general theory of medical reasoning and the concepts of "diagnosis" and "symptom") [1,25],
• Abound in general theories (e.g., guidelines) which are "lossy" (i.e., ignore individual context variation) and which are evaluated statistically by their practical success relative to existing ones (e.g., cancer therapy),
• Attempt to make up for the knowledge gap between general knowledge and the reality where knowledge is applied, by employing experienced clinicians who require extensive training and information technology (e.g., decision support), and, in addition,
• Are compounded by time-constrained circumstances and largely unsolved ethical issues (e.g., privacy and confidentiality, genomics research).
Given the special circumstances of our applied science in the context of other sciences and the increasing recognition of the importance of knowledge processing to Medical Informatics [28], we propose, as part of the thesis of this paper, that Medical Informatics should complement the traditional quest for general biomedical knowledge with the advance of acquisition, storage, communication and use of individual knowledge. By doing so, Medical Informatics will provide a solution to the problems that arise during the use of general knowledge and, in the same time, will enable clinical research as well as advanced decision support and education of both healthcare providers and patients.
Individual knowledge processing equates to a case-based reasoning (CBR) approach that employs collections of patient cases. Currently, such collections are the focus of research on Electronic Health Records (EHR). Envisioned as "womb to tomb" collections of patient-specific data, EHR contain a wealth of data that could be used to support case-based decisions. If EHR are to be used in a CBR context, the issues pertinent to the design of case-bases automatically become pertinent to the EHR design, and the CBR paradigm becomes important to Medical Informatics.
The overall knowledge processing capacity of healthcare systems can be thought to be distributed between two sources: human resources (i.e., healthcare professionals) and information technology (Medical Informatics). An ideal CBR approach would increase this knowledge processing capacity by allowing for the automatic processing (acquisition, representation, storage, retrieval and use) of individual knowledge present in increasingly rich knowledge media such as natural language artifacts, images, videos and computer simulations of reality (Figure 4). The storage and communication of knowledge are well advanced by current information technology. However, most of the acquisition, retrieval and knowledge use are, and will continue to be the task of professionals until advanced processing (e.g., real-time computer vision, scene understanding and synthesis, image understanding, robotics, natural language understanding) are applicable. Given the widespread use of natural languages as knowledge representation and communication media, it follows that natural language processing (NLP) research is a very important component of Medical Informatics, required to advance the organization and processing of individual knowledge in reusable case-bases. Further, the goal to advance processing of increasingly complex knowledge representations (e.g., natural language, sounds, images, simulations) and create intelligent machines that can hear, see, think, adapt and make decisions, brings Informatics even closer to what traditionally was the concern of Artificial Intelligence (AI).
Finally, because the knowledge processing capacity of human resources tends to remain relatively constant, moving towards the ideal of individual knowledge processing, no matter how slowly, may also have ethical implications because it proves that medical informaticians are trying to do everything they can in order to serve the interest of the individual.
Discussion
In order to support our thesis, the following discussion will focus initially on fundamental aspects of medical decision-making and biomedical knowledge creation from the standpoint of the knowledge spectrum. This will lead to a discussion of fundamental knowledge representation and processing principles and the proposal of a CBR perspective on EHR, including challenges and potential solutions.
Human and computer knowledge processing
Decision making in medicine
Medicine is a knowledge intensive domain where time-constrained decisions based on uncertain observations are commonplace. In order to successfully cope with such situations, health professionals go through a tedious learning process in which they gain the necessary domain knowledge to evolve from novices to experts. As experts, health professionals have attained, among other things, two important, highly interrelated abilities:
• To be able to reduce knowledge complexity by determining efficiently what is relevant for solving a problem in a particular situation, and,
• To be able to reduce the knowledge gap between knowledge facts and reality which translates into being able to reduce the uncertainty of knowledge instantiation to a particular context.
For example, both the presence and the absence of a past appendectomy are relevant and contribute (potentially unequally) to reducing the uncertainty of instantiation of the biomedical knowledge of an expert to a particular context of a patient with right lower abdominal pain. Fundamental to decision making, relevance judgments and uncertainty reduction seem both closely connected with the quality and quantity of knowledge available for solving a problem as well as with the nature of knowledge processing mechanisms. Studies of expert-novice differences in medicine [29] have shown that the key difference between novices and experts is the highly organized knowledge structures of the latter, and not the explicit strategies or algorithms they use to solve a problem. This is supported by expert system development experiences which showed that a system's power lies in the domain knowledge rather than in the sophistication of the reasoning strategies [14]. Studies of predictive measures of students' performance indicate that tests which measure the acquisition of domain knowledge are the best predictors [30]. The work on naturalistic decision-making (NDM) and the development of psychological models of "recognitional decision-making" such as the Recognition-Primed Decision (RPD) [31-33], suggest the heavy dependence of decision makers on their previous experience of problem-solving and also on their ability to perform mental simulations.
The discussion around the amount of problem solving experience of a decision maker becomes critical in time-constrained decision circumstances. The exhaustiveness of the knowledge base and the efficiency of retrieval mechanisms now become paramount to the decision speed. Empirical evidence that shows the existence of "systematic changes of cognitive processes" related to time stress, comes from the studies on the psychology of decision-making under time constraints [34]. Although most of these studies attest the overall negative effect of time stress on the "effectiveness of decision-making processes" [35], others [31,33] argue that even extremely time-constrained situations could be handled successfully by human subjects, given enough expertise (i.e., enough problem solving experience).
Since humans are able to make sound relevance judgments and reduce instantiation uncertainty of knowledge most of the times, the following questions arise: What is their strategy for increasing the exhaustiveness of their knowledge base while managing its exponential complexity? How do they represent and organize their knowledge and how do they manage time-constrained situations? At least some of these questions have been under intense scrutiny that has resulted in important empirical work on naturalistic decision-making [32,33,36,37]. Important insights have been gained at the individual but also at the organizational and social levels. Coherent with the importance of the social aspects of decision-making, Armstrong [38] builds an interesting argument about the Darwinian evolution, social networking and the drive for knowledge discovery of the humanity as being some of the reasons that contribute to the human decision making potential.
From the perspective of the knowledge spectrum, it seems reasonable to associate expert decision makers with individual knowledge and novices with the more abstract general knowledge about a subject, available in explicit knowledge artifacts (e.g., textbooks, guidelines). It is also conceivable that mental models of experts span a great length of the knowledge spectrum, causing them to efficiently perform implicit processing (feature selection, pattern recognition, associative recall) and also just-in-time explicit reasoning (Figure 5). The ability to move freely across the knowledge spectrum causes experts to efficiently reduce data to abstractions and to create hypotheses and micro-theories through sound relevance judgments. The powerful mental simulations that experts can perform allow them to construe appropriate meanings of concepts and to verify their hypotheses against contexts of reality.
Novices, on the other hand, have limited mental models of reality situated towards the abstract region of the spectrum. This causes them to have difficulties with construing appropriate meanings of concepts due to the increased knowledge gaps between their mental models and reality. Novices are therefore unable to make sound relevance judgments and limited in their ability of interpreting data and creating abstractions. They are also usually overwhelmed by the explicit, general knowledge present in textbooks and guidelines and unable to fully construe the meanings of concepts present in such knowledge artifacts.
In conclusion, in information and knowledge intensive domains such as medicine, explicit reasoning is important but individual knowledge acquisition (i.e., experience) and processing (i.e., CBR) are crucial for decision-making. Because the nature of expertise seems largely connected with individual knowledge processing, it follows that the evolution of novices into experts is unattainable only by the provision of extensive general knowledge. In addition, not only the individual learning but also the collective sharing of experiences (e.g., case records, personal stories, etc.) between individuals and between generations, contribute to the way humans deal with decision problems.
Patient-centered vs. population-centered healthcare
The major driving force of science is universally applicable knowledge (i.e., general knowledge). While creating and communicating new knowledge, scientists move across the knowledge spectrum from the data that captures the reality of their experiments and observations towards abstract representations that allow them to communicate their theories. In biomedical research, such an example is the randomized controlled trial (RCT), currently regarded as the gold standard for knowledge creation. The correct design of an RCT is crucial for the validity of the medical evidence obtained. A correct randomization process in RCTs will limit the bias and increase the chance for applicability of the evidence obtained, to a specifically selected group of patients (e.g., "women aged 40–49 without family history of breast cancer"). However, at the same time, the randomization process removes the circumstances of individual cases and creates a knowledge gap between the RCT evidence and future application instances. As with any statistical approach, the RCT-based evidence is best applicable at the population level rather than at the individual level.
This depersonalization of medical knowledge and evidence was also noted by others [39,40] and could also be illustrated by the observation that most patients feel relieved when told that the chances of being successfully treated for a certain condition are 99%, for example. Although this is psychologically very positive, the patients should not necessarily be relieved, as they could very well happen to fall among the 1%, for whom things could go wrong and for whom, usually, the RCT-based evidence does not provide additional information. An experienced physician and, from a CBR perspective, a highly efficient case-based reasoner, is most of the times able to individualize the medical decision for a particular patient for whom things are likely to go wrong and fill in the knowledge gap between the RCT evidence and the medical problem at hand. This could lead to avoiding a therapeutic procedure recommended by the medical evidence. The individual knowledge that this decision is based on is usually not provided by the RCT, but is acquired through a tedious process of training. This decision is often so complex that it cannot be easily explained as it becomes heuristic in nature and is motivated by the individual knowledge that a decision maker possesses.
Others [41] have also pointed out that when physicians manage their cases (e.g., diagnosis and treatment), their previous experience allows them to make informed decisions based on heuristics rather than on a sound, complete and reproducible reasoning, such as logical inference based on a predicate calculus representation of a problem. In addition, human experts often disregard probabilistic, RCT-type of evidence and consistently detach themselves from the normative models of classical decision theory (e.g. probability theory, Bayes theory) in favor of heuristics-based approaches. Although prone to occasional failures, heuristics-based decisions are much more efficient in time-constrained and uncertain situations [33].
From the perspective of the knowledge spectrum, the driving forces of Health Informatics and RCT methodology seem to have opposite directions: while Informatics aims towards individual knowledge and personalized health care, the general knowledge gained through populational studies (e.g., RCTs) targets the ideal of universal applicability (Figure 6). The value of a single bit of data (e.g., a Yes/No answer to a specific question such as a past appendectomy) can be very relevant in a decision-making context if it reduces the overall uncertainty of knowledge. However, such individual bits of data are inevitably lost during the creation of general knowledge.
Rigorously and expensively collected, general, populational level knowledge is useful only in situations where individual knowledge lacks (e.g., new drugs), providing the decision makers have access to it and are able to apply it to specific situations. However, general knowledge is unlikely to be used as such in many naturalistic decision-making processes, because it does not support the way expert decision makers think. The knowledge gap and inherent instantiation uncertainty manifested in the application of general knowledge does not fully enable the education of providers and patients which would require additional knowledge about individual contexts of successful or unsuccessful application instances. Informatics, on the other hand, by advancing individual knowledge processing, provides an alternative solution to the problems that arise from the use of general knowledge that targets universal applicability. An integral part of individual knowledge, genomic data is already recognized [39,40]as being of extreme importance for a solution to the problems of general knowledge.
Knowledge representation by formal methods
The application of formal knowledge representations to real problems suffers from a fundamental shortcoming: the frame problem. As explained above (see "The frame problem"), the frame problem can be recast as a problem of relevance. Given the capability of relatively effortless human relevance judgments, the frame problem seems a rather "artificial" creation, difficult to grasp and which usually goes unnoticed. In order to circumvent its abstract nature, Dennett uses a story-telling approach. However, the frame problem also applies to and could be illustrated from the perspective of humans, who in their first years of life, learn and can easily and efficiently reason about the side effects and the implicit changes of the complex four-dimensional spatio-temporal physical world in which they live. As this learning gradually becomes common sense knowledge, it causes us to efficiently determine the relevant implicit changes while ignoring the non-relevant ones for a given situation. For example, such facts as that the clothes we are wearing are moving with us while walking or traveling are most of the times irrelevant given the context of a planned trip. However, if the trip involves some rapid movement through the air such as riding a motorbike, suddenly wearing a sombrero becomes a relevant fact. As experts at managing our physical world, we are able, through an effortless but powerful mental simulation, to determine the relevance of such a particular fact. The recall of our personal experiences of moving fast through the air and of the dragging force of the air becomes paramount. Therefore, intelligent agent must be endowed with efficient mechanisms for determining the relevance of particular facts for a decision.
We suggest that what made the robots vulnerable was their creators' choice for knowledge representation and reasoning: the robots did not have quick access to implicit knowledge about the relevance of particular facts (i.e., records of problem solving instances) but only to explicit facts in frames which had to be employed in time-consuming, immense number of explicit relevance judgments about the effects of particular actions. Although they were supposed to be experts at their task, the robots were behaving like novices. The frame problem is not a problem of the knowledge representation per se, but a problem of the choice for representation of knowledge needed to solve time-constrained decisions. In other words, formal representations and logic reasoning work, but not in time constrained, complex situations.
From the perspective of our knowledge spectrum, explicit, formal representations sit on the abstract side of the spectrum (Figure 7). The retrieval of explicit knowledge representation is currently the subject of the increasingly important field of research of information retrieval (IR). It is commonly accepted that IR is strongly coupled with the notion of intended meaning of concepts: a retrieved document is considered to be relevant to a query if the intended meanings of the authors of a document are relevant to the intended meaning of that query. We propose that "meaning," a property that characterizes all concepts present in explicit knowledge, is intimately connected (if not identical) with the notion of context. According to this rather paradoxical view, meaning, a property which characterizes the abstract side of the knowledge spectrum, is strongly coupled with context which, by definition, is a feature of the reality side of the knowledge spectrum. Therefore, in order to construe meaning appropriately one needs to be able to efficiently move from abstractions towards richer representations of reality. This movement on the knowledge spectrum is necessary in order to fill the knowledge gap between abstract concepts and the richer mental representations required for construing their meaning.
Explicit, formal representations attempt to capture general truth and generally applicable problem solving strategies, but become too abstract in nature. Through the abstraction process, which is essentially a reduction driven by the relevance judgments of knowledge creators, the context of a problem is lost. Losing context creates difficulties with construing meaning (which is context-dependent by definition) and widens the knowledge gap between the representation itself and the reality of a future problem-solving instance. The knowledge gap translates into the instantiation uncertainty that characterizes the application of general knowledge to specific problems. Making up for the knowledge gap through explicit relevance reasoning becomes time consuming and consequently takes its toll on the applicability of the representation. In sensitive applications such as medical decision-making and health research, general knowledge may potentially be harmful (e.g., prescribing an highly recommended drug to which a patient has a undocumented allergy). In addition, abstractions and general methods and theories of problem solving and decision making (e.g., guidelines) do not fully enable the education of individuals and the learning from successes and mistakes.
Knowledge representation approaches must therefore preserve to the extent possible, the context of a problem-solving instance. By efficiently recalling similar past instances of problem solving and their contexts, intelligent agents are immediately provided with implicit knowledge about relevance, encoded in the retrieved contexts and, in the same time, with more possibilities to reduce the instantiation uncertainty of general knowledge when applied to specific problems. To enable this, informatics research must advance the processing of rich representations of the knowledge encoded in past problem solving cases: this is the definition of CBR research.
Knowledge representation by natural language
Similar to formal specifications (e.g., predicate calculus) natural language uses abstractions, i.e., concepts. Its richness and power of expression place it in the knowledge spectrum to the left side of formal specifications but to the right side of rich descriptions consisting of images, sounds, video-clips and simulations of reality (Figure 4). Natural language has power of expression but loose semantics and inherent ambiguity. However, despite its abstract nature, it remains the indispensable, main knowledge representation and transfer medium between humans.
In order to illustrate our point about ambiguity we direct the reader to the previous, natural language definition of the concept of "a brick." Although the definition may look unequivocal, there are subtle ambiguities that make a difference in the predicate calculus representation. The first condition of an object to be "a brick" (i.e., "the brick is on something that is not a pyramid," highlighted in the equations 2 and 3) is an ambiguous natural language construction and could have slightly different formal representations:
In (2) this condition has been interpreted as: "the brick being on something IMPLIES that that something is not a pyramid" and was therefore represented as "for all Y, if X is on Y, this implies that Y is not a pyramid." In (3), which is identical to (1) but is repeated to the benefit of the reader, this condition was interpreted as "the brick MUST BE (or is always) on something that is not a pyramid" and that was represented as "there exists Y such as X is on Y and Y is not a pyramid."
The first definition is therefore more "relaxed" as it allows the possibility that a brick sits on nothing. The second definition is more restrictive, because it requires the brick to be on something that is "not a pyramid" or otherwise X is not a brick anymore. Therefore, the first definition is more general and defines the concept of "a brick" in such a way that the definition would be true even in a world with no gravity (i.e., the brick is on nothing). In addition, definition (3) does not reject the possibility that an object sits on both another brick and a pyramid, at the same time (Figure 8).
The point is that, most often, humans receive and transmit knowledge without the deep understanding and completeness required by an exact mathematical representation of the knowledge to be transmitted. This shallowness has also been recognized by others [42] who are trying to draw natural language processing researchers' attention to the fact that humans are rather superficial in their knowledge acquisition and processing and often make use of "underspecified" representations. Although, since the early days of science, scientists have fallen in love with the pure reasoning approaches, as they were reproducible, unambiguous means to express new knowledge, the problems with the use of classical predicate calculus as a knowledge representation method and of the classical logic inference as a reasoning strategy are discouraging. This is due to the requirements of complete, unequivocal representations, which prevents them from dealing with the messiness of the real world problems.
If possessing the necessary knowledge, humans are able to effortlessly fill the knowledge gaps between natural language representations and their richer representations of reality (i.e., mental models), and to easily construe the appropriate meaning of potentially ambiguous concepts. Although current technology allows for its storage, knowledge present in richer media (e.g., images, videos, simulations) is currently very difficult to process (e.g., real-time computer vision, scene understanding and synthesis, image understanding) using today's technology.
Because natural languages are used by people universally and allow rich representations that no other language specification can attain, natural language processing (NLP) research is a first step that Informatics should take in order to advance the organization and processing of individual knowledge in case-bases that can be reused. The insights gained will advance knowledge processing towards richer knowledge representation media, will reduce the knowledge processing gap and consequently increase the knowledge processing capacity currently supported largely by human knowledge processors.
Memory-based knowledge processing
One of the main features of information processing systems is their memory. It is accepted that storage and manipulation of information are necessary for complex cognitive activities in humans [43]. Memory is also considered crucial for both the "situation recognition" and mental modeling processes which are part of naturalistic decision models [33].
From a computational point of view, one could easily argue that without a random access memory structure there can be no effective processing. In the context of "the computational architecture of creativity," this argument is clearly outlined in [44]. It is based on the examination of the classes of computational devices, in the ascending order of their computational power, ranging from finite-state machines to pushdown automata and linear automata. These are paralleled by their corresponding grammars, arranged similarly in the Chomsky hierarchy, consisting of regular grammars, context-free grammars, context-sensitive grammars and of the unrestricted transformational grammars for machines with random access memory [44].
Recent natural language processing (NLP) research stresses the importance of memorization of individual natural language examples [45]. The importance of memory is also emphasized in earlier [46] and more recent models of language processing in humans [47-49]. These converge on the idea that natural language processing, regardless of the processor, is memory-based (i.e., case-based). Additional evidence comes from the fact that most language constructs (e.g. words, phrases) have very low frequencies. In fact, the very low frequency of most words in the English language (i.e., Zipf's law) is known from the 1940s since Zipf's famous book "Human Behavior and the Principle of Least Effort" [50] which is discussed in [51]. The main implication of "Zipf's law" is that purely statistical approaches or language processing algorithms that do not memorize training examples will either lose important information or may need extensive data (potentially impossible to collect) in order to be able to retain important features which have extremely low frequencies [52] and which may be crucial for construing the appropriate meanings of a language's concepts.
The tradeoff between learning effort and communication efficiency seems to be biased naturally towards memorization rather than towards logical reasoning. The processing complexity of natural language might therefore not be an intrinsic quality of the algorithms, but rather a function of the memorization capabilities of the language processor, given the sparseness of natural language pattern space. By analogy, the advanced knowledge processing in humans might not be the result of very sophisticated reasoning strategies, but rather the utilization of a limited reasoning apparatus on a huge knowledge base, consisting of rich representations of one's experience. The limitations in reasoning are balanced by complex spatio-temporal pattern recognition capabilities operating on a case base built from years of experience. This case base includes common-sense knowledge.
Furthermore, people and computers memorize information differently. Both have a short term, working memory and long-term memory for storing data and information. However, the memory access is carried out in different ways. Computers can reliably store large streams of data, which most of the times have a very well defined spatial and temporal structure (e.g., a movie clip). In contrast, people can only store information and knowledge rather than data and their storage is unreliable, temporally fragmented and spatially incomplete. Computers have very reliable memories capable of error checking at the bit level while the human memory supports only a high-level semantic consistency check. Finally, computers access their memory in a random seek fashion, being able to position their "reading heads" at any position in the data streams in order to extract a certain block of data. People can access their memory by content, by being provided with an incomplete description of a potentially complex, spatio-temporal pattern serving as a retrieval key. Therefore, one of the main differences between computers and humans is that computers have address-based random access memories, while humans possess content-addressable memories.
In conclusion, from a case-based reasoning perspective, humans seem to be naturally endowed with the necessary structures for efficient case base acquisition, organization and retrieval while computers do not directly support this way of processing information and knowledge.
Pattern recognition, comparison and analogy-making
Pattern recognition is an undisputed feature of human cognitive abilities and a research area in its own right. However, it does not seem to be as pervasive as it should, in the information processing systems in current use. Natural language, as a product of human cognition, offers compelling evidence that people are naturally inclined toward processing information using pattern recognition and similarity principles. This evidence is supported by the widespread use of language devices such as the simile and the metaphor. These are examples of comparison and analogy making that humans perform without effort, in contrast to the difficulty of implementing them in the artificial information processing systems [53]. Analogy making is essential to generating new knowledge and new artifact designs [54-56], as well as to problem solving and inductive reasoning [57,58]. In a case-based reasoning context, the essential tasks of case matching and retrieval rely on pattern recognition, comparison and analogy making. In a decision making process, these mechanisms provide the immediate, implicit access to information about relevance stored in the contexts of similar instances of problems solving.
The patterns and analogies that humans are able to handle are often represented by complex spatio-temporal events with a potentially multi-sensorial impact. For example, while humans have no difficulty in understanding a metaphor like "the computer swallowed the disk," an artificial information processing system that has no visual input sensors and which lacks the capability of image understanding, would probably never be able to perceive this particular analogy with the same speed, because of the extensive reasoning and amount of explicit knowledge needed to bring the swallowing process, as it occurs in living things, close to the action of inserting a disk into a computer's disk drive.
In addition to operating on high dimensional, spatio-temporal complex patterns, analogy making in humans may also possess a dynamic component that could yield different relevance judgment outcomes, depending of context. A very illustrative example is given by French and Labiouse in [59], using the concept of a "claw hammer." According to its designed purpose, the "claw hammer" is semantically close to other concepts like "nail," "hit" and "pound." However, it may be dynamically "relocated" or reassigned in the semantic space, through a complex spatio-temporal mental simulation and analogy-making process, to the dynamically created class of "back-scratching devices," in the semantic neighborhood of the "itch," "scratch" and "claw" concepts. Similarly, one could think about the concept of a wooden decoy duck, which inherits properties from at least the "wooden object", "animal duck", "toy" and "hunting gear" classes. This concept may also be dynamically relocated into the semantic neighborhood of any of the classes, depending on the context of use that may be focused on themes such as "combustibles" or "hunting" for example. In the medical domain, the contextual dependence of relevance judgments, classifications and analogies is even more important, as these are often based on uncertain information and may be dynamically reevaluated in the light of new information about the patients or about their diseases.
Polyhierarchy and multiple inheritance are indisputable desiderata of terminology systems [60]. However, building multiple inheritance mechanism using current technology seems very difficult, simply because the number of possible alternative classifications increases exponentially with the number of concepts. It is also very unlikely that this kind of taxonomic dynamicity (e.g., the claw hammer circumstantially classified as a back-scratching-device) of the human semantic space could work on such fixed conceptual structures which are constructed beforehand through learning, in human semantic memory. A more plausible hypothesis is that such ad-hoc classifications are circumstantially created using mechanisms that are closer to a distance calculation between high dimensional, distributed, vector representation of concepts. This is in agreement with neurolinguistic evidence from functional brain imaging studies of the human semantic memory. These studies suggest the existence of distributed feature networks for the representation of object concepts [61] and help the case for less structured approaches to capturing and representing semantics such as compositional terminology schemes (e.g. as in GALEN-GRAIL [62] and SNOMED-RT [63]), latent semantic indexing (LSA) [64-70] and connectionist models [49,71,72]. These approaches allow for a multidimensional semantic space where concept features can vary in importance, evolve or change dynamically, accounting for many possible classifications and subtle variations of concept meaning, including the new and the less plausible ones. This contrasts with the fixed or highly structured semantic representation schemas (e.g. fixed knowledge frames, semantic networks, ontologies), which fail to capture concept semantics in a way that provides richness, dynamicity and reusability.
The dynamicity of concept meanings and relevance judgments may offer at least one of the reasons why fixed classification schemes, controlled terminology systems or open domain ontologies have not turned out satisfactory. It may also explain why existing lexical databases based on carefully handcrafted knowledge such as WordNet [73] often contain either too fine-grained or too coarse-grained, "static" semantic information [64]. In information intensive domains like medicine, concept dynamicity may account for why the development of a universal (i.e., one size fits all) clinical terminology system is so difficult [74].
From a case-based reasoning perspective, humans are naturally equipped with powerful pattern matching and classification capabilities which allow them to cope with complex, time-constrained relevance judgments, to easily construe meaning of concepts and to tolerate the ambiguity of natural language. Only relatively recently have computers come close to this functionality with the introduction of data mining and machine learning techniques such as self organizing maps and clustering algorithms based on similarity metrics [75]. In such machine learning approaches, the important problem of feature selection equates to a problems of relevance.
CBR enabled EHR – Proposals, Challenges and Solutions
Iatrogenic causes are said to be important causes of death in the US [76]. The reported incidence of adverse effects among patients in Canadian acute care hospitals is 7.5% [77]. A proposed means to counteract such medical errors is information technology, through the education and decision support offered to health care professionals.
One very effective form of medical education is the retrospective analysis of case records where health professionals, both experienced or novices, learn from their own and from others' successes and failures [78]. Providing that legal and ethical implications such as provider and patient protection are dealt with appropriately, the efficacy of this teaching method can be improved if case records are continuously created, enriched, accumulated and organized on similarity principles. This is possible through a CBR approach of the EHR which, from this perspective, could serve as a comprehensive case base of managed patients that will evolve asymptotically towards an exhaustive knowledge base.
Medical errors are also connected with the complex human cognitive task of planning [79]. CBR approaches, devised originally as a solution to automated planning tasks [80], have been since used in various applications including healthcare, legal and military (e.g., battle planning) [21]. This demonstrates a particularly good fit of a medical decision support based on CBR with its human users, the healthcare professionals.
Providing that the privacy and confidentiality issues, which are even more stringent in this case, are dealt with appropriately, opening EHR to patients could benefit them [81]. It is perfectly conceivable that patients could learn from the history of other cases similar to theirs, which could be presented in an anonymized, story-telling format and organized on case similarity principles. It is also possible that patients may be willing to directly provide some of their own case information in order to be matched with previously managed cases, for example in the context of online chronic disease support groups. These principles are already realized in form of bulletin boards, mailing lists and forums, where actual patients interact with each other and occasionally with health professionals and exchange information regarding health related problems ([82] and [83]). The unstructured, textual exchange of information in such resources would ideally be moderated by knowledgeable individuals (e.g., providers). Although the automatic processing of text still is not readily available, case matching is possible so far and is performed by the very individuals who are able to offer useful information and knowledge to others, based on the similarity of their own experiences (i.e., their own story).
Medicine has always and will always be a case oriented profession. Medical Informatics has recognized this early through the works of various researchers who pioneered the area of decision support systems [84]. Relevant to CBR work are also the attempts to enhance early decision support systems with domain knowledge from simulated patient cases [85]. Currently, the exploration of CBR in medical contexts is increasing [86-94]. Regardless of the problem nature, the most important components of a CBR expert system are
• The case base, the memory of past problem-solving instances
• The case matching or pattern matching procedure which retrieves the relevant cases for a certain problem
While humans seem to possess a natural support for these two components, there is still work to be done in order to make the computer support this kind of knowledge acquisition and processing. We envision four important challenges in advancing towards CBR enabled EHRs:
1. Case record comprehensiveness
2. Organization on similarity and associative principles (associative memory) and development of advance data visualization techniques
3. Development of pattern recognition and similarity measures between heterogeneous records
4. Solving ethical issues and provision of privacy and confidentiality measures
1) Case record comprehensiveness
EHR comprehensiveness is required because the exhaustiveness of a case base is not only a function of the number of records but also of the richness of each case record. Current knowledge processing technology limits the acquisition and especially the processing of comprehensive EHR records which incorporate structured data, images, video-clips, bio-signals, genomic data, unstructured textual data covering clinical findings, detailed patient history, etc. However, as knowledge processing technology advances and knowledge acquisition bottlenecks are overcome, it might be possible to overcome the heterogeneity and sparseness of EHR and allow the creation of representative case-bases and the organization of knowledge on principles that facilitate similarity based retrieval.
Temporal knowledge is also a good example of a heterogeneously represented type of knowledge in the form of potentially non-interoperable standards for dates and times and temporal knowledge of various degrees of precision, embedded in knowledge facts such as "soon after receiving the drug, the patient developed a rash." Currently, for many people, the problem may seems to boil down to devising yet another standard which encompasses all the different temporal representations of dates, times and temporal concepts into a unified, common representation. From a knowledge engineering standpoint, and again currently for many researchers, this may equate to the creation of a comprehensive ontology of temporal knowledge. However, the problem of representing time starts to look like a somewhat limited version of another burning problem of Medical Informatics: that of medical terminologies. The fact that all these issues remain largely unsolved, can only help the case for CBR and for adaptive, empirical methods and approaches to knowledge processing. We believe that such approaches have the potential to cope and overcome the problems with redundant, possibly ambiguous representations, which have arbitrary degrees of precision. Thereby we are specifying a goal towards which the development of EHR should proceed.
2) Organization on similarity and associative principles (associative memory) and development of advanced data visualization techniques
Similarity based retrieval is difficult with current database technology. For example, queries to retrieve cases which are similar to a textual description of a given case are difficult to answer. The comprehensiveness of EHR must be complemented with the possibility of indexing its records on similarity principles. Conceptually, the functionality of EHR will be that of an associative memory of cases that will enable the CBR paradigm. The organization of a case-base must be complemented by the development of advanced data visualization techniques that comply with the principles of organization of information by similarity. One example of such data visualization techniques are self-organizing maps [75]. These models are able to perform cluster analyses on high dimensional data sets and provide a visual display which can help with the navigation through and retrieval of similar cases. For instance, the self-organizing map obtained from the analysis of the Wisconsin Breast Cancer Dataset [95] used to cluster and classify cases based on their similarity in [96], could also be used for data visualization and navigation purposes, in a CBR context (Figure 9). It also demonstrates how high level abstractions (i.e., benign tumors forming the green cluster on the map) can be derived through an entirely automatic, data driven approach.
3) Development of pattern recognition and similarity measures between heterogeneous records
CBR relies on the proper management of the case base and on appropriate mechanisms for matching and retrieval of these case records. All similarity retrieval mechanisms are based on some sort of distance calculation between the problem at hand and the records in the case base, followed by the retrieval of the most relevant ones. Clinical narratives and other EHR components containing unrestricted text represent a particularly difficult challenge for semantic similarity measures. The development of terminology systems based on less structured (e.g., latent semantic indexing, connectionist models) and data-driven approaches will provide the semantic richness, dynamicity and reusability needed for such complex tasks.
A concrete example for the potential feasibility of such approaches, is the automated knowledge induction based on contextual similarity modeling ranging from morphological to sentential context [97] (Figure 10). An experimental knowledge processing system can induce automatically the new knowledge fact that Ayercillin, an item unknown to the system and hence not appearing in Figure 10, is most likely to be a drug, precisely a penicillin. The decision is based on morphological (e.g., "-cillin"), semantic (e.g., six of the similar items are known to be drugs, precisely, penicillins) and pragmatic (e.g., the six, semantically similar items are consistent with the use in a medical context) similarities that help in filtering out the non-relevant information (e.g., book of common prayer). On the same basis, the system can also induce that surgical procedures ending in "-tomy" (e.g., perineotomy, valvulotomy, myringotomy, strabotomy) are usually incisions while those ending in "-ectomy" (e.g., myringectomy, tonsillectomy, splenectomy, nephrectomy) are usually removals, that concepts containing the morpheme "leuco" (e.g., leucocyte, leucothoe, platalea leucorodia) are usually associated with color white while those containing "eryth" (e.g., erythroblast, erythema, erythrina) with color red.
However, despite such proof-of-concept applications and other progress in data mining and knowledge extraction from heterogeneous databases, case matching remains largely an open research question.
4) Solving ethical issues, provision of privacy and confidentiality measures
We discuss this challenge last, not because it is less important but, on the contrary, because of its potential to become the most important obstacle to individual knowledge processing. The very fact that individual knowledge has the potential to contribute to solving future problems instances, raises the important ethical issue whether such knowledge should be made available to decision makers and researchers. Because the definition of individual knowledge implies the possibility to match it in time and space with an application context, i.e., with a patient, sharing individual knowledge is counterbalanced by the need for privacy and confidentiality. In addition, to further complicate matters, it may turn out that some of the most useful records for future instances of decision making are instances of medical errors or other unexpected events that are unique in their course of events and therefore easily identifiable together with their contexts of development (i.e., patients, providers, family members).
The high complexity of individual knowledge renders explicit, manually controlled access to individual knowledge cases and their components unfeasible. The only solution to this problem seems to be of technological nature. Current privacy and confidentiality measures which include de-identification, de-nominalization and scrambling of the unique personal identifiers automated or semi-automated seem insufficient to counteract the potential to identify patients from unique, individual knowledge patterns.
As a general approach, we propose that the accurate measuring of similarity of individual knowledge could form the basis of a confidentiality risk assessment. This could be intuitively understood by considering that:
• very similar individual knowledge patterns which are in great numbers are a very low threat to the privacy and confidentiality infringement, and, at the other extreme,
• stand-alone patterns which possess unique features or combinations of features, are at high risk of privacy and confidentiality breaches.
In addition, the provision of privacy and confidentiality could be regarded as a special case of knowledge processing, which involves knowledge about the proper use (e.g., access, modification, transfer) of individual knowledge. This potentially complex, particular case of meta-knowledge processing could be implemented and managed using the principles of CBR paradigm itself, by building case-bases with examples of both proper and improper (simulated, not necessarily real) individual knowledge accesses and that can be compared with future access instances.
Overcoming this very important challenge hinges on the possibility to effectively measure the similarity between heterogeneous records and on the advancement of knowledge processing on CBR principles.
If successful, CBR research might therefore fulfill a longstanding need for intelligent information processing and advance informatics towards the ideal of individual knowledge processing. This calls for further investigation of information processing models that are, similarly to human experts, capable to efficiently move across the knowledge spectrum. One class of such models is represented by artificial neural networks [14,23], which are highly adaptive information processing models able to create high-level abstractions from raw data, completely automatically [75] and "learn by themselves" new information processing functions from data. From this perspective, Informatics aligns closely to the goals of AI to create intelligent machines that can hear, see, speak, think, adapt and make decisions.
Conclusions
CBR provides potential solutions to important problems that, among other, stymy the usefulness of EHR. The natural integration of learning with reasoning and the CBR resemblance to the cognitive models of human decision-making hold the promise to overcome the "brittleness" and "knowledge acquisition bottleneck" of classical expert systems. The CBR applications to the medical field have the potential to offering the training and decision support needed by health professionals and the means towards a true patient-centered healthcare.
With a CBR theoretical foundation still in its infancy and with limited medical applications in existence, more research is needed for providing proofs of the feasibility of practical CBR-EHR applications. Challenges in the way to accomplish this include the increasing complexity, ethical issues as well as the paradigm shift that our current computing devices must undergo in order to support the CBR principles of knowledge processing.
Summary
1. Science is twofold and is driven by two opposite forces: that of creating theories (theoretical sciences), and that of applying theories to practical applications (applied sciences). Medical Informatics is fundamentally an applied science that should be committed to advancing patient-centred medicine through individual knowledge processing.
2. Case-based reasoning is the technical solution that enables a continuous individual knowledge processing that could be integrated with the Electronic Health Records.
3. Medicine is an information and knowledge intensive domain where time-constrained decision problems can only be solved effectively based on the recollection of similar problems and their solutions (i.e., a case-based reasoning strategy). The collective sharing of experiences is important for making future decisions as well as for learning how to make decisions.
4. Unlike computers, human decision makers possess the components necessary to perform case-based reasoning naturally (i.e., a content addressable memory to organize a case base efficiently by similarity principles, as well as the capability to perform pattern recognition, comparison, and analogy-making).
5. Applying the CBR approach to EHR might be a way to overcome the important obstacles of EHR acceptance and use, providing that technical challenges and ethical issues arising are addressed appropriately.
List of abbreviations
AI Artificial intelligence
AIT Algorithmic Information Theory
CBR Case Based Reasoning
E Explicit knowledge
EHR Electronic Health Records
G General knowledge
LSA Latent Semantic Indexing
I Individual knowledge
NDM Naturalistic Decision Making
RCT Randomized Controlled Trial
RPD Recognition-Primed Decision
U Implicit (Unobvious) knowledge
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
Before the reviews
SP researched the paper and provided a first draft. JA, JM critically revised the manuscript three times each and provided their own additions to the initial draft. JA provided more feedback on the cognitive aspects and decision-making as well as writing style and missing references. JM additions were with regard to the writing style, clarity, missing references and the overall organization of the paper.
After the reviews
SP and JM worked on the responses to reviewers' comments. SP wrote a first revision of the paper. JM provided extensive feedback as well as new references and suggested a major revision that includes recent ideas. JA also commented and made suggestions on the knowledge spectrum model and on the meta-level view on Medical Informatics. SP overhauled the entire paper. JM revised the new version and provided feedback. SP operated the changes and proposed new modifications. JM revised the second draft. JA provided feedback on the second draft of the paper with regard to fundamental aspects of knowledge modalities.
SP and JM incorporated the minor changes suggested by the last review.
All authors read and approved the final version of the paper.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We would like to thank Dr. André Kushniruk, Scarlette Verjinschi, Dr. Jim McDaniel and Dr. Yuri Kagolovsky for their excellent suggestions on the first version of this paper. Also, special acknowledgements are addressed to Dr. Mahmood Tara for observations and interesting discussions on many aspects of the material presented in the paper. We also want to thank the others individuals who provided informal feedback on the initial stages of the knowledge spectrum idea.
Finally, we like to acknowledge the contributions of Dr. James Cimino and Dr. Stefan Schulz. Their reviews contributed substantially to the improvement of this article.
Figures and Tables
Figure 1 The knowledge spectrum.
Figure 2 A blocks world example. In this particular example expressions such as: on(a, c), on(c, table), on(b, table), pyramid(a), brick(b), brick(c), ¬same-as(a, c), same-as(b, c), etc., are true.
Figure 3 The relationships between the knowledge modalities.
Figure 4 Knowledge representation media on the knowledge spectrum. The storage and transmission of knowledge are more advanced compared to the knowledge acquisition, retrieval and use capability of current technology.
Figure 5 Knowledge representation and processing in novices and experts.
Figure 6 Biomedical knowledge on the knowledge spectrum.
Figure 7 Representations of "brick" on the knowledge spectrum. Such representations range from rich (e.g., images, mental models) to less complex (sketches and diagrams) and to symbolic descriptions (textual, formal and conceptual).
Figure 8 A blocks world example. In this particular example brick(b), brick(c), pyramid(a), on(c, b), on(c, a) are true and therefore not rejected by definition (3): the condition that "c" MUST sit on something that is not a pyramid in order to be a brick is met by on(c, b).
Figure 9 Example of self organizing map (associative memory). Each one of the 9-dimensional, 683 individual cases is associated with a region on the 2-dimensional map and highlighted using a 3rd dimension (an "activation bubble" with elevation and colour). The organization of individual descriptions of cases obeys similarity principles: similar cases are closely mapped and very similar cases form clusters (e.g., the green area contains most of the benign cases).
Figure 10 Example of similarity based retrieval and knowledge induction. Ayercillin, a relatively new drug unknown to the system, is likely to be a penicillin because of its high contextual similarities with known penicillines.
Table 1 Implicit and explicit knowledge
Implicit knowledge (U) Explicit knowledge (E)
Example The implicit knowledge used to recognize the face of a specific person. The explicit knowledge (e.g., textual descriptions) that would allow to recognize faces of people (including a specific person).
Complexity, Context retention Rich, grounded in reality.
High retention of context in form of salient features. Lean, more abstract, symbolic.
Variable amount of context retention.
Acquisition Detection, learning of correlations and regularities of environment. Explicitation of one's implicit knowledge.
Explicit acquisition of knowledge (e.g., through reading).
Representation Unstructured, present implicitly in data recordings of the environment (e.g., image of a person). Varies from less structured (e.g., natural language) to very structured (e.g., formal descriptions).
Transferability Transferable only in implicit form through the data recordings (i.e., representations) of the environment. Transferable through languages (natural or formal) and communication (e.g., verbal).
Applicability Very well applicable to specific problem instances. Applicable to both, specific and more generic problems.
Processing mechanisms Pattern recognition, feature selection, associative memory. Reasoning.
Table 2 Individual and general knowledge
General knowledge Individual knowledge
Example Explicit general propositions, rules, algorithms, guidelines and formal theories for recognizing faces of people (e.g., a formal theory of human face recognition). The implicit knowledge used to recognize and the explicit knowledge (e.g., textual description) that would allow recognizing the face of a specific person.
Complexity Very lean, abstract, symbolic. Varies from rich to lean.
Acquisition Identical to acquisition of explicit knowledge. Identical to acquisition of both implicit and explicit knowledge.
Representation Transferability Very structured, highly transferable, explicitly as general propositions, rules and guidelines. Varies from unstructured to less structured. Transferable in both implicit and explicit form.
Context retention Applicability Does not retain context.
Easy applicable to generic problems, difficult to apply to specific problem instances (e.g., recognition of the face of a specific person). Retains context.
Well applicable to specific problem instances, especially if context retention is high.
Processing mechanisms Logic reasoning. Pattern recognition, feature selection, associative recall, case-based reasoning.
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| 15533257 | PMC544898 | CC BY | 2021-01-04 16:03:41 | no | BMC Med Inform Decis Mak. 2004 Nov 8; 4:19 | utf-8 | BMC Med Inform Decis Mak | 2,004 | 10.1186/1472-6947-4-19 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1567816610.1371/journal.pbio.0030027Research ArticleNeuroscienceHomo (Human)A Functional Neuroimaging Study of Sound Localization: Visual Cortex Activity Predicts Performance in Early-Blind Individuals Sound Localization in Early-Blind PersonsGougoux Frédéric
1
Zatorre Robert J
2
Lassonde Maryse
1
Voss Patrice
1
Lepore Franco franco.lepore@ umontreal.ca
1
3
1Centre de Recherche en Neuropsychologie et Cognition, Département de PsychologieUniversité de Montréal, Montréal, QuébecCanada2Neuropsychology/Cognitive Neuroscience Unit, Montreal Neurological InstituteMcGill University, Montreal, QuébecCanada3Institut Universitaire de Gériatrie de MontréalMontréal, QuébecCanadaRaichle Marcus Academic EditorWashington University in St. LouisUnited States of America2 2005 25 1 2005 25 1 2005 3 2 e276 6 2004 16 11 2004 Copyright: © 2005 Gougoux et al.2005This 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 author and source are properly credited.
Loss of Sight and Enhanced Hearing: A Neural Picture
Blind individuals often demonstrate enhanced nonvisual perceptual abilities. However, the neural substrate that underlies this improved performance remains to be fully understood. An earlier behavioral study demonstrated that some early-blind people localize sounds more accurately than sighted controls using monaural cues. In order to investigate the neural basis of these behavioral differences in humans, we carried out functional imaging studies using positron emission tomography and a speaker array that permitted pseudo-free-field presentations within the scanner. During binaural sound localization, a sighted control group showed decreased cerebral blood flow in the occipital lobe, which was not seen in early-blind individuals. During monaural sound localization (one ear plugged), the subgroup of early-blind subjects who were behaviorally superior at sound localization displayed two activation foci in the occipital cortex. This effect was not seen in blind persons who did not have superior monaural sound localization abilities, nor in sighted individuals. The degree of activation of one of these foci was strongly correlated with sound localization accuracy across the entire group of blind subjects. The results show that those blind persons who perform better than sighted persons recruit occipital areas to carry out auditory localization under monaural conditions. We therefore conclude that computations carried out in the occipital cortex specifically underlie the enhanced capacity to use monaural cues. Our findings shed light not only on intermodal compensatory mechanisms, but also on individual differences in these mechanisms and on inhibitory patterns that differ between sighted individuals and those deprived of vision early in life.
Blind individuals who localize sound better than sighted subjects recruit visual cortical areas in the process
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Introduction
Animals and humans deprived of vision have been shown to have enhanced nonvisual perceptual abilities. Indeed, many blind individuals are extremely efficient in tactile processing, including Braille reading; the talent manifested by some well-known blind musicians, singers, and even piano tuners is often in part attributed to the fact that they were blind since their youth. One may conclude that blind persons should be better in nonvisual tasks since they compensate for their lack of vision by focusing on their remaining modalities. Many studies have in fact shown that some early-blind human subjects outperform sighted persons in nonvisual tasks, such as speech perception [1,2,3], unfamiliar voice recognition [4], verbal memory [5,6,7], and musical abilities [8,9,10]. Of particular relevance to the present study are data suggesting that some blind individuals show better auditory spatial discrimination [11] or localization of sound sources than sighted subjects [2,12,13]; however, other studies have failed to show this advantage [14,15], raising the question of what may underlie individual differences. In general, the nature of any behavioral enhancement, its extent, and its neural bases are still matters of considerable debate.
Animal studies provide some insight as to the neural substrates underlying such enhanced capacities (reviewed in [16]). For instance, in cats that had been visually deprived for several years by eyelid suture shortly after birth, the auditory cortical representation expanded into visual areas [17], and auditory spatial tuning was sharpened in the auditory cortex [18]. Similarly, in neonatally enucleated rats, electrophysiological recordings showed somatosensory responses in the visual cortex [19], and the somatosensory cortex showed an enlargement of receptive fields of the cells in some barrels together with an increase of angular sensitivity for deflection in another barrel [20]. Thus, those experiments indicate a recruitment of the visual cortex for nonvisual tasks, but do not conclusively prove that the enhanced perceptions of the blind rely on the visual cortex.
Several studies using neuroimaging techniques have also established that posterior visual areas in blind individuals may be active during the performance of nonvisual tasks such as Braille reading [21,22], memory retrieval [7], and auditory localization [23,24] as well as other auditory functions [25,26,27,28,29]. It remains to be established whether recruitment of visual cortices necessarily reflects functional reorganization, or whether it indicates a nonspecific or even pathological response. Indeed, despite numerous studies showing activation in visual areas during nonvisual tasks, the functional significance of this phenomenon has been questioned by some investigators who suggest that the occipital cortex might be nonspecifically coactivated [30]. If the visual cortex participates in nonvisual functions in the blind, then its activity level should be related to individual differences in behavior, and in effect predict behavioral outcome.
Localization of sound, a very important function for the blind, is one domain in which it is particularly useful to study the cross-modal interactions that may occur following visual deprivation. This task entails integration of binaural and monaural cues to derive spatial information. In accordance with the idea that nonvisual processing can be enhanced in the blind, a prior study demonstrated that a subset of early-blind subjects was more accurate than sighted controls (SIG) at localizing sound sources, specifically when using monaural cues [12]. These findings provide a clear opportunity to study the nature of visual cortical recruitment, and the extent to which it relates to behavioral improvements.
Thus, in the present study, subjects were first studied in an anechoic chamber using binaural and monaural sound localization tasks. Depending on their performance at the monaural task, they were divided into three groups: (i) early-blind participants who could localize the sounds more accurately than control subjects (early blind with superior performance [EBSP]); (ii) early-blind participants who were unable to localize the sounds any more accurately than controls (early blind with normal performance [EBNP]); and (iii) SIG. The same localization task was next adapted so that it could be carried out within the positron emission tomography (PET) apparatus, using a speaker array which permitted pseudo-free-field presentations [31]. Two control conditions, for monaural sound localization (MSL) and binaural sound localization (BSL), were used to control for the auditory input and motor responses.
The hypothesis tested was that blind persons showing supranormal performance do so because they recruit visual cortical areas to carry out the task. We therefore predicted that they would show activation in visual areas specifically during the MSL task, and not during the binaural or control tasks. The other blind group, which does not have enhanced MSL ability, should not show this activation pattern. We further hypothesized that the degree of visual cortical activity would be predictive of individual differences in the behavioral performance of the monaural task.
Results
Anechoic Chamber Experiments
In the anechoic chamber experiments, only five of the 12 early-blind subjects could accurately localize the sounds monaurally, whereas most of the sighted subjects could not (Figure 1). In order to differentiate two groups of early-blind participants on the basis of their performance at this monaural task, we computed their mean absolute error score and set the cut-off at a score of 45° (see Materials and Methods). Individuals with scores below this value formed the EBSP group, while those above it constituted the EBNP group. The results of the former group are illustrated on the right side of Figure 1B. As can be seen, in these subjects the regression line was very close to the dashed line representing ideal performance. By contrast, the other subgroup of early blind subjects, EBNP, and the SIG could not localize the sounds correctly, and the regression line is quite distant from that representing ideal performance. To confirm this localization performance, the mean absolute error score was compared for the three groups; a group X position interaction was observed (F30, 225 = 6.299, p < 0.01), localization being more accurate in the EBSP group, especially on the side of the obstructed ear. The performance of the EBNP did not differ from that of the SIG. These findings confirm the previous study of Lessard et al. [12] with a different group of early-blind participants.
Figure 1 Monaural Sound Localization in PET Experiments Performed in the Three Groups of Subjects
(A) CBF increases. Activations of the right striate and extrastriate cortices are observed in EBSP but not in the two other groups for the contrast of MSL minus its control task. Upper image series, sagittal slices; lower image series, coronal slices. X and Y coordinates refer to standardized stereotaxic space.
(B) Behavioral data. Behavioral results in MSL task (with SE bars). The dashed lines represent the ideal performance, whereas the solid lines indicate the best linear fit to the observed localization performance. Negative angles on the abscissa correspond to the obstructed ear, while positive angles correspond to the unobstructed ear. Note the better performance of the EBSP group compared to the EBNP and SIG.
In contrast, all 19 subjects tested, whether sighted or early blind, could correctly localize sounds binaurally (Figure 2). Despite an overall good performance, as measured by the absolute error score, the EBNP group was somewhat less efficient than the other two groups, especially for more lateral positions (group X position: F30,225 = 4.058, p < 0.01; see Figure 2C). The latter finding is reminiscent of the undershooting performance also found in visually deprived cats [32].
Figure 2 Binaural Sound Localization in PET Experiments Performed in the Three Groups of Subjects
(A) CBF decreases. In the sagittal (upper image series) and coronal (lower image series) slices, a decreased CBF is observed in the visual cortex of SIG (striate and extrastriate cortices), for the contrast of BSL minus its control task. X and Y coordinates refer to standardized stereotaxic space.
(B) CBF increases. In the sagittal (upper image series) and coronal (lower image series) images, a CBF activation peak is seen in the right ventral extrastriate cortex for the EBSP group, but not for the other two groups, for the contrast of BSL minus its control task.
(C) Behavioral data. Behavioral results in the BSL task are presented (with SE bars). The dashed lines represent the ideal performance, and the solid lines indicate the best linear fit to the observed localization performance. All three groups were able to localize sounds accurately.
PET Scanner Experiments
Binaural sound localization
During binaural localization of the sound, when compared to the control task (Figure 2A and Table 1), cerebral blood flow (CBF) decreased in the extrastriate and striate cortex of SIG, suggesting inhibition between visual and auditory areas. However, neither group of early-blind subjects showed this deactivation. A further confirmation of these results was obtained by carrying out a direct intergroup comparison of the activation of each of the blind groups to that of the sighted one. These differences are presented in Figure 3. The two blind groups show what appears to be an increase in CBF relative to the SIG, confirming that the CBF response of these groups differ in this region. However, given the deactivation observed within this region in the SIG in the first analysis, as opposed to the lack of CBF difference in the blind, the more likely interpretation is that this effect reflects a decrease in CBF in the sighted.
Figure 3 Intergroup Contrasts in Binaural Sound Localization Minus Control Task
Sagittal (top) and coronal (bottom) images showing the contrasts between EBNP (left) compared to SIG, and EBSP (right) compared to SIG. These contrasts confirmed the differences in occipital areas between the SIG and the two other groups, which are likely attributable to a decrease in CBF activity in the sighted relative to the control task (see Figure 2). X and Y coordinates refer to standardized stereotaxic space.
Table 1 Stereotaxic Coordinates and t Values of Activation and Deactivation Foci in Occipital Areas
Empty cells indicate that there is no activation above the threshold for this group. Coordinates x, y, and z refer to standardized stereotaxic space [75]. See Table S1 for complete list of foci
Another finding from the binaural versus control task contrast was a small region of activation in the visual cortex (ventral extrastriate) of the EBSP group, but not in the other two groups (Figure 2B and Table 1). Finally, all three groups also showed activation in several other cortical regions (see Table S1 for further details); among the most relevant of these is a focus in right inferior parietal cortex (Figure S1).
Monaural sound localization.
Of greatest relevance to our hypotheses, during monaural stimulation (one ear plugged), as compared to control task, right-hemisphere striate and ventral extrastriate areas showed increased CBF only in the EBSP subset of blind subjects (Figure 1A and Table 1). For the EBNP subset, occipital activation was not significant, as was also the case with the SIG. Once again, direct intergroup contrasts confirmed the differences in activation in occipital areas during MSL between the EBSP group and the two other groups (Figure 4).
Figure 4 Intergroup Contrasts in Monaural Sound Localization Minus Control Task
Sagittal (top) and coronal (bottom) images showing contrasts between the EBSP and EBNP (left), and between the EBSP and SIG (right). These contrasts confirmed the differences in occipital areas between the EBSP group and the two other groups. X and Y coordinates refer to standardized stereotaxic space.
While parietal and frontal activations were also seen in the three groups, temporal activations were not found for monaural stimulation, the EBNP group showing even some deactivation in temporal areas. The EBSP group also showed some activation in the right cerebellum (see Table S1 for further details). Significant differences between groups were not observed in temporal and parietal cortices, however.
Correlation analysis
In order to assess whether occipital activations have a functional role in auditory localization, independent voxel-wise covariation analyses were carried out across the entire group of blind individuals. Irrespective of the group to which they had been assigned, the individual absolute error score was entered as a regressor in the analysis examining covariation with CBF change between overall accuracy at the localization tasks and activation across the entire brain volume, following the procedure outlined by Paus et al. [33]. For MSL, a negative and significant correlation was observed between the absolute error score and CBF in some areas of the visual cortex (especially extrastriate but also striate [Figure 5 and Table 1]). It follows from these results that the degree of activation (percent CBF change) predicted behavioral performance in MSL. The highest correlation observed was in the right ventral extrastriate cortex (lingual gyrus; Brodmann area [BA]18, r = –0.81, p < 0.01) but two other significant foci were found in right dorsal extrastriate cortex (superior occipital gyrus; BA19, r = –0.77, p < 0.01), and striate cortex (BA17, r = –0.68, p < 0.05). Two of these foci are close to the ones identified in the analyses presented for MSL. These findings support the hypothesis that the visual cortex is directly involved in localizing a sound stimulus in the monaural condition.
Figure 5 Correlational Analysis for Monaural Sound Localization in Blind Persons
These data show the correlational analysis between performance (mean absolute error) in pointing task to monaurally presented sounds and CBF in a group of blind subjects. The two columns of brain images (left image series, sagittal sections; right image series, coronal sections) illustrate the statistical parametric map of the correlation, which is maximal in the ventral extrastriate cortex (A) but also significant in dorsal extrastriate (B) and striate (C) cortices. The red arrows in the coronal slices indicate the focus selected for the respective sagittal slices. The scattergram shows the individual values extracted from each of these regions; closed circles indicate blind subjects; open circles indicate SIG. The dotted vertical line represents the cutoff in performance for the a priori classification of blind subjects into those with low error rates (EBSP) and those who do not show the enhancement (EBNP). X and Y coordinates refer to standardized stereotaxic space.
Discussion
The imaging results of this study support our hypothesis that blind persons recruit occipital areas in the context of auditory localization and, more importantly, the correlation observed with MSL performance strongly suggests that individual differences in reorganization of the occipital cortex have behavioral consequences. Hence, this relationship does not support the possibility that the recruitment is a nonspecific coactivation or a pathological response. Instead, these results suggest that visual cortex is specifically recruited to process subtle monaural cues more effectively.
Functional activation of the visual cortex by nonvisual stimulation in the blind has already been shown in several previous studies. Activation of primary and secondary visual areas was observed during Braille reading and other tactile discrimination tasks in early-blind persons [21]. This tactile-induced activation in the occipital cortex was also confirmed by a series of subsequent studies [7,22,34]. The hypothesis proposing a functional role for this activation in the visual cortex was supported by the study of Cohen et al. [35] who showed, using transcranial magnetic stimulation, that this occipital area is required for Braille reading in blind subjects. This phenomenon was further illustrated in the case of a proficient Braille reader, blind since birth, who became unable to read Braille, despite normal somatosensory perception, after bilateral occipital damage resulting from an ischemic stroke [36]. Moreover, in a speech processing study in the blind, it was shown that occipital activity (striate and extrastriate) varied as a function of semantic or syntactic content [29]. Activation of area V1 has also been found to correlate with performance in memory tasks [7].
Of greatest relevance for the present findings are the results of Weeks et al. [24] in which CBF was measured during BSL in blind and sighted individuals, and reported activation in posterior parietal areas and also in association areas in the right occipital cortex only in the blind. In addition, interregional covariations observed between the right parietal and occipital (ventral, dorsal, and parieto-occipital) cortices were interpreted as reflecting parts of a functional network for auditory localization [24]. These data and the present findings converge on the conclusion that the visual cortex is recruited during auditory localization in the blind. However, whereas the study of Weeks et al. [24] reports extensive recruitment of visual areas during binaural processing, we observed only a small area of activation in the ventral visual area in the binaural condition. Instead, our data point to the importance of visual regions in successful localization under monaural conditions, which we interpret as reflecting a recruitment of these areas for processing of spectral cues. One possible interpretation of the discrepancy is that the BSL task of Weeks et al. [24] may have involved spectral cues, provided by the head-related transfer functions used in that study to simulate extrapersonal space.
Functional Significance of the Recruitment of Visual Areas in the Blind: Better Use of Spectral Cues?
It is interesting to consider the function of the right occipital areas, which seem to be important for MSL. Monaural cues (spectral cues and head shadow effect) are involved in the localization of sounds when one ear is obstructed, or in unilaterally deaf subjects [37]. However, spectral cues also contribute to BSL, particularly for vertical and front-back discrimination [38], but also for azimuth localization [39,40]. Moreover, some authors suggest that spectral cues based on head-related transfer function templates are sensitive to experience [41,42]. In this vein, Doucet and coworkers [43] showed that the supranormal performance of early-blind persons in MSL was decreased by occlusion of the pinna or by high-pass and low-pass filtering of the stimuli, again suggesting that use of spectral cues is important for this task. However, because performance was not completely abolished, head shadowing cues [37] might also have operated. Similarly, the study by Röder and collaborators [13] suggests that blind persons might be more sensitive to spectral cues, since their blind participants were better at localizing at lateral positions. Finally, a recent study showed superior binaural spatial discrimination performance in both early- and late-blind subjects compared to sighted subjects, when the stimuli were presented in peripheral field [44]
Combination of Intramodal and Cross-Modal Plasticity?
Cross-modal plasticity may not necessarily be the only mechanism to explain the present results. Some studies have also shown intramodal plasticity in auditory cortex of the blind. For example, the tonotopic region of area A1 in blind persons seems to be enlarged compared to that of sighted subjects [45], presumably reflecting greater use of auditory cues by the blind. Enhanced recruitment and sharpening of spatial tuning of auditory cortical neurons has also been found in binocularly deprived cats [17,18]. Thus, a combination of intramodal plasticity in auditory cortex and cross-modal plasticity involving visual cortex may have contributed to the superior performance seen in our early-blind subjects. In the present study, a significant difference in activation in auditory cortical areas was not observed among the three groups. However, we cannot exclude the possibility of plasticity at this level, as CBF responses might not be sensitive to effects such as better spatial tuning properties of auditory neurons.
Experience-driven improvements in auditory localization can occur without necessarily invoking cross-modal recruitment. Indeed, some studies have shown that in MSL tasks, practice may lead to increased performance in the case of unilaterally deaf patients [46] or even normal subjects [47]. Excellent performance in MSL was also observed in juvenile ferrets when they were raised with one ear plugged. Even in adult ferrets, changes were seen in the performance during MSL after regular practice [48]. Moreover, adult humans seem to be able to calibrate auditory cues after their pinnae were modified with moulds, showing good performance with their “new ears” after a few weeks [49]. These results would favor the hypothesis that, instead of becoming supranormal in their remaining senses, blind persons may use them more efficiently within normal limits [50]. Nonetheless, our data suggest that this efficiency gain in the blind is achieved at least in part via recruitment of visual cortical areas.
Why do some blind persons and not others acquire superior monaural sound localization skills? It may be that these changes are entirely experience-driven. That is, some blind persons may have had more practice navigating or using auditory cues to explore their environments. On the other hand, the individual differences we observed in the degree of cross-modal plasticity could reflect innate factors that remain to be identified. An additional explanation may be that the blind persons who are not better than normal at sound localization may be superior in other nonvisual tasks, such as Braille reading or other somatosensory discrimination, because the visual cortex was preferentially recruited to carry out these tasks instead of auditory ones. If this is the case, it is possible that cross-modal plasticity is limited to a certain extent, such that recruitment of these areas by one modality inhibits recruitment by another. These speculations will have to be explored systematically in future studies.
How Does the Visual Cortex of the Blind Process Auditory Information?
What is the nature of the mechanism implied in the processing of auditory stimuli by visual cortical areas? The specific areas of visual cortex recruited may provide a clue. The analyses yielded one peak in the right V1 area. V1 has already been shown to be activated in other studies examining Braille reading [21,22,34], verbal memory [7], verb generation [7,28], and speech processing [29]. These findings therefore suggest that V1 may play a very general role in a variety of nonvisual tasks in the blind. However, a right lingual gyrus peak was the main focus revealed by the analyses in the present study. This cortical region is known to form part of the ventral visual pathway, which is important for identifying visual objects [51]. If this region is important for the processing of visual object features, such as contour or texture [52,53], we may speculate that the same area is possibly used in the blind to process analogous features for auditory stimuli such as spectral contour. These cues to auditory object identity, which are normally processed in anteroventral regions of the auditory cortex [54], might be processed in the occipital ventral stream in the blind when they are relevant for spatial position.
Contribution of Parietal Cortex to Binaural Sound Localization
The parietal activation observed in all groups during BSL suggests that these areas are important when carrying out the task used in the present experiment (see Figure S1). This finding agrees with a previous study, which reported a right-sided inferior parietal activation that positively correlated with absolute error score in normal sighted subjects with the same procedure as in our study [31]. Parietal activation of both hemispheres, or a right hemisphere advantage, has been shown in several other neuroimaging studies of auditory localization and spatial discrimination experiments with sighted subjects [24,55,56,57,58,59,60]. In the study of Weeks et al. [24], a strong right-hemisphere recruitment of parietal and occipital regions was shown for blind subjects. Our findings therefore agree with these studies and with the well-known right-hemisphere advantage for spatial processing. However, we did not observe preferential activation in this parietal region in the blind as compared to the sighted, nor did CBF correlate with behavioral performance in this region. Based on those findings, we conclude that parietal area activation is related to the sensory-motor integration and spatial coordinate transformation required by the pointing task [31,61] at some stage after sensory processing has occurred. Thus, we propose that in those blind subjects who have specifically learned to use monaural cues, parietal regions receive additional input from the ventral visual cortex, but that no reorganization within the parietal cortex itself has occurred.
Different Inhibitory Patterns for the Visual Cortex in Blind and Sighted Persons
During BSL, the sighted control group (SIG) showed a deactivation in both extrastriate and striate areas of the occipital lobe, a phenomenon that was not observed in either subset of early-blind individuals. Many previous studies with sighted subjects have shown that following stimulation in one modality, cross-modal inhibition occurs in the unattended modalities [62,63,64,65,66] or even in some areas within the same modality [66,67]. Interestingly, an imaging study with sighted subjects carried out by Zatorre and coworkers [57] reported a visual deactivation in tasks of pitch and location discrimination. Because deactivation is not seen in all studies, the phenomenon may be related to the nature of the task [64,65,68].
Deactivation of primary visual areas has also been seen in sighted subjects during a tactile discrimination task, whereas in blind subjects activation was shown in the same area [21]. Within the context of auditory localization, Weeks and coworkers [24] also reported some occipital deactivation in sighted subjects, while the blind showed activation in the same area. All these results suggest that cross-modal inhibitory processes could be different in blind and sighted subjects, at least under some experimental conditions. Blind subjects might not have to inhibit the normally competing visual cortex when they perform some of the same nonvisual tasks as sighted people do. By contrast, the specific recruitment of the same cortex in order to complete a difficult task might permit them to compensate for their handicap.
Conclusion
The present study establishes for the first time in certain early-blind persons a clear relationship between monaural sound localization performance and increased CBF in occipital areas. Indeed, some of the blind persons showed occipital activation that appeared to be functional, since this phenomenon was correlated with a supranormal performance in MSL. This finding suggests that visual deprivation from an early age could lead to important cross-modal plasticity and give blind persons an advantage in using spectral cues to carry out a crucial everyday task, sound localization. Moreover, we report that inhibitory patterns differ between early-blind and sighted individuals. Under binaural conditions, the SIG seemed to inhibit part of the occipital areas when localizing sounds, but this was not the case for either group of blind persons. This differential pattern may provide clues as to how different parts of the brain normally interact during unimodal stimulation, and further suggests that these interactions may be modified in the absence of a sensory modality.
It may also be important in future studies to investigate whether blind persons can recruit visual areas in other auditory tasks, for example in a task in which spectral and level cues are relevant but in a nonspatial context. Along the same lines, a spatial discrimination task not requiring the explicit localization of the sounds may also be of interest. One can thus verify that this special competence of some blind persons can be generalized in different auditory contexts other than MSL. Indeed, it would be interesting to know whether this ability is related to more complex tasks such as navigation, obstacle detection, or analysis of sound flow, for example when the subject moves or objects move around the subject. Similarly, it may be pertinent to investigate whether special training or substitution devices, frequently described in the literature, not only improve the relevant behavior but also facilitate cross-modal plasticity.
Materials and Methods
Subjects.
The participants were seven healthy sighted volunteers and 12 early-blind subjects who had lost their vision before puberty, most of them in the first few years of life (see Table 2). In each case, the visual deficit was of peripheral origin and led to total blindness except for some light perception in a few subjects (categories 4 and 5, according to the World Health Organization classification [69]). All participants underwent audiometric testing to ensure good hearing, equal in both ears. They gave their written informed consent in accordance with guidelines approved by the Ethics and Research Committees of the Montreal Neurological Institute and the Nazareth and Louis Braille Institute for the Blind.
Table 2 Characteristics of Blind and Sighted Subjects
Anechoic chamber experiments.
Subjects were asked to localize sounds binaurally or monaurally while they were seated in the anechoic room. The acoustic apparatus used to test sound localization, previously described in detail [70], consisted of 16 loudspeakers mounted on a graduated semicircular perimeter with a radius of 50 cm (positions: ±5°, ±16°, ±26°, ±37°, ±47°, ±58°, ±68°, and ±78°). The subject was seated in the center of the perimeter, the head placed on a headrest attached to the chair with the speakers positioned at ear level.
The stimuli were broadband noise bursts that lasted 30 ms (10-ms rise and fall times, and a 10-ms plateau). The sound pressure level (SPL) was maintained at 40 dB. A stimulus was delivered through a randomly selected loudspeaker and repeated five times for each position. A buzzer warned the subjects that a sound was about to be presented and that they should maintain a stable head position and fixate straight ahead. Compliance with all instructions was ascertained by an experimenter remaining in the chamber behind the subject. The response consisted of pointing with the dominant hand toward the apparent source of stimulation. Lines graduated in 1° steps were drawn on the perimeter, and the response of the subject was recorded by the experimenter.
For monaural testing, one ear was plugged with a combination of an ear plug (mean attenuation = 37.5 dB SPL) and a hearing protection muff (mean attenuation = 29 dB SPL). In order to compare the overall accuracy in localization between the subjects, the absolute error score was utilized in both the anechoic chamber and scanner experiments. This value is the average of the difference (in absolute value) between the correct position and the response for each trial. To allow combining of data from subjects with left or right ear plugged, the behavioral results were transformed such that the left side was arbitrarily assigned to correspond to the obstructed ear. Thus, in the behavioral data presented, negative angles on the abscissa correspond to the obstructed side, while positive angles correspond to the unobstructed side.
Scanner experiments.
In this part of the experiment, subjects were asked to localize sounds binaurally or monaurally while they were lying within the scanner. Monaural testing was carried out using the same ear attenuation procedure as used in the anechoic chamber. All conditions, localization tasks, and their specific control task, were part of a larger study. These conditions were counterbalanced across subjects for the order of scan conditions. Approximately half of the subjects within each group received the ear plug in the left and the other half in the right ear during the monaural part of the scanning session. Auditory stimuli were presented using a circular array of nine speakers, positioned 15° apart from ±60°, and having a radius of 24 cm [31]. The array was placed inside the PET scanner such that the head was in the center of the array, with speakers positioned on the horizontal plane relative to the subject's head, at the level of the ears. In order to ensure stable head position, the head was maintained by a Velcro band, and its position was checked frequently by means of three laser pointers included in the scanner. Background noise in the scanner room was 56 dB SPL.
The stimuli were two broadband noise bursts that lasted 30 ms and were separated by a 0.5-s intrapair interval while the intertrial interval was 2.5 s. Each pair of stimuli was presented from a single speaker at 60 dB SPL, as measured at the center of the array. Each of the nine speakers was utilized 12 times in random order for a total of 108 trials for each condition. The behavioral tasks were started around 15 s before the beginning of data acquisition with the scanner. The response consisted of pointing with a joystick, placed at the subject's side, to the apparent source of stimulation. It was ascertained before the experiments that all subjects were familiar with the use of the joystick and with the task requirements. In a series of preliminary experiments, it was verified that subjects (n = 6) wearing binaural earplugs and ear muffs could not localize any of the stimuli from the speakers. The control task consisted of pointing in alternation to the left and right (−90°, +90°, −90°, and so on), after hearing a stimulus pair presented always in the frontal (0°) position. Two control tasks were tested, one binaural and one monaural; in the monaural case the same ear was plugged as was used for the localization task for that individual. Thus, four conditions were tested in all subjects:BSL, binaural control task, MSL, and monaural control task.
PET scans were obtained with a Siemens Exact HR+ tomograph (Forchheim, Germany) operating in three-dimensional acquisition mode. The distribution of CBF was measured during each 60-s scan using the H2O15 water bolus method [71]. MRI scans (160 1-mm slices) were also obtained for each subject with a 1.5T Philips ACS system (Andover, Massachusetts, United States) to provide anatomical detail. CBF images were reconstructed using a 14-mm Hanning filter, normalized for differences in global CBF, and co-registered with the individual MRI data [72]. Each matched MRI/PET dataset was then linearly resampled into a standardized stereotaxic coordinate system based on the MNI305 target (a sample of 305 normal subjects) via an automated feature-matching algorithm [73], resulting in a normalized brain space similar to the Talairach and Tournoux atlas (for additional information, see: http://www.mrc-cbu.cam.ac.uk/Imaging/). Statistical analysis was performed applying the method described by Worsley et al. [74]; covariation analysis followed the procedure outlined by Paus et al. [33]. A t value of 3.5 was used for significant changes in CBF during exploratory searches. However, a t value of 3.0 was used for a priori regions of interest, such as occipital areas.
Supporting Information
Figure S1 Parietal Activation Foci in Binaural Sound Localization Task
Sagittal and coronal images contrasting BSL to the control task. All three groups showed increased CBF in the right inferior parietal lobe (as shown by the red arrows), consistent with other neuroimaging studies of auditory localization. X and Y coordinates refer to standardized stereotaxic space.
(2.2 MB TIF).
Click here for additional data file.
Table S1 Stereotaxic Coordinates and t Values of Activation and Deactivation Foci
(35 KB DOC).
Click here for additional data file.
FG was supported by a postgraduate fellowship from the Natural Sciences and Engineering Research Council of Canada and the Fonds de la Recherche en Santé du Québec. The work was supported by grants from the Canadian Institutes of Health Research to ML, RZ, and FL and the Canada Research Chairs programs to ML and FL. We thank Kate Hanratty, Pierre Ahad, Marc Bouffard, Francine Giroux, Sylvain Milot, Robert Lisbonna, Gary Sauchuck, Rick Fukasawa, Alan Evans, Bruce Pike, the McConnell Brain Imaging Center, the Nazareth and Louis Braille Institute (Pierre Rondeau), the Montreal Association for the Blind (Patricia Ferrarezi, Maria Moschopoulos), the Regroupement pour les Aveugles et Amblyopes de Montréal (Serge Poulin), Charles Leclerc, Marie-Eve Doucet, Nadia Lessard, and all the participants.
Competing interests. The authors have declared that no competing interests exist.
Author contributions. FG, RZ, ML, and FL conceived and designed the experiments. FG, RZ, ML, PV, and FL performed the experiments. FG, RZ, ML, PV, and FL analyzed the data. FG, RZ, ML, and FL contributed reagents/materials/analysis tools. FG, RZ, ML, and FL wrote the paper.
Citation: Gougoux F, Zatorre RJ, Lassonde M, Voss P, Lepore F (2005) A functional neuroimaging study of sound localization: Visual cortex activity predicts performance in early-blind individuals. PLoS Biol 3(2): e27.
Abbreviations
BABrodmann area
BSLbinaural sound localization
CBFcerebral blood flow
EBNPearly blind with normal performance
EBSPearly blind with superior performance
MSLmonaural sound localization
PETpositron emission tomography
SIGsighted control group
SPLsound pressure level
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| 15678166 | PMC544927 | CC BY | 2021-01-05 08:28:10 | no | PLoS Biol. 2005 Feb 25; 3(2):e27 | utf-8 | PLoS Biol | 2,005 | 10.1371/journal.pbio.0030027 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1567816710.1371/journal.pbio.0030033Research ArticleEcologyEvolutionZoologyArthropodsThe Indirect Benefits of Mating with Attractive Males Outweigh the Direct Costs Indirect Benefits Outweigh Direct CostsHead Megan L [email protected]
1
Hunt John
1
Jennions Michael D
2
Brooks Robert
1
1School of Biological, Earth and Environmental Sciencesthe University of New South Wales, SydneyAustralia2School of Botany and Zoology, The Australian National UniversityCanberraAustraliaHarvey Paul Academic EditorUniversity of OxfordUnited Kingdom2 2005 25 1 2005 25 1 2005 3 2 e337 9 2004 22 11 2004 Copyright: © 2005 Head et al.2005This 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 author and source are properly credited.
Why Bad Boys Always Get the Girl and Other Tales of Evolutionary Madness
The fitness consequences of mate choice are a source of ongoing debate in evolutionary biology. Recent theory predicts that indirect benefits of female choice due to offspring inheriting superior genes are likely to be negated when there are direct costs associated with choice, including any costs of mating with attractive males. To estimate the fitness consequences of mating with males of varying attractiveness, we housed female house crickets, Acheta domesticus, with either attractive or unattractive males and measured a variety of direct and indirect fitness components. These fitness components were combined to give relative estimates of the number of grandchildren produced and the intrinsic rate of increase (relative net fitness). We found that females mated to attractive males incur a substantial survival cost. However, these costs are cancelled out and may be outweighed by the benefits of having offspring with elevated fitness. This benefit is due predominantly, but not exclusively, to the effect of an increase in sons' attractiveness. Our results suggest that the direct costs that females experience when mating with attractive males can be outweighed by indirect benefits. They also reveal the value of estimating the net fitness consequences of a mating strategy by including measures of offspring quality in estimates of fitness.
Experiments reveal that female crickets that choose an attractive mate have lower survival, but that this can be cancelled out or even outweighed by the increased fitness of her offspring
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Introduction
Whether mate choice can be maintained by indirect selection when females incur direct costs by being choosy is the subject of ongoing theoretical controversy [1,2,3,4,5]. This is particularly true when the principal or only benefit of mating with attractive males is that they sire attractive sons. Weatherhead and Robertson [6] suggested 25 y ago that the genetic benefits of mating with an attractive male could outweigh the cost of reduced investment in parental care that such a male makes. This suggestion has been opposed by several important theoretical models [5,7,8]. More generally, some recent theoretical work has suggested that because of the weakness of indirect selection relative to direct selection, genetic benefits of choice are likely to have little effect on the evolution of costly mate choice [2,3]. This assertion has been contested by other theoretical work [1].
In order to understand how mate choice evolves, it is necessary to estimate the overall effect of mate choice on female fitness [9,10,11,12]. The effect of mating with males of differing attractiveness on total female fitness depends on both positive and negative effects on a variety of fitness components. The signs and strengths of these effects are paramount to distinguishing between the relative importance of various models of mate-choice evolution.
Evidence from studies that have measured one or a few fitness components has been invoked to support direct benefits [13,14], “viability genes” [15,16,17,18], “Fisherian runaway” [19,20], and “sexually antagonistic coevolution” [21,22] models of mate-choice evolution. Similar evidence has also been used in tests for differential allocation of reproductive effort to offspring sired by attractive males [14,23,24]. Understanding the relative significance of these processes, however, requires measuring as complete a set of fitness components as possible [12,19] and estimation of the multigenerational effects of mate choice on fitness [11,25] through both sons and daughters [12,26]. To date, only two studies have compared the number of grandchildren produced when females mate with attractive or unattractive males [10,16]. Unfortunately, neither study accounted for the beneficial effects of heritable male attractiveness, an important consideration in most models of mate-choice evolution.
How fitness should be estimated is controversial [27,28]. Measuring total fitness is logistically preclusive, but rate-insensitive estimates, such as the number of grandchildren, or rate-sensitive estimates, such as the intrinsic rate of increase, may offer reasonable approximations [10,11,25]. The key difference between these two estimates is that rate-sensitive estimates take into account both the timing of reproduction and the developmental time of offspring, whereas rate-insensitive measures do not. To date, most empirical studies have employed rate-insensitive estimates, whereas theoretical models tend to focus on the intrinsic rate of increase [28].
Here, we measured both direct and indirect fitness components of female house crickets, Acheta domesticus, mated to either attractive or unattractive males for the term of their adult life span. We present a female's total fitness as both a rate-sensitive (the intrinsic rate of increase) and a rate-insensitive estimate of fitness (the total number of grandchildren) in interpreting our findings.
Results
Our treatment did not affect the number of grandchildren produced via daughters, via sons, or in total (Table 1). Thus there was no difference in the rate-insensitive estimate of fitness for females mated to males of differing attractiveness. Females that mated with attractive males did, however, experience higher relative intrinsic rates of increase (rest) than females mated with unattractive males (Table 2).
Table 1 The Effects of Mating with Either Attractive or Unattractive Males on a Number of Fitness Components
Table 2 The Sensitivity of rest to Variation in Individual and Combined Fitness Components
In each reduced model individual females' scores for the component(s) listed were replaced with experiment-wide mean scores. r¯a and r¯u are the mean rest for females mated with attractive and with unattractive males, respectively. Test 1 indicates the significance of the r¯a versus r¯u comparison within the reduced model (based on 10,000 randomizations). Test 2 assesses the significance of the change in effect size (based on 10,000 jackknifed pseudoestimates) between the reduced model and the full model
The overall difference between the treatments on rest was not due to any single fitness component (Table 2). When looking at the fitness components individually, the strongest effects were a survival cost experienced by females mated to attractive males (Figure 1), and an indirect benefit because sons of attractive males were more than twice as likely to mate as those of unattractive males (see Table 1). However, neither of these components alone can explain the significant difference in rest between females mated to attractive or to unattractive males (see Table 2). Treatment differences in other fitness components, although individually not significant, still influenced our estimates of the overall fitness consequences of mating with attractive males. In particular the combined effect of sons' attractiveness and daughters' fecundity had a significant effect on our model (see Table 2).
Figure 1 Female Survival in Relation to Experimental Treatment
Females housed alone (black line) survived longer than females housed with either type of male (Cox regression Wald1 = 29.636, p = 0.000). Females mated to unattractive males (blue line) survived longer than females mated to attractive males (red line) (Wald1 = 10.802, p = 0.001) n = 40, 40, 40.
When we combined a female's egg number, egg width, and egg length (from the first week of egg laying) into a single index of reproductive effort, we found that females mated to attractive males exerted greater reproductive effort in the first week of the experiment than those mated to unattractive males (principal component 1: attractive = 0.239 ± 0.116, unattractive = −0.233 ± 0.199, randomisation test p = 0.043). Of the constituent measures of week 1 reproductive effort, only egg width differed significantly between treatments (egg number: attractive = 129.07 ± 15.08, unattractive = 108.17 ± 18.84, p = 0.382; egg width: attractive = 0.618 ± 0.008, unattractive = 0.568 ± 0.014, p = 0.005; egg length: attractive = 2.71 ± 0.017, unattractive = 2.68 ± 0.025, p = 0.373).
Discussion
To provide an inclusive estimate of the total fitness consequences of mating with an attractive or unattractive male, we quantified both the direct costs to females and the indirect benefits to their offspring. We made two main findings. First, the mating-associated costs borne by females are greater when mating to attractive males throughout their life than when they are mated to unattractive males. Second, these costs are cancelled out (when we use the rate-insensitive measure of the number of grandchildren) and may be outweighed by (when we use the rate-sensitive estimate of the intrinsic rate of increase) the benefits of having offspring with elevated fitness (i.e., indirect benefits).
Contrary to some theoretical predictions [2,3,5], but see [1,6], our results suggest that it may be possible for female mate choice to evolve via indirect benefits, despite the presence of direct costs. Whether this is the case or not will, however, depend on the magnitude of other costs of choice not measured here, such as the costs associated with being choosy, as well as the accuracy of female choice [3].
The costs of choice, including the costs of mating with attractive males, are of central importance to theoretic models of mate-choice evolution [1,2,3,4,5,29]. In many species females incur survival or fecundity costs due to being courted or harassed by males [30,31], mating [32,33], and allocating resources to egg laying, gestation, and/or parental care [34]. Female Drosophila melanogaster mated to large (and thus presumably attractive) males incur a greater survival cost than females mated to smaller males [21,22], and this appears to be due to a higher mating rate with large males [22]. A potential criticism of such studies is that they are based on single traits that are taken to be an indirect measure of a male's attractiveness. By using a direct biological measure that incorporates all traits that contribute to a male's ability to induce a female to mate during short-range courtship, our results provide the first direct evidence that females sustain greater direct costs when mating with males that are more attractive in this context.
While we do not know the exact mechanisms driving the survival cost seen in our experiment, our finding that females mated to more attractive males experience lower survival is consistent with sexual conflict between males and females over mating decisions [25,35], and with differential allocation [34]. Females mated to attractive males exerted greater reproductive effort in the first week of the experiment. This could be the result of male manipulation, for example, increased mating rate [32], or stimulants in seminal fluids [36,37,38] whereby more attractive males manipulate females to invest more in their offspring than is optimal for the females. The possibility of male manipulation is also supported by a study by Murtaugh and Denlinger [39], which shows that in A. domesticus, males pass substances in their ejaculate that promote higher rates of short-term oviposition. Alternatively, it may be adaptive for females to invest more in the offspring of attractive males [34,40]. Differential allocation is only likely to be adaptive if there is an indirect fitness benefit to allocating greater reproductive effort when mated to attractive males [34]. The indirect fitness benefits that we report here, particularly the benefit of having more attractive sons, may provide an adaptive basis for differential allocation by females to the offspring of more attractive males.
Several studies have reported fitness benefits of mating with attractive males. Females mated to such males have been reported to have offspring that have greater longevity [15,41], faster growth rate [16,17,42], increased fecundity of daughters [16,42], and increased attractiveness of sons [19,20,42,43,44]. In our study, the net fitness benefit of mating with attractive males is not due to any single indirect benefit but to a combination of fitness components. This illustrates the importance of measuring net fitness, especially if fitness components act in opposition to each other.
A number of studies have proposed the use of an aggregate measure of male attractiveness rather than a single morphological indicator [44,45]. Our use of time to mounting allows us to gain a measure of male attractiveness that is based on all traits that contribute to male mating success (hence ‘attractiveness') during short-range courtship interactions [46]. It is the use of such a measure that may explain the high correlation between fathers' and sons' attractiveness in this experiment and others based on similar measures [12,44]. The greater attractiveness of sons sired by attractive males may also be explained by differential allocation; studies have shown that maternal effects may enhance the heritability of male traits [47]. An important role for maternal effects is unlikely in our experiment, however, because no other fitness components of sons or daughters differ significantly between the treatments. Regardless of whether sons' greater attractiveness is due to additive genetic variation for attractiveness per se or to the ability to manipulate females into allocating more resources to the offspring, such a trait will increase a female's net fitness if it increases the reproductive success of her sons sufficiently.
Due to the nature of our experimental design we were unable to measure all the costs and benefits associated with choosing and mating with attractive males. First, we did not measure sons' ability to compete with other males for access to females. However, in this population of A. domesticus, fighting ability has been shown to be positively correlated with attractiveness as we have measured it here [48,49]. Thus, if anything we may have underestimated the fitness benefit gained through having attractive sons. Second, we did not measure long-range attraction of males through advertisement calling. Third, our design simplifies the way mating takes place for females paired with attractive or unattractive males. Pairing females with a single male for 7 d at a time may decrease or increase the costs associated with mating with males. For instance, costs may be increased because females are unable to escape male harassment, or they may be decreased because there is no male–male competition. Despite these limitations, we believe that our estimate of the intrinsic rate of increase offers a reasonable approximation of net fitness.
The fitness estimate of choice in empirical studies may depend on the importance of reproductive timing in the system in question [28]. Brommer et al. [27] compared estimates of lifetime reproductive success and intrinsic rate of increase to real long-term data from two species of bird. They showed that lifetime reproductive success was a better estimate of genetic contribution to future generations. However, their estimates did not include measures of offspring quality, and as they point out, their results may depend on the species life history, and the generality of their conclusions thus remains to be tested.
There are several reasons why reproducing early and having short maturation times is likely to be advantageous in crickets. First, extrinsic mortality of crickets in the wild is likely to be high. Second, females become less choosy [50], lose condition, and produce fewer eggs as they age (M. L. Head, unpublished data). Also, individuals with shorter generation times will contribute their genes to future generations more rapidly [51].
Our research constitutes one of the first attempts to directly and simultaneously test the combined direct and indirect effects of mating with males that differ in attractiveness. Only by following the effects of mating with attractive or unattractive males through at least two generations, and through both sons and daughters, is it possible to observe the combined direct effects on female lifetime fecundity and the genetic effects on offspring fitness [11,12,25]. Although the need to conduct such a study under laboratory conditions may constrain our ability to definitively answer this question, our results suggest that indirect genetic benefits have the potential to outweigh direct costs of mating with attractive males. Moreover, this effect comes about largely, but not exclusively, due to the production of more attractive sons.
Materials and Methods
Study species
We obtained approximately 1,000 final-instar A. domesticus nymphs from a commercial cricket breeder (Pisces Enterprises, Phoenix, Arizona, United States). Nymphs were separated into single-sex culture tubs (4 × 80 l containers per sex) to ensure their virginity, and reared with constant access to food (Friskies Go-Cat senior) and water until eclosion. At eclosion, adults were maintained in single-sex cultures for a further 10 d to ensure sexual maturity.
In the cultures from which the insects have been derived, crickets are raised in densities ranging from 23,000–34,000 m−3 and fed grain ad libitum. In these conditions males and females mate multiply. Males fight with other males and court females, and there is a positive relationship between male dominance and attractiveness [49]. Despite high densities, female cooperation is needed for mating to occur because a female must actively mount the male and align her genitalia with his to mate. Mate choice in both culture and wild populations of A. domesticus is generally sequential. That is, females choose males by either mating or rejecting males one at a time, rather than choosing between males simultaneously. We chose to work on cultured A. domesticus because our laboratory conditions closely resemble the culture conditions under which they have recently evolved. This similarity maximises the evolutionary relevance of our measures. Our experimental design, however, requires that females be kept alone, creating an important environmental difference from the culture conditions to which females have been adapted.
Male attractiveness
The attractiveness trials throughout our experiment were based on latency to mounting for pairs of crickets. While this protocol does not allow all elements of female choice to be measured, in A. domesticus a female mounting a male is a reliable predictor of mating success (in a previous study 46 out of 50 mountings led to successful transfer of a spermatophore [49]). Also, females have been shown to produce more eggs for males that they choose quickly (M. L. Head, unpublished data). This indicates that latency to mounting is representative of other aspects of choice in this species.
To obtain males that were either attractive or unattractive to females we ran a two-round tournament. In round one, each male was placed in a clear plastic container (7 × 7 × 5 cm) with a single randomly assigned female, at night, under red lighting. When a female mounted a male, but before spermatophore transfer, they were separated. Once half of the females had mounted, all remaining pairs were separated. Round two commenced with a new female assigned at random to each male. The first half of first-round mounted males to be remounted became our “attractive” treatment males. The half of first-round unmounted males that remained unmounted longest in round two became the “unattractive” treatment males. Only males that courted females during the tournament were used. This biological assay of male attractiveness incorporates all traits that make a male attractive during short-range courtship, rather than a single trait correlated with attractiveness (see [10,11,44]).
Experimental design
Forty females were randomly assigned to each of three treatments: attractive, unattractive, and an unmated control. Females were weighed and placed individually in a small plastic container (as above) with food, water, and a petri dish of moist sand for egg laying. Males from the appropriate treatment were randomly assigned to a female. Every 7 d, or whenever a male died, a new male from the same treatment (but from a new tournament) was placed with the female. This allowed us to measure the fitness consequences of the strategy of mating with attractive or unattractive males, rather than the consequences of mating with a given individual male. Food, water, and sand were replaced every 7 d.
Fitness measures
Female survival was monitored daily, and the number of eggs laid was counted weekly. Hatching success was estimated as the proportion of eggs that hatched within 14 d of the first egg hatching in each collection. Hatchlings were collected every 3 d, and their mean weight was recorded. Each week, 50 hatchlings per female were separated into two boxes (20 × 13 × 13 cm), each containing 25 nymphs. We monitored offspring survival every 7 d and recorded the time to mature and sex and body weight at eclosion.
If a female had fewer than 50 hatchlings in a given week these were discarded. For these females, the actual number of hatchlings was multiplied by the overall experimental mean for each subsequent offspring fitness measure, to predict the number of grandchildren produced. This is a conservative approach to missing values because it reduces the difference between the treatments.
Offspring generation times were calculated from the time females were first placed with a male until the offspring matured. This takes into account not only the time it takes for the offspring to mature, but also the timing of egg laying. Mature offspring were housed individually, and their survival monitored daily. Ten days after eclosion each son's attractiveness was estimated by placing him with a stock virgin female in a small plastic container for 90 min. Mounted males were separated from females before spermatophore transfer occurred. We used the proportion of a female's sons that were successful in this assay as our measure of sons' average attractiveness (e.g., if 8 of 16 sons were mounted, we assumed that, on average, each son had a 50% chance of mating per encounter with a female).
Ten days after eclosion daughters were placed with a stock male for 12 h to allow mating. Afterwards, survival of sons and daughters was again monitored daily, and sand was collected from daughters weekly. Eggs from daughters were counted to estimate lifetime fecundity.
Statistical analysis
We calculated two estimates of female relative net fitness when mating with either an attractive or unattractive male. A rate-insensitive estimate, the relative number of grandchildren produced by a female (gest), and a rate-sensitive estimate, rest.
To estimate the absolute number of grandchildren each female had (Gest), we added the number of grandchildren she had through daughters, estimated as
to the number she had through sons, Gsons, estimated as
The attractiveness of a female's sons was estimated as the proportion of her sons that were mounted in attractiveness trials; longevity is the mean adult life span of a female's sons and c is the ratio of the total number of grandchildren through daughters in the experiment to the total number of sons mounted in the attractiveness trials. This correction factor converts an attractiveness score into units of the number of grandchildren. Using this correction factor ensured that mean son and daughter reproductive success across the entire experiment was equal, satisfying the assumption that mean reproductive success for males and females is the same in populations with an equal sex ratio [52].
Gest for each female was then divided by the experimental mean to give the relative gest.
We estimated the absolute intrinsic rate of increase for each female as
where t is the generation time from parental first mating to offspring maturity in a particular lineage. We converted our rate-sensitive measure into a measure of relative intrinsic rate of increase (rest), by dividing each female's Rest by the experiment-wide mean.
Due to the non-normal distributions of many fitness components, we tested the significance of treatment differences for each fitness component using two-tailed randomisation tests. In each randomisation test the observed data was randomly assigned to the two treatments 10,000 times. P-values are based on the proportion of randomisations in which the absolute value of the estimated difference was greater than that observed in the original data.
To explore the sensitivity of our estimates of rest to variation in each fitness component we used a model-building approach. We removed the variance of each fitness component from our full model, in turn, by assigning every female the overall experimental mean value of that component. We similarly excluded every combination of two fitness components. We then ran a randomization test (as above, 10,000 randomizations) for each reduced model to test whether the treatment effect remained. We also obtained 10,000 jackknifed estimates of the difference between the treatments for each reduced model (by randomly omitting 20% of the sample in each estimate), to test whether the reduced model resulted in a significantly different effect size than the original full model. P-values are based on the proportion of jackknifed estimates in which the absolute value of the difference between the treatments was greater than the absolute difference in the full model.
We used principal components analysis to investigate the effects of mating with attractive or unattractive males on week 1 reproductive effort via egg number, egg width, and egg length. All three measures showed a strong positive loading on the first principal component, which explained 66% of the variation in the constituent measures. We then tested for differences in female reproductive effort between the treatments using a randomisation test.
We thank L. Bussière, J. Kelley, K. Savage, J. Evans, S. Griffith, H. Kokko, A. Lindholm, K. Monro, S. Zajitschek, F. Zajitschek, and M. Blows for valuable discussion and comments on the manuscript. This research was funded by an ARC grant to JH, RB, and MDJ, and an Australian Postgraduate Award to MLH.
Competing interests. The authors have declared that no competing interests exist.
Author contributions. MLH, JH, and RB conceived and designed the experiments. MLH and JH performed the experiments. MLH, MDJ, and RB analyzed the data. JH and RB contributed reagents/materials/analysis tools. MLH, JH, MDJ, and RB wrote the paper.
Citation: Head ML, Hunt J, Jennions MD, Brooks R (2005) The indirect benefits of mating with attractive males outweigh the direct costs. PLoS Biol 3(2): e33.
Abbreviation
restrelative intrinsic rate of increase
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| 15678167 | PMC544928 | CC BY | 2021-01-05 08:28:13 | no | PLoS Biol. 2005 Feb 25; 3(2):e33 | utf-8 | PLoS Biol | 2,005 | 10.1371/journal.pbio.0030033 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1567816810.1371/journal.pbio.0030042Research ArticleEvolutionGenetics/Genomics/Gene TherapyZoologyHomo (Human)PrimatesRattus (Rat)Mus (Mouse)Evidence for Widespread Degradation of Gene Control Regions in Hominid Genomes Genomic Degradation in HominidsKeightley Peter D [email protected]
1
Lercher Martin J
2
Eyre-Walker Adam
3
1School of Biological Sciences, University of EdinburghEdinburghUnited Kingdom2Department of Biology and Biochemistry, University of BathBathUnited Kingdom3Centre for the Study of Evolution and School of Life Sciences, University of SussexBrightonUnited KingdomSlatkin Monty Academic EditorUniversity of California at BerkeleyUnited States of America2 2005 25 1 2005 25 1 2005 3 2 e4230 8 2004 1 12 2004 Copyright: © 2005 Keightley et al.2005This 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 author and source are properly credited.
Hominids Lose Control
Although sequences containing regulatory elements located close to protein-coding genes are often only weakly conserved during evolution, comparisons of rodent genomes have implied that these sequences are subject to some selective constraints. Evolutionary conservation is particularly apparent upstream of coding sequences and in first introns, regions that are enriched for regulatory elements. By comparing the human and chimpanzee genomes, we show here that there is almost no evidence for conservation in these regions in hominids. Furthermore, we show that gene expression is diverging more rapidly in hominids than in murids per unit of neutral sequence divergence. By combining data on polymorphism levels in human noncoding DNA and the corresponding human–chimpanzee divergence, we show that the proportion of adaptive substitutions in these regions in hominids is very low. It therefore seems likely that the lack of conservation and increased rate of gene expression divergence are caused by a reduction in the effectiveness of natural selection against deleterious mutations because of the low effective population sizes of hominids. This has resulted in the accumulation of a large number of deleterious mutations in sequences containing gene control elements and hence a widespread degradation of the genome during the evolution of humans and chimpanzees.
A comparison of hominid and rodent lineages reveals that the gene control regions of hominids are not conserved and are accumulating mutations, suggesting widespread degradation of the hominid genome
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Introduction
Functionally important sequences are expected to evolve more slowly than neutrally evolving sequences. This is because long periods of selection for functional efficiency lead to sequences in which most advantageous mutations have already become fixed. The majority of new mutations in a sequence are then deleterious, because they perturb the highly adapted state. Studies of protein-coding DNA evolution have supported this expectation by showing that rates of amino acid substitution are substantially lower than rates of synonymous substitution in the majority of genes (e.g., [1]).
Recently there has been great interest in using sequence conservation to detect functionally important regions of the genome outside of protein-coding sequences. However, detecting conservation has proven difficult because noncoding DNA sequences often appear to be weakly conserved. In mammals, for example, more than 98.5% of the genome is believed to be intron or intergenic DNA [2], and at least 40% of this is composed of the remnants of transposable element insertions that are presumably decaying in a neutral fashion. Comparisons of the rates of evolutionary divergence between species have revealed evolutionary constraints in certain classes of intergenic DNA, particularly in DNA close to coding sequences [3], regions in which gene expression control motifs are believed to be prevalent. For example, on the basis of comparisons between rodent genomes, it has been suggested that there are at least as many selectively constrained sites outside, as within, protein-coding sequences [4,5]. Between mouse and rat, comparisons of rates of evolutionary divergence of non-protein-coding DNA imply that about 17% of sites in the 2 kb upstream and downstream from genes and in first introns are selectively constrained sites [6]. There is also evidence for highly conserved DNA sequences at locations distant from coding sequences [6,7,8,9,10,11].
However, the extent to which constraint in noncoding regions varies among species is unclear. In this paper, we investigate sequence conservation in introns and intergenic DNA in interspecific comparisons of mouse and rat (murids) and human and chimpanzee (hominids). To estimate the levels of constraint in segments of non-protein-coding DNA, we compare the observed numbers of substitutions to the number expected from substitution rates at linked sequences assumed to be neutrally evolving. Unexpectedly, we find that selective constraints are essentially absent in hominids in regions upstream of genes and in first introns, in contrast to murids, in which these regions are subject to moderate levels of constraint.
Results/Discussion
Selective Constraints in Hominids and Murids
We investigated the frequency of deleterious mutations and the pattern of sequence conservation in regions containing gene expression control sequences in hominids by compiling a dataset of 1,000 well-annotated, randomly chosen human genes. The genomic sequences upstream and downstream of each coding sequence and samples of their introns were aligned against the draft chimpanzee sequence. We compared the pattern of evolution in these hominid sequences against that in a previously compiled dataset of murid sequences [6]. Numbers of nucleotides sampled and other summary statistics for the sequences are shown in Table 1. As others have noted previously, intron sequences evolve slightly faster (10%–25%) than 4-fold degenerate synonymous sites, when hypermutable CpG dinucleotides are excluded. (CpG dinucleotides are more frequent in coding sequences than intergenic DNA.) This could imply the presence of selection on synonymous sites [12,13]. Of the genomic sequences surveyed, intron sequences, other than intron 1, appear to be the fastest-evolving sequences in mammalian genomes and are therefore used as our neutral standard.
Table 1 Summary Statistics for Sequences Sampled from Hominid and Murid Genomes
Numbers of bases sampled and aligned in different categories from the human and chimpanzee genomes and mouse and rat genomes, levels of sequence divergence (K), and proportions of non-CpG-prone sites are shown
We calculated levels of selective constraint in blocks of 500 bp, averaged over loci, using the aligned human–chimpanzee and mouse–rat genome sequence datasets. We corrected our constraint estimates for the decline in GC content that appears to be occurring in most mammalian genomes, including hominids and murids [14], although this correction made little difference, since the GC content of the flanking regions is similar to that of introns. Because different parts of a sequence can differ markedly in their frequency of CpG dinucleotides, and most CpG dinucleotides are hypermutable and are saturated by nucleotide substitutions between mice and rats, we excluded potentially CpG-prone sites by excluding sites preceded by C or followed by G. Simulation results ([6]; D. J. Gaffney and P. D. Keightley, unpublished data) indicated that failure to exclude CpG-prone sites leads to biased estimates of constraint.
Selective constraints in hominid sequences in the regions 6,000 bp upstream and downstream of coding sequences and in the 5′ regions of first introns are clearly far lower than in murid sequences (Figures 1 and 2). In particular, for the first 2,000 bp of the 5′ flanking region and the first 2,000 bp at the 5′ end of intron 1, constraint estimates (± standard error) are 0.0016 (± 0.019) and −0.0029 (± 0.019), respectively, for hominids, and are 0.17 (± 0.016) and 0.16 (± 0.018) for murids (Table 2). The differences in constraint between hominids and murids in these regions are therefore about six times larger than the standard errors of differences between constraint estimates. Constraint is significantly different from zero near the 3′ end of hominid coding sequences, but still more than 50% lower than in murids (Table 2). The standard errors for these estimates are very similar in murids and hominids, indicating that we have similar power to detect constraint in the two datasets. Differences in mean levels of constraint between hominids and murids are somewhat smaller if all nucleotide sites (including CpG-prone sites) are analyzed (Table 2), but constraint in 5′ flanking regions and first introns is still very low in hominids (approximately 0.03) and significantly higher in murids. Such estimates of constraint are likely to be downwardly biased in murids, owing to saturation at CpG dinucleotides, giving an underestimate of the substitution rate in introns. This may be partly offset by the fact that some gene control regions are in unmethylated CpG islands, which would tend to increase estimates of constraint in both hominids and murids.
Figure 1 Selective Constraint Plotted against Distance from the Coding Sequence in 5′ Flanking Regions and 3′ Flanking Regions
Results for (A) hominids and (B) murids. The 5′ flanking regions are shown left of the origin, and 3′ flanking regions, right. Segments of 500 bases starting from the start or stop codon were analyzed. Bootstrap 95% confidence limits are shown in light grey.
Figure 2 Selective Constraint at the 5′ End of First Introns Plotted against Distance from the Intron Start
Results for (A) hominids and (B) murids. Segments of 500 bases starting from the 5′ end of intron 1 were analyzed. Bootstrap 95% confidence limits are shown in light grey. Constraint at the 3′ end of first introns is nonsignificant and close to zero in both hominids and murids.
Table 2 Constraint in Hominids and Murids Calculated for Datasets Excluding CpG-Prone Sites and for Datasets Including All Sites
The first 2,000 bases upstream and downstream from the coding sequence and at the 5′ end of intron 1 were analyzed
There are a number of possible explanations for the apparent absence of constraint in hominid 5′ flanking and first intron sequences (and for the lower constraint in the 3′ flanking region), which we examine below.
Sequencing Errors, Pseudogenes, and Genome Reorganization
Because the mean sequence divergence of hominids is an order of magnitude lower than that of murids, sequencing errors are expected to disproportionally downwardly bias estimates of constraint in hominids. To estimate the extent of sequencing error in our data, we investigated how many conserved intron splice junctions had been mis-sequenced in the chimpanzee—the chimpanzee sequence has been produced and assembled without reference to the human sequence, so this should provide an unbiased estimate of the error rate. We observed 25 differences in 21,048 intronic splice donor/acceptor nucleotides, giving a maximum error rate (ɛ) of 1.19 × 10−3. This is expected to reduce constraint by approximately one-tenth since the relationship between the true level of constraint (Ctrue) and observed level (Cobs) is Cobs = Ctrue/(1 + ɛ/k), and the human–chimp divergence (k) is approximately 1%. If the true levels of constraint in the 5′, 3′, and intron 1 sequences closest to hominid genes were 30%, as we find in murids, the observed constraint in hominids would be 27%. Sequencing errors can therefore explain only a small proportion of the difference in constraint between hominids and murids. Further evidence that our data do not have unusually high rates of sequencing errors is that the divergences (see Table 1) are very similar to values that have been reported previously [12,15,16,17]. The results are also unlikely to be explained by polymorphism since levels of diversity in hominids are about 0.1% [18].
A second possibility that could explain the differences in constraint between hominids and murids is that our dataset of hominid genes contains large numbers of pseudogenes. However, several lines of evidence argue against this. First, the genes are well annotated and contain no stop codons. Second, the intronic splice donor/acceptor nucleotides are highly conserved. Third, the exons of our gene sample show strong conservation: constraint at second positions of codons estimated in the same manner as for noncoding sequences is 0.750 ± 0.013. And finally, a very substantial proportion of genes would have to be pseudogenes to explain our data. For example, to reduce the constraint by one-half would require 50% of the hominid genes to be pseudogenes.
A third possibility is that the lower constraints in hominid 5′ flanking and first intron regions could be a consequence of a reorganization of gene regulation such that murids have a concentration of regulatory sequences in 5′ regions and in first introns and hominids have regulatory sequences concentrated in introns outside intron 1. However, two lines of evidence suggest that this is unlikely. First, there is remarkable conservation of syntenic blocks [19] and per locus intron/exon number [4,5] between murids and human. Second, the vast majority of known mammalian gene expression regulatory regions are situated within 2 kb of promoters [4].
Adaptive Evolution
The lower level of constraint in hominids could be due to higher rates of adaptive substitution in the 5′, 3′, and first intron regions in hominids, masking constraint on other sites. If this is the case then we expect reduced nucleotide diversity in the 5′, 3′, and first intron regions for two reasons. First, the level of diversity in a region is largely determined by the number of effectively neutral mutations, because adaptive substitutions contribute little to polymorphism. This implies that levels of polymorphism will be lower if some sites are subject to constraint. Second, levels of diversity are expected to be lower in regions undergoing adaptive substitution because adaptive substitutions can remove variation by genetic hitchhiking [20]. To test for reduced levels of variation, we analyzed single nucleotide polymorphism data from the 5′ flanking, 3′ flanking, and intron sequences of 305 human genes compiled from the Environmental Genome Project (http://www.niehs.nih.gov/envgenom/home.htm). This dataset was chosen because it represents the most extensive and consistently sampled database of human single nucleotide polymorphisms. The genes were sampled as being “environmentally responsive,” and are therefore not a random sample, but they show average levels of constraint in all regions similar to those of our sample of 1,000 hominid genes. The analysis revealed no reduction in diversity in the 5′, 3′, and intron 1 regions when compared to the levels of diversity in intron sequences outside first introns (Figure 3). In contrast, the level of diversity is significantly lower at nonsynonymous sites. To test the adaptive hypothesis further, we aligned the polymorphism dataset of 305 human genes against the chimpanzee genome sequence, measured divergence, and tested for adaptive evolution by an extension of the McDonald–Kreitman test [21] under the assumption that introns other than intron 1 are evolving neutrally (Table 3). There was no evidence of adaptive evolution in these tests or in tests in which polymorphisms segregating at less than 10% were excluded—excluding rare polymorphisms has the effect of removing any slightly deleterious mutations that might be segregating in the 5′, 3′, and intron 1 sequences [22]. Interestingly the estimate of proportion of substitutions driven by adaptive evolution (α) was significantly negative for the 3′ flanking region. This is likely due to the segregation of slightly deleterious mutations, which is consistent with the low but significant level of constraint we observed in this region.
Figure 3 Mean Nucleotide Diversity in Human Intergenic DNA in Blocks of 500 Bases Upstream and Downstream of Genes
Data shown separately for first introns, introns excluding first introns (“Introns > 1”), and nonsynonymous sites.
95% confidence limits are indicated.
Table 3 Tests of Adaptive Evolution for 5′, 3′, and Intron 1 Sequences in Hominids
Fixation of Mildly Deleterious Mutations
Two lines of evidence suggest that many mutations that affect gene expression may be under only weak purifying selection. First, sequences involved in gene regulation often appear to evolve rapidly [23]. Second, the rate of divergence in gene expression of primate genes is as fast as that of expressed pseudogenes [24]. These observations suggest another explanation for the low constraint in hominids: selection on mutations in the 5′ and 3′ flanking and intron 1 sequences that affect gene expression may be ineffective in hominids, since hominids have low effective population sizes (Ne). Based on polymorphism data and estimates of nucleotide mutation rates (u), estimates of human and chimpanzee Ne are typically in the range 10,000–30,000 for nuclear sequences [25,26,27], and this is likely to have been the case for much of their evolution, since the ancestral Ne for both species is estimated to be approximately 20,000 [28]. Unfortunately, we have little data from murids with which to estimate effective population sizes. A recent survey of nucleotide diversity in Mus musculus domesticus yielded an estimate of 4Neu of 0.0054 [29]. Combining this with as estimate of the nucleotide mutation rate of between 1.67 × 10−9 and 2.98 × 10−9 [25], we estimate Ne for the house mouse to be between 450,000 and 810,000. The fate of a deleterious mutation depends on the product Nes; if Ne|s| > 1, then the fixation probability for a deleterious mutation starts to become appreciable. Therefore, deleterious mutations, whose strength of selection falls within the range 1/Ne(murids) < |s| < 1/Ne(hominids), will tend to be removed by natural selection in murids, but can drift to fixation in hominids. Under the assumption that selection coefficients against deleterious mutations are equivalent in all taxa, the levels of selective constraint in noncoding DNA of murids and hominids imply that approximately 83%, 17%, and 0% of mutations in the first 2,000 bp of 5′ flanking DNA and intron 1 have a strength of selection such that |s| < 1/Ne(rodents), 1/Ne(rodents) < |s| < 1/Ne(hominids) and |s| > 1/Ne(hominids), respectively. For the the first 2,000 bp of 3′ flanking DNA we estimate that about 81% of mutations have |s| < 1/Ne(rodents), 12% are in the range 1/Ne(rodents) < |s| < 1/Ne(hominids) and 7% have |s| >1/Ne(hominids). In contrast to noncoding DNA, the fraction of slightly deleterious mutations fixed in hominid coding sequences is quite low. Constraint estimates at second codon positions outside CpG-prone sites in hominids and murids are 0.750 ± 0.016 and 0.900 ± 0.0085, respectively. Taking into account sequencing errors in hominids, the predicted “true” constraint value in hominids is 0.84, and this implies that only about 6% of mutations are in the slightly deleterious class.
Gene Expression Divergence
The lower level of constraint in hominid 5′, 3′, and intron 1 sequences leads to a testable prediction about the evolution of gene expression. Since the flanking regions of genes contain a high concentration of cis-acting gene control sequences [4], we expect gene expression to be evolving more rapidly in hominids than in murids, relative to the rate of neutral sequence evolution (i.e., the rate of mutation). To test this prediction, we used the microarray data of Enard et al. [30], who examined gene expression profiles in brain and liver tissue for humans, chimpanzees, M. domesticus, and M. spretus. From an analysis of 3,801 orthologous genes across all four species, we found that levels of divergence in expression between human and chimp are very similar to levels of divergence in expression between the two mouse species (Table 4). At the same time, the level of nucleotide divergence in introns, Ki, between the two hominid species is about 55% that of the mouse species (Table 4). Thus, when measured relative to the level of intron nucleotide divergence, the divergence in gene expression d is almost 1.8-fold higher in hominids than it is in murids. This acceleration is significant both for liver (hominid/murid ratio of d/Ki, 1.71; 95% confidence interval, 1.46–2.12) and for brain (hominid/murid ratio of d/Ki, 1.79; 95% confidence interval, 1.53–2.21).
Table 4 Expression Divergence (d) and Intron Nucleotide Divergence outside CpG-Prone Sites (Ki) for Hominids and Murids
Comparisons are human–chimpanzee and M. musculus–M. spretus
CI, confidence interval
As demonstrated in an analysis across different primate species, substantial increases in expression distances are still observed when going beyond the evolutionary distances examined here [30]. Thus, our finding of similar expression distances between the two species pairs cannot be due to expression distances reaching saturation. Because the Euclidean expression distances in Table 4 are of comparable magnitude, our conclusions are independent of the exact relationship between expression divergence and sequence divergence; similar results are obtained when using the mean squared gene-wise difference in log-expression (data not shown), as used, for example, by Khaitovich et al. [24].
A potential source of error in this analysis is the use of microarrays designed for humans on chimpanzee samples, and of microarrays designed for M. musculus on M. spretus samples. Sequence differences between the species will lead to lower hybridization efficiencies in chimpanzee and M. spretus, and will consequently exaggerate expression distances. However, this problem will be more pronounced in the mouse comparisons, since sequence divergence between M. musculus and M. spretus is higher than between human and chimpanzee. Thus, this would bias our results towards higher rates of gene expression evolution in mice, making our test conservative.
Conclusion
The lack of conservation of regions containing expression control sequences demonstrated above is consistent with the observation that the vast majority of Mendelian genetic disease mutations are located in coding sequences [31]. Our results have a number of interesting repercussions. The virtual absence of constraint in 5′ flanking regions and in 5′ regions of first introns, in which the majority of mammalian gene expression control sequences are believed to reside [4], implies that there has been widespread degradation of regions containing gene control sequences in hominids. We estimate that humans and chimpanzees have accumulated approximately 140,000 slightly deleterious mutations each, mutations that would have been eliminated by selection in murids. These mutations have small effects, since it can be inferred that they have selection coefficients less than 1/Ne for hominids, i.e., less than 10−4. It should be noted that it is unlikely that the mutation accumulation is due to a recent relaxation of natural selection in humans due to an improvement in our living conditions [32], since the time of this improvement is short relative the overall length of human evolution. We would not expect the decline in fitness to continue indefinitely, since the absolute strength of selection on new mutations, both advantageous and deleterious, may increase as fitness declines [33]. Furthermore, this accumulation of deleterious mutations may have been compensated in part by adaptive substitutions in gene expression control regions and elsewhere in the genome.
We have also demonstrated that gene expression evolution is significantly accelerated in hominid brain and liver compared to the respective murid tissues. This result has important implications for theories of neutral gene expression evolution [24,34]. First, our results are consistent with the view that most variation in gene expression level found between human alleles is neutral [30,35,36]. However, the difference in expression divergence, relative to nucleotide divergence, between hominid and murid genomes implies that the proportion of gene expression changes that are under natural selection varies between different lineages, and that many of the mutations that affect gene expression in murids may in fact be under selection. Consequently, the strict notion of a gene expression clock [24], like the molecular clock, may only apply within closely related species. This is true irrespective of whether time is measured in units of sequence divergence (See Table 1) or in years: the divergence time between human and chimp is approximately 6 million years, while between M. musculus and M. spretus it is approximately 1.8 million years [24], so the absolute rate of gene expression divergence is much slower in hominids than murids, whilst it is substantially faster compared to the rate of neutral sequence evolution.
The importance of effective population size in influencing the organization and complexity of genomes has recently been highlighted [37]. Our findings support the idea that microevolutionary processes are also strongly influenced by population size, and are evidence for the nearly neutral model of molecular evolution [38] in mammalian genomes.
Materials and Methods
Sampling of hominid genomic sequences
DNA sequences of 1,000 annotated loci were compiled from the reference sequence (build 33) of the human genome. In a preliminary analysis of a smaller dataset, we determined that such a dataset of 1,000 loci would provide standard errors on constraint estimates of less than 2%. We randomly sampled loci by the criterion that each record contained the description of at least one mRNA. We extracted all exons, up to eight introns, including first, second, last, and second last introns, and up to 6 kb of intergenic DNA 5′ and 3′ from the start and stop codon, respectively. Intergenic DNA was extracted up to the midpoint between the sampled coding sequence and the start or stop codon of the following or preceding locus in the genomic contig. We extracted complete introns if they were less than 30 kb in length, otherwise the first and last 10 kb. For more than 80% of the hominid loci sampled, the 6-kb 5′ region includes all annotated untranslated exons and introns.
We used reciprocal best-hits BLAST [39] to identify sequences orthologous to the human sequences in the reference assembly of the whole genome shotgun assembly of the chimpanzee genome. If a DNA segment exceeded 2 kb in length, this was subdivided into approximately 1-kb segments for analysis via BLAST. Sequences were aligned using MCALIGN [40] under a model of indel evolution appropriate to hominid intronic DNA. Parts of the chimp genome are of relatively low sequencing coverage, and errors in the assembly are expected. We therefore masked off sequence segments containing more than ten mismatches in a stretch of 100 bp and more than five mismatches in a stretch of 25 bp. Assuming independently distributed substitutions, the first level of nonhomology is expected almost never to appear by chance in our dataset, and the second level is expected to occur approximately four times in the approximately 27 Mb surveyed (see Table 1).
Sampling of murid sequences
Orthologous genes from 300 well-annotated loci were randomly sampled from the whole-genome assemblies of mouse and rat from GenBank. Loci were chosen for which annotation evidence included at least one complete mRNA sequence in both species. Further details of the sampling are given in [6]. Coding sequences, a sample of up to three introns (including first and last introns), and up to 6 kb of intergenic DNA 5′ and 3′ from the start and stop codon, respectively, were extracted from both species. Sequences were aligned by MCALIGN [40] using a model of indel evolution appropriate to rodent intronic DNA as described previously [6].
Calculation of evolutionary constraint
We estimated selective constraints (C) for each of the above categories of sites by comparing the observed numbers of substitutions (O) with numbers expected (E) if substitution rates were equal to that of a class of putatively neutral sites. In this analysis, these putatively neutral sites were intronic sites excluding intronic splice control regions (bases 1–6 and 1–16 at the 5′ and 3′ ends, respectively) and first introns, since these show evidence of moderate selective constraint [6]. If the effect of higher substitution rates within the CpG dinucleotide context is removed, mean substitution rates in both murid and hominid lineages are somewhat higher at introns than at 4-fold degenerate sites ([5]; see Table 1); synonymous sites are believed to be under weak selection in mammals [1]. The level of evolutionary constraint for a specific category of sites in n loci is
[41]. Constraint was calculated excluding nucleotides preceded by C or followed by G. Such CpG-susceptible sites have a high probability of being part of a hypermutable CpG dinucleotide, which approach saturation between mouse and rat, and have multiple substitutions sufficiently frequently between human and chimp as to induce bias in estimating substitution rates. The mutation process at microsatellite loci differs radically from that for nucleotide substitutions, so these were excluded from the analysis [6].
A nonequilibrium model of base composition evolution was used to calculate E, as described previously [42], assuming an equilibrium GC content of 0.4. For coding sequences, constraint was calculated for second positions of codons, where substitutions always lead to an amino acid change, using intronic sequences as the neutral reference. In noncoding DNA, constraint was calculated for blocks, typically of 500 bp, and averaged over loci using equation 1. Standard errors and confidence intervals were computed by bootstrapping over loci.
The number of slightly deleterious mutations fixed in hominids was calculated from the product of 25,000 (genes) × 4,000 bp (of 5′ and intron 1 sequence) × 0.17 (difference in constraint between murids and hominids) × 0.012/2 (human–chimp divergence/2) + 25,000 (genes) × 2,000 bp (of 3′ sequence) × 0.12 (difference in constraint between murids and hominids) × 0.012/2 = 138,000.
Test of adaptive evolution
Single nucleotide polymorphism data for 335 genes were compiled from the Environmental Genome Project (http://www.niehs.nih.gov/envgenom/home.htm). Of these, 305 had more than two intron sequences, and were suitable for further analysis. The sequences were aligned against the chimpanzee genome sequence, as described above, and the number of substitutions estimated by counting the number of differences, with no correction made for multiple substitutions (humans and chimps are sufficiently close as to make corrections for multiple substitutions unnecessary, particularly when CpG dinucleotides are excluded). We only considered sites that were not preceded by C or followed by G to maintain consistency with the other analyses reported here. For each gene we calculated the numbers of 5′ (or 3′ or intron 1) substitutions (Dn) and polymorphisms (Pn), along with the equivalent figures for the other introns, which acted as our neutral standard (respectively Di and Pi). To test for adaptive evolution we estimated the proportion of substitutions that were driven by adaptive evolution using the method of Smith and Eyre-Walker [21]:
where Ln and Li are the numbers of putatively selected and intron sites, respectively. Note that the number of sites appears in this formula because the number of sites for the polymorphism and divergence data are slightly different, since not all the sequence could be aligned against the chimpanzee genome. Figures for the divergence data are indicated by a prime. To obtain confidence limits for α, we bootstrapped the data by gene.
Gene expression data
Affymetrix oligonucleotide microarray data for brain cortex and liver tissue samples from three individuals each of human, chimpanzee, M. musculus, and M. spretus were obtained from Enard et al. [30]. To have comparable data from all species, only the first replicate of each human and chimp was used. Raw hybridization intensities were converted to expression levels using the Affymetrix MAS5 function as implemented in the BioConductor package [43], and then log2-transformed. The primate expression data were restricted to probe sets contained in both the HG-U95Av2 microarray (used for liver) and the HG-U95A microarray (used for cortex).
We restricted the expression analysis to orthologous genes as follows. From Ensembl (www.ensembl.org), we obtained a mapping of probe sets to Ensembl gene IDs, and a list of human–mouse orthologs. If more than one probe set matched the same Ensembl gene, we averaged expression levels over all probe sets for this gene. We retained only one-to-one orthologs (i.e., genes where human and mouse IDs uniquely match each other), further requiring that each sequence cover at least 70% of the other. Expression matrices for the individual experiments were scaled to the same mean. All mouse experiments (and, separately, all primate experiments) were then normalized relative to each other by means of a quantile normalization [44]. For each species/tissue combination, these normalized expression values were averaged over the three individuals, resulting in expression vectors for 3,801 genes orthologous between human and mouse.
Expression distances between species were calculated as Euclidean distances between expression vectors. Bootstrap analysis (resampling of genes; 1,000 datasets) was used to estimate standard errors (standard deviation of bootstrap distances) and confidence intervals (2.5% and 97.5% quantiles of bootstrap distances).
We compared expression differences to intronic nucleotide divergence levels calculated without correction for multiple substitutions, excluding CpG-prone sites as described above. We analyzed the complete 1,000 gene chimp–human intronic dataset described above and a dataset of 39 introns from 24 orthologous loci of M. spretus and M. domesticus, compiled from GenBank. In order to obtain confidence intervals for the ratio of expression divergence d over sequence divergence Ki, we used a bootstrap analysis of 1,000 datasets, each combining d values obtained from resampling genes from the expression analyses, and Ki values obtained from resampling genes from the divergence analyses.
We are grateful to the genome sequencing centers for the genome sequences used in our analysis. We thank Jay Taylor, Bill Hill, Dan Halligan, Donald Smith, Brian Charlesworth, and three anonymous referees for constructive comments on the manuscript, and Itai Yanai for helpful discussions. We are grateful to Dan Gaffney for advice on Perl programming and Dan Halligan for providing a reciprocal best-hits BLAST script. MJL acknowledges support from a Royal Society University Research Fellowship.
Competing interests. The authors have declared that no competing interests exist.
Author contributions. PK, ML, and AEW conceived and designed the experiments, analyzed the data, and wrote the paper.
Citation: Keightley PD, Lercher MJ, Eyre-Walker A (2005) Evidence for widespread degradation of gene control regions in hominid genomes. PLoS Biol 3(2): e42.
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| 15678168 | PMC544929 | CC BY | 2021-01-05 08:21:19 | no | PLoS Biol. 2005 Feb 25; 3(2):e42 | utf-8 | PLoS Biol | 2,005 | 10.1371/journal.pbio.0030042 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0030048SynopsisNeuroscienceHomo (Human)Loss of Sight and Enhanced Hearing: A Neural Picture Synopsis2 2005 25 1 2005 25 1 2005 3 2 e48Copyright: © 2005 Public Library of Science.2005This 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 author and source are properly credited.
A Functional Neuroimaging Study of Sound Localization: Visual Cortex Activity Predicts Performance in Early-Blind Individuals
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Stevie Wonder and Ray Charles are often cited as evidence that blindness confers superior musical ability. Wonder lost his sight after an incubator-related oxygen overdose during infancy; Charles lost his as a boy to glaucoma. It's impossible to know whether sight would have compromised their success, but many gifted musicians, from Jose Feliciano to Rahsaan Roland Kirk, lost their sight at an early age.
A number of human studies show that blind persons perform nonvisual tasks better than those with sight. Neuroimaging studies of blind persons performing nonvisual tasks, including hearing, show activity in brain areas normally associated with vision. But much remains to be learned about the nature and extent of this phenomenon: how these “visual areas” are used, the mechanisms that generate individual differences (not all blind persons can localize sounds better than the sighted, for example), and the neural processes that underlie it.
The task of localizing sound—which requires integrating information available to one ear only (monaural sounds available, for example, when one ear is plugged) or information derived from comparing sounds binaurally—is particularly suited to investigating the neural remapping that seems to follow vision loss. In a previous study, Franco Lepore and colleagues showed that people who lost their sight at an early age could localize sound, particularly from monaural cues, better than those who could see. These findings suggested that areas of the brain normally dedicated to processing visual stimuli (the visual cortex, located at the back of the brain in the occipital lobe) might play a role in processing sound in these individuals. In a new report, Lepore and colleagues use functional imaging studies to investigate the functional relationship between neural activity and enhanced hearing abilities in the blind, and find a strong correlation between superior sound localization skills and increased activity in the brain's visual center.
The authors hypothesized that if visual cortex recruitment bolstered auditory function in some individuals, then visual cortex activity would correlate with individual differences in performance, and the degree of activity should predict such differences. Nineteen people—seven sighted and twelve who lost their sight at an early age—were placed in an echo-free chamber and asked to indicate where a sound was coming from, using either one (monaural) or both (binaural) ears. The participants then performed the same tasks within a positron emission tomography (PET) machine, which measures brain activity through changes in cerebral blood flow (CBF).
Five of the blind participants could accurately localize sounds monaurally; most of the sighted could not. (All 19 participants had no trouble localizing binaural sounds.) Only the blind individuals with superior localization skills showed increased CBF in the visual cortex while performing monaural localization tasks. Interestingly, during binaural localization, the sighted participants showed decreased CBF in visual cortical areas. This decrease comports with previous studies showing that engaging one brain center—say, the temporal lobe, which processes sound—inhibits activation of others—such as the occipital lobe, which processes visual cues. These inhibitions appear to be absent in blind persons, though it's not clear why. It could be that blind persons don't need such inhibitions, the authors speculate, or maybe unrestricted access to the visual center serves to compensate for vision loss by boosting nonvisual senses.
Whether the enhanced auditory performance reported here simply reflects increased efficiency of auditory processing or indicates “supranormal” powers, Lepore and colleagues argue that their results show that the visual cortex is “specifically recruited to process subtle monaural cues more effectively.” It will be interesting to learn whether blind persons can recruit visual centers for other auditory tasks or to help them navigate the world without sight. Such studies would be vital for tailoring sensory support to suit individual needs and maybe even suggest ways to facilitate the neural cross talk that enhances auditory performance. But don't expect such innovations to recreate the likes of Rahsaan Kirk or Ray Charles anytime soon.
| 0 | PMC544930 | CC BY | 2021-01-05 08:21:23 | no | PLoS Biol. 2005 Feb 25; 3(2):e48 | utf-8 | PLoS Biol | 2,005 | 10.1371/journal.pbio.0030048 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0030057SynopsisEcologyEvolutionZoologyArthropodsWhy Bad Boys Always Get the Girl and Other Tales of Evolutionary Madness Synopsis2 2005 25 1 2005 25 1 2005 3 2 e57Copyright: © 2005 Public Library of Science.2005This 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 author and source are properly credited.
The Indirect Benefits of Mating with Attractive Males Outweigh the Direct Costs
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Biologists studying the evolution of mate choice have their work cut out for them. Not only is there little agreement on how best to determine the interplay between mate choice and fitness, there's not even consensus on how to estimate fitness. (Fitness being an individual's success in passing their genes on to future generations.) Why females fall for captivating males that give them nothing but trouble is especially puzzling. Such males tend to contribute their genes and little else, leaving the female to spend precious resources bearing and raising her young, efforts that often cut her life short. Of course, females are not all innocence and light in the mating game: some black widow species, for example, famously make dad a post-coitus snack. Still, females typically incur more costs than males in rearing offspring, especially when they choose flashy mates. So why do they do it?
One model holds that females put up with deadbeat dads because the benefits, though indirect, outweigh the costs: that is, attractive males are more likely to grace their offspring with good genes that increase survival (“I'm so fit I can afford to waste energy on this excessively exuberant tail”) or with sexy traits that confer mating success (“my magnificent plumage may decrease my survival, but I get lots of dates”). Another model argues that selection for such indirect benefits is much weaker than direct selection on genes that affect mate preference and thus is likely to exert little influence on mating preference.
In a new study, Megan Head and her colleagues navigate this intellectual minefield by studying the mating behavior of crickets. The authors paired females with either “attractive” or “unattractive” males (see below) and measured a variety of fitness components to estimate the overall fitness consequences of the various unions. Female crickets, they found, pay a high price for mating with attractive males. But when the fitness consequences for their sons and daughters are taken into account, mate costs are balanced by, and may even be outweighed by, the indirect benefits of spawning offspring with elevated fitness. This benefit stems in large part, the authors argue, from siring sexy sons.
How does one distinguish lothario from loser in the cricket world? By running a cricket tournament, of course. For crickets to mate successfully, the female must mount the male so their genitalia align. Noting that females produce more eggs for males they mount quickly, the authors use time to mount as a measure of male attractiveness.
An attractive cricket?
In the first round of the tournament, Head and colleagues paired males with a randomly assigned female; after mounting, but before copulation, the couples were separated. This continued until half of all females had mounted a male. (Under tournament rules, crickets had to be in the first half of a given category to qualify for the next round.) In round two, a new female was randomly assigned to each male. Males that had been mounted in the first round and remounted in the second were deemed “attractive.” Males rebuffed in the first round that remained unmounted longest in round two were “unattractive.” Females were randomly assigned to males that were either attractive or unattractive. Equivalent males were swapped out every seven days to control for any individual quirks that might bias the results.
To estimate the total fitness of the participants, the authors measured both direct and indirect fitness components, such as female hatching success and reproductive effort (egg number and size), as well as sons' attractiveness and the number of eggs laid by daughters. Females that mated with attractive males produced daughters that laid more eggs within a given time and sons that were more attractive, though they had lower survival. Thus, by evaluating both the direct effects of female lifetime fecundity and the indirect effects of offspring fitness, the authors determined the net consequences of a mating strategy. And once again, it's mom's sacrifices that keep things on track.
With this approach, Head and colleagues bridge the gap between empirical studies of mating choice evolution, which rely largely on rate-insensitive measures (such as counting grandchildren), and theoretical studies, which typically use rate-sensitive measures. Their results suggest that there may be selection for choosing costly mates and that generating a reliable analysis of the fitness consequences requires a long view: look at the reproductive success of mom's sons and daughters before judging her bad taste in mates.
| 0 | PMC544931 | CC BY | 2021-01-05 08:21:19 | no | PLoS Biol. 2005 Feb 25; 3(2):e57 | utf-8 | PLoS Biol | 2,005 | 10.1371/journal.pbio.0030057 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0030073SynopsisEvolutionGenetics/Genomics/Gene TherapyZoologyHomo (Human)PrimatesRattus (Rat)Mus (Mouse)Hominids Lose Control Synopsis2 2005 25 1 2005 25 1 2005 3 2 e73Copyright: © 2005 Public Library of Science.2005This 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 author and source are properly credited.
Evidence for Widespread Degradation of Gene Control Regions in Hominid Genomes
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What makes us human? From a philosophical perspective, the answer may lie in part in our apparently unique need—and self-awareness—to ask the question in the first place. From a biological perspective, the answer lies in part in the sequence of our DNA. While fossil evidence has provided a rough draft of the story of human evolution, much more remains to be learned about the path our genes followed, a path that diverged millions of years ago from our closest living hominid relatives, the chimp and bonobo. Charting differences between human genomes and those of our evolutionary relatives—both near and distant—has become a powerful tool for filling in the gaps in the human fossil record.
Comparing the human genome to the genomes of other great apes can provide a window into the molecular changes that may ultimately spell the difference between human and nonhuman primates. That task was recently aided by the release of the draft sequence of the chimpanzee genome. Comparing the protein-coding sequences of human and chimp has identified molecular dissimilarities between us, which is to be expected. Though many differences between species can be explained at the molecular level by differences in protein structure, where and when a given protein is produced can be just as or even more important. Differences in protein expression arise from sequences in non-coding DNA that influence the timing and regulation of protein production and action.
In a new study, Peter Keightley and colleagues conduct parallel comparative genomics studies—comparing regulatory regions in the chimp and human genome with those of mouse and rat—and make a startling discovery. The hominid lineages show a surprising lack of selective constraint—deleterious mutations have apparently accumulated—compared to the rodents, racking up an estimated additional 140,000 harmful mutations fixed, or retained, in the human and chimp lineages since they diverged. Such mutations have been selectively eliminated in mouse and rat.
The authors focused on DNA sequences making up the bulk of gene-regulating elements—regions immediately preceding or following protein-coding sequences, as well as the first intron of each gene (an intron is a non-coding DNA sequence squeezed between two adjacent coding fragments). The degree of conservation in these areas was weighed against the conservation in other nearby non-coding sequences, which were assumed to be free of selective constraints. Keightley and colleagues found marked conservation in the regulatory regions between mice and rats, but nearly none between humans and chimps. This result suggests that the gene-regulating elements of hominids are subject to nearly unfettered mutation accumulation, likely due to an absence of natural selection forces strong enough to stabilize the ancestral sequences common to both human and chimpanzee.
How can one explain these puzzling results? Keightley and colleagues propose that selection is ineffective against mildly unfavorable mutations in the gene-regulating regions because of the small effective population size in the evolutionary history of hominids.
What do these results suggest for the future of human evolution? It's unlikely that the regulatory gatekeepers of our genome will allow mutations to spin out of control. Even if the number of unwanted mutations were to increase, stronger natural selection against them is likely to develop in parallel, Keightley and colleagues explain, protecting our fitness from a downward spiral. The authors' results support the notion that population size exerts a powerful influence on evolutionary changes at the molecular level and that many changes in gene control regions are under weak selection. With each new sequenced genome added to the comparative genomics lexicon, scientists are becoming increasingly conversant in the grammar and syntax of gene sequences—and filling in more and more gaps in the human story, letter by letter.
| 0 | PMC544932 | CC BY | 2021-01-05 08:21:19 | no | PLoS Biol. 2005 Feb 25; 3(2):e73 | utf-8 | PLoS Biol | 2,005 | 10.1371/journal.pbio.0030073 | oa_comm |
==== Front
PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0030073SynopsisEvolutionGenetics/Genomics/Gene TherapyZoologyHomo (Human)PrimatesRattus (Rat)Mus (Mouse)Hominids Lose Control Synopsis2 2005 25 1 2005 25 1 2005 3 2 e73Copyright: © 2005 Public Library of Science.2005This 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 author and source are properly credited.
Evidence for Widespread Degradation of Gene Control Regions in Hominid Genomes
==== Body
What makes us human? From a philosophical perspective, the answer may lie in part in our apparently unique need—and self-awareness—to ask the question in the first place. From a biological perspective, the answer lies in part in the sequence of our DNA. While fossil evidence has provided a rough draft of the story of human evolution, much more remains to be learned about the path our genes followed, a path that diverged millions of years ago from our closest living hominid relatives, the chimp and bonobo. Charting differences between human genomes and those of our evolutionary relatives—both near and distant—has become a powerful tool for filling in the gaps in the human fossil record.
Comparing the human genome to the genomes of other great apes can provide a window into the molecular changes that may ultimately spell the difference between human and nonhuman primates. That task was recently aided by the release of the draft sequence of the chimpanzee genome. Comparing the protein-coding sequences of human and chimp has identified molecular dissimilarities between us, which is to be expected. Though many differences between species can be explained at the molecular level by differences in protein structure, where and when a given protein is produced can be just as or even more important. Differences in protein expression arise from sequences in non-coding DNA that influence the timing and regulation of protein production and action.
In a new study, Peter Keightley and colleagues conduct parallel comparative genomics studies—comparing regulatory regions in the chimp and human genome with those of mouse and rat—and make a startling discovery. The hominid lineages show a surprising lack of selective constraint—deleterious mutations have apparently accumulated—compared to the rodents, racking up an estimated additional 140,000 harmful mutations fixed, or retained, in the human and chimp lineages since they diverged. Such mutations have been selectively eliminated in mouse and rat.
The authors focused on DNA sequences making up the bulk of gene-regulating elements—regions immediately preceding or following protein-coding sequences, as well as the first intron of each gene (an intron is a non-coding DNA sequence squeezed between two adjacent coding fragments). The degree of conservation in these areas was weighed against the conservation in other nearby non-coding sequences, which were assumed to be free of selective constraints. Keightley and colleagues found marked conservation in the regulatory regions between mice and rats, but nearly none between humans and chimps. This result suggests that the gene-regulating elements of hominids are subject to nearly unfettered mutation accumulation, likely due to an absence of natural selection forces strong enough to stabilize the ancestral sequences common to both human and chimpanzee.
How can one explain these puzzling results? Keightley and colleagues propose that selection is ineffective against mildly unfavorable mutations in the gene-regulating regions because of the small effective population size in the evolutionary history of hominids.
What do these results suggest for the future of human evolution? It's unlikely that the regulatory gatekeepers of our genome will allow mutations to spin out of control. Even if the number of unwanted mutations were to increase, stronger natural selection against them is likely to develop in parallel, Keightley and colleagues explain, protecting our fitness from a downward spiral. The authors' results support the notion that population size exerts a powerful influence on evolutionary changes at the molecular level and that many changes in gene control regions are under weak selection. With each new sequenced genome added to the comparative genomics lexicon, scientists are becoming increasingly conversant in the grammar and syntax of gene sequences—and filling in more and more gaps in the human story, letter by letter.
| 15679902 | PMC544933 | CC BY | 2021-01-04 16:38:33 | no | Front Zool. 2004 Sep 29; 1:1 | latin-1 | Front Zool | 2,004 | 10.1186/1742-9994-1-1 | oa_comm |
==== Front
Front ZoolFrontiers in Zoology1742-9994BioMed Central London 1742-9994-1-21567990410.1186/1742-9994-1-2MethodologyWWW design code – a new tool for colour estimation in animal studies Berggren Åsa [email protected]ä Juha [email protected] Ecology Group, Massey University, Private Bag 11 222, Palmerston North, New Zealand2 Department of Biological and Environmental Sciences, P.O. Box 65, FI-00014 University of Helsinki, Finland3 Present Address: Department of Entomology, P.O. Box 7044, Swedish University of Agricultural Sciences, SE-75007 Uppsala, Sweden2004 6 10 2004 1 2 2 1 9 2004 6 10 2004 Copyright © 2004 Berggren and Merilä; licensee BioMed Central Ltd.2004Berggren and Merilä; 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 colour of animals' skin, fur, feathers or cuticula has been estimated in a large number of studies. The methods used to do so are diverse, with some being costly and not available to all researchers. In a study to measure plumage colour in a bird species, a new method of creating a colour chart was developed. While colour-charts have their own limitations, these can be minimised when they have the following properties: 1) being readily available to the majority of biologists, 2) containing a large array of colours to allow accurate recording and differentiation of subtle colour differences, 3) low cost, 4) adhering to a world-wide standard, and 5) being available in both hard-copy and digital formats to allow for various analytical methods. The method described below satisfies all of these requirements.
Results
Colour charts estimated to fit the range of the species' plumage colours were created on the computer screen using web software that allowed for HTML-coding (in this case Dreamweaver™). The charts were adjusted using feathers from dead specimens until a satisfying range of darker and lighter colours were found. The resulting chart was printed out and was successfully used in the field to determine the plumage colour of hand-held birds.
Conclusion
Access to a computer and printer, and the software to enable the creation of a chart, is within the reach of the vast majority of biologists. The numbers of colours that can be generated should suit most studies, with the advantage of the method being that the chart can be individually tailored to the species under study. HTML colour coding is a worldwide standard, thus the colours used in studies can be described in the methods section of journal articles using the six-digit alphanumeric code. We believe this method is very useful as a low-tech method for future estimation of individual colour.
==== Body
Background
Animal studies from diverse fields – such as morphology, physiology, behaviour, population dynamics, genetics, ageing and sexing – often require the estimation or classification of colour in skin, fur, feathers or cuticula. To make meaningful comparisons within and between studies, and effectively communicate these findings, a standardised method of assigning a colouration is required. Colour can be categorised, scored and ranked without recourse of any aid other than the observer's eye, as has been successfully demonstrated in many studies [1-4]. Aids to indexing and labelling colours have traditionally compared animals to published colour-standard cards, charts or books. These were often generated for other purposes, such as paint making or characterising soil types [5-8]. Such charts have been employed since the early 1800's, with Charles Darwin using Werner's colour charts [9] on the Beagle expedition [10]. Newer electronic measuring tools, using a reflectance spectrophotometer to give a high precision estimate of hue, saturation and brightness of the colour [11-14], have been recommended as a reliable and objective way of acquiring detailed data on different aspects of colour [15]. Lately, photo-processing software (e.g. Adobe Photoshop® – Adobe Systems Inc., San Jose, CA, USA) has been used in estimating colour brightness, saturation and hue from digitised photos [11,16].
The benefit of using highly technical methods is the ability to gain very detailed and precise information on different aspects of colour that may be impossible to detect using the human eye. However, in many studies such a high level of precision is not required, and the equipment may be impractical in the field or beyond the budget of the research group. Thus despite the option of high-tech measurement methods, visual comparisons to standardised colour charts are still practical and valuable for many field biologists. While colour-charts have their own limitations, these are minimised when they have the following properties: 1) availability to the majority of biologists, 2) large array of colours to allow accurate recording and differentiation of subtle colour differences, 3) low cost, 4) adherence to a world-wide standard, and 5) availability in both hard-copy and digital formats to allow for various analytical methods. Below we describe a method that satisfies all of these requirements.
Results and Discussion
A starting point for development of this new method was a study on plumage colour in an endemic New Zealand passerine – North Island robin (Petroica longipes) – where there was a need for a reliable, species-specific and cost-efficient method to estimate colour (Fig. 1; Å Berggren unpublished data). The birds were to be caught and handled in the field, but could not be moved and no samples from the plumage were to be taken. Hence, it was decided to create a specific colour chart, unique for the colours to be compared in this study, and easy to use under fieldwork conditions. It was decided that a computer-generated colour-chart would ideally suit the purposes of the study.
Figure 1 The making of a HTML coded colour chart for estimation of animal colour in the field. First the colours relevant for the study are estimated from samples from the species of interest (in this case the New Zealand robin (Petroica longipes)) (a). From the sample, a set of colours is coded using HTML coding (b), which has a specific code for every colour. In this case the software Dreamweaver™ was used to create nine different shades ranging from light grey (888888) to black (000000) (c). When the chart is made it is printed out and can be enclosed within a plastic covering for protection. This procedure worked well for colour estimation of the species in focus, with individuals' colours being easy to index under field conditions (d).
The light emitted from a computer monitor is composed of a particular combination of red, green, and blue light. The proportion of each of these components in the visual colour spectrum can be expressed as a number unique for each specific colour or hue. These colours are coded for using the HTML (Hypertext Markup Language) computer language. This system of colour coding was developed for the Netscape® web-browser (Netscape Communication Corp.) and has since become the industry standard [17]. Today, colour coding is one of the most important features in web page design when creating informative and graphically appealing sites [18]. Each HTML colour-code specifies the composite of a colour with a six-digit alphanumeric code where the first two digits represent the amount of red, the middle two the amount of green, and the last two the amount of blue. Each character may be represented in one of 16 ways (0 – 9 and A – F), creating a vast array of potential colours [17]. The actual number of colours that can be produced for viewing on the screen is limited by the computer, ranging from 256 colours in older computers to 16.7 million in newer models. These colours can be generated in web browser software such as Netscape®, by using the composer feature, or through web-design programmes (e.g. Dreamweaver™ – Macromedia Inc. or FrontPage© – Microsoft Corp.). The colours can be displayed on the computer screen and fine-tuned by adjusting the HTML code. For example, between the colours "0000FF" for blue, "00008B" for dark blue and "00C78C" for turquoise blue, a large number of other blues can be created and viewed. When a suitable set of colours have been decided upon, they can be printed out as they present themselves on the screen and saved for future use.
The focus species of the plumage study, the North Island robin, has a brown-grey-black plumage (Fig. 1) [19]. Using HTML coding, a colour chart was developed to match the natural variance in the species' brown to black plumage. To ensure the colour range of the feathers was accurately represented in the chart, feathers from a dead specimen were used and compared to the computer generated colours. From this point, an equal number of brighter and darker colour gradations were created, centring on the colour of the feather specimens. The HTML-coding numbering from 0 to 8 (000000 – 888888) resulted in nine shades from black to light grey (Fig. 1). This allowed a progression in equal steps from lighter to darker to be displayed sequentially on a printout. This made it possible to hold the bird next to the chart and move it until a colour match was made (Fig. 1). The technique worked well and it was possible to get an accurate colour ranking of the darkness of the plumage for the captured 32 birds (Å Berggren unpublished).
Conclusions
The method of colour-chart creation utilising HTML code satisfies the criteria listed above. Access to a computer and printer, and the software to enable the creation of a chart, is within the reach of the vast majority of biologists. The numbers of colours (and patterns) that can be generated should suit most studies, with the advantage of the method being that the chart can be individually tailored to the species under study. HTML colour coding is a worldwide standard, thus the colours used in studies can be described in the methods section of journal articles using the six-digit hexadecimal code. Comparisons are not limited to a printout of a colour chart, digital images can also be compared and their colours scored using this method. Drawbacks may include identifying the right colours for the chart to accurately match the animals as encountered in the field, a problem equivalent to other printed colour charts. It is also possible that when printing, the printed colours differ from the colour range as displayed on the computer screen. With some people still using older computers, which are not able to display colours coded in newer machines, there is a risk that there is a discrepancy between computers in the colour presented on the screen. This may be a problem when the aim is to compare specific colours between different studies, but not an issue within studies. Fortunately, this problem will decrease with more computer system being able to display the full range of 16.7 million colours. When using the colour-charts in the field, the usual care of indexing individuals under the same lighting conditions should be taken [15]. As the technique is easy to refine and adjust to the requirements needed for the species, we believe it is very useful as a low-tech method for future estimation of individual colour. We encourage other researchers and field workers to try the method in future colour studies.
Authors' contributions
JM came up with the initial idea of using web designer tools for creating colour charts. ÅB developed the colour charts using HTML coding and used them in the field as a research tool. ÅB and JM wrote the manuscript, with ÅB doing the major part. All authors read and approved the manuscript.
Acknowledgements
We thank Matthew Low for help in applying this method in the field. We are also thankful to Matthew Low and two anonymous referees for comments on an earlier version of this manuscript.
==== Refs
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Colvin J Cooter RJ Diapause induction and coloration in the senegalese grasshopper, Oedaleus senegalensis Physiological Entomology 1995 20 13 17
Lindenmayer DB Viggers KL Cunningham RB Donnelly CF Morphological variation among populations of the mountain brushtail possum, Trichosurus caninus Ogilby (Phalangeridae: Marsupialia) Australian Journal of Zoology 1995 43 449 458
Dickman CR Parnaby HE Crowther MS King DH Antechinus agilis (Marsupialia: Dasyuridae), a new species from the A. stuartii complex in south-eastern Australia Australian Journal of Zoology 1998 46 1 26 10.1071/ZO97036
Merilä J Sheldon BC Lindström K Plumage brightness in relation to haematozoan infections in the greenfinch Carduelis chloris : bright males are a good bet Ecoscience 1999 6 12 18
Roulin A Dijkstra C Genetic and environmental components of variation in eumelanin and phaeomelanin sex-traits in the barn owl Heredity 2003 90 359 364 12714980 10.1038/sj.hdy.6800260
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McGraw KJ Mackillop EA Dale J Hauber ME Different colors reveal different information: how nutritional stress affects the expression of melanin- and structurally based ornamental plumage The Journal of Experimental Biology 2002 205 3747 3755 12409501
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| 15679904 | PMC544934 | CC BY | 2021-01-04 16:38:33 | no | Front Zool. 2004 Oct 6; 1:2 | utf-8 | Front Zool | 2,004 | 10.1186/1742-9994-1-2 | oa_comm |
==== Front
Front ZoolFrontiers in Zoology1742-9994BioMed Central London 1742-9994-1-31567993110.1186/1742-9994-1-3ResearchSecondary neurons are arrested in an immature state by formation of epithelial vesicles during neurogenesis of the spider Cupiennius salei Stollewerk Angelika [email protected] Abteilung fuer Evolutionsgenetik, Institut fuer Genetik, Universitaet zu Koeln, Weyertal 121, 50931 Koeln, Germany2 Department of Zoology, University of Cambridge, Downing Street, Cambridge, CB2 3EJ, uk2004 25 10 2004 1 3 3 19 10 2004 25 10 2004 Copyright © 2004 Stollewerk; licensee BioMed Central Ltd.2004Stollewerk; 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 the spider Cupiennius salei about 30 groups of neural precursors are generated per hemi-segment during early neurogenesis. Analysis of the ventral neuromeres after invagination of the primary neural precursor groups revealed that secondary neural precursors arise during late embryogenesis that partially do not differentiate until larval stages.
Results
In contrast to the primary groups, the secondary invaginating cells do not detach from each other after invagination but maintain their epithelial character and form so-called epithelial vesicles. As revealed by dye labeling, secondary neural precursors within epithelial vesicles do not show any morphological features of differentiation indicating that the formation of epithelial vesicles after invagination leads to a delay in the differentiation of the corresponding neural precursors. About half of the secondary neural precursor groups do not dissociate from each other during embryogenesis indicating that they provide neural precursors for larval and adult stages.
Conclusions
Secondary neural precursors are arrested in an immature state by formation of epithelial vesicles. This mechanism facilitates the production of larval neural precursors during embryogenesis. I discuss the evolutionary changes that have occured during neural precursor formation in the arthropod group and present a model for the basal mode of neurogenesis.
neural precursorsinvaginationepithelial vesiclesglial cellschelicerateCupiennius salei
==== Body
Background
The arthropods form a diverse group with a correspondingly high variation of neural structures adapted to the specialized behaviour and lifestyles of individual species. This raises the question of how developmental processes have been modified during evolution to generate the wide diversity of nervous systems seen in adult arthropods. Evolutionary modifications that lead to variations in neural structures can occur during different processes of neurogenesis. The establishment of neural networks can be influenced by changes in the generation of neural precursors, modifications of cell fates or elimination of individual neurons as well as changes in axonal guidance. A comparative analysis of neurogenesis in chelicerates and myriapods has revealed that although the developmental program is genetically conserved, there is a major difference in the recruitment of neural precursors as compared to insects and crustaceans [1-5]. Groups of neural precursors invaginate from the ventral neuroectoderm in a regular, strikingly similar pattern in spiders (chelicerates) and myriapods, while in insects and crustaceans single neural precursors are selected. This modification may be the basis for variations in the functions of spider and myriapod neurons, since a comparison of early segmentally repeated neurons that pioneer the major axon tracts in crustaceans and insects has not revealed any similarities in cell body positions or axonal outgrowths to myriapod neurons [6,7].
In the spider 30 to 32 groups of neural precursors are generated per hemi-segment during neurogenesis. As in Drosophila melanogaster, the neural precursors arise at stereotyped positions that are prefigured by a proneural gene (CsASH 1), while the neurogenic genes Delta and Notch restrict the proportion of cells that adopt the neural fate at each wave of neural precursor formation [1,2]. In Drosophila melanogaster, the Delta/Notch signalling pathway is used for a decision between two cell fates in the ventral neuroectoderm: delaminating cells become neural precursors, while cells that remain apical give rise to epidermis. This decision does not take place in the central neurogenic regions of the spider [2]. The epidermal cells are derived from lateral regions that overgrow the neuromeres after invagination of the neural precursors.
Since each invagination group consists of five to nine neural precursors, it can be estimated that an embryonic hemineuromere consists of about 220 neurons on average, similar to Drosophila. However, in the adult spider Cupiennius salei the subesophageal ganglion consists of 49,000 neurons [8] indicating that over 40,000 neurons must be generated during late embryonic and larval stages. In Drosophila melanogaster, 'embryonic' neuroblasts proliferate again and give rise to larval and adult lineages after a phase of cell cycle arrest from late embryogenesis to first larval instar [9-11]. An analysis of the mitotic pattern during neurogenesis has revealed that neuroblasts are missing in the spider [1]. In addition, most of the neural precursors do not divide after invagination. This raises the question of how additional neurons are generated that contribute to the larval and adult CNS of the spider.
Results
In the spider Cupiennius salei the germband develops from aggregations of cells that form the cephalic lobe and the caudal lobe [12]. One to three prosomal segments are generated by a subdivision of the cephalic lobe, while the remaining segments arise sequentially from the caudal lobe, the so-called posterior growth zone [12,13]. At the beginning of neurogenesis (about 130 hours of development; stages after Seitz [12]) a longitudinal furrow forms that divides the germband into left and right parts that remain connected only at the cephalic lobe and the posterior growth zone. The two halves of the embryo move laterally until they finally meet at the dorsal midline (ca. 300 hours of developement). This process is called inversion [12]. The formation of neural precursors and the invagination of these cells occurs during inversion [1].
Secondary invagination sites form after invagination of the primary neural precursors
Although the invagination sites in the ventral neuroectoderm of the spider are generated in four subsequent waves over a time period of three days, the neural precursors detach form the apical surface at about the same time between 200 and 230 hours of development. Fig. 1A shows the final arrangement of the invagination sites shortly before the neural precursors loose contact to the apical surface. After invagination, the neural precursors differentiate and a neuropil develops at the basal side of the neuromeres (Fig. 1B, arrow). Since the epidermis arises lateral and medial to the ventral hemi-neuromeres (Fig. 1C, arrow), all cells of the central neurogenic region are eventually incoporated into the ganglion, i.e., the ventral neuroectoderm does not give rise to epidermoblasts and neuroblasts as in Drosophila melanogaster (see above). A detailed analysis of the morphology of the ventral neuromeres after invagination of the neural precursor groups revealed that the cells that remain apical form secondary invagination sites (Fig. 1B, asterisks).
Figure 1 (A-E): Morphology of the secondary invagination sites. Confocal micrograph (A, inverted) of a flat preparation of an embryo stained with phalloidin-rhodamine and light micrographs (B-D) and electron micrograph (E) of transverse sections through prosomal hemi-neuromeres. The midline is to the right. (A) Final pattern of the primary invagination sites in the opisthosomal segments 1 and 2. The invagination sites are arranged in 7 rows. The black dots correspond to the constricted cell processes of the individual precursor groups that are attached to the apical surface (arrow). (B) Morphology of the secondary invagination sites. At 250 hours the secondary invaginating cell groups (asterisks) are still attached to the apical surface. The individual groups are isolated by brighter sheath cells (arrowhead). The primary precursor groups have dissociated (arrow) and form basal cell layers. The longitudinal connective (lc) is already visible at the basal side. (C) The secondary invagination sites (asterisks) loose contact to the apical surface, when the epidermis (arrow) overgrows the ventral neuromeres. (D) After invagination the secondary neural precursors (asterisks) remain attached to each other forming epithelial vesicles. The cell processes run parallel to each other and extend to a lumen (arrow). (E) The cell processes (o) of the invaginating cells of a group are opposed to each other and the lumen between the cell processes is filled with microvilli (arrow). Cell junctions connect the individual processes (arrowheads). lc, longitudinal connective; o2 to o3, opisthosomal hemi-segments 2 to 3.
The secondary invagination sites can be distinguished from the primary invagination groups by several morphological features. (1) Each secondary invagination group contains up to 40 cells as compared to 5 to 8 cells that form the primary invagination sites (Stollewerk et al., 2001; Fig. 1B,1D; see also Fig. 4F). (2) The cell processes of the secondary neural precursors do not extend straight to the apical surface as the primary invaginating cells, but face each other (Fig. 1D,1E). Microvilli extend into the lumen between the opposite cell processes (Fig. 1E, arrow). (3) While the primary neural precursors detach from each other after invagination, the secondary invaginating cell groups remain attached to each other and maintain their epithelial character. (Fig. 1D,1E) The individual cell processes are connected by cell junctions (Fig. 1E, arrow heads). (4) In contrast to the primary precursors, the secondary invaginating cell groups are surrounded by sheath cells. These cells are visible in the light and electron microscope as brighter cells that separate the individual invagination sites (Fig. 1B, arrowhead; Fig. 2, asterisks; Fig. 3B). They extend long cell processes that ensheath each cell group (Fig. 2A, arrowhead). Interestingly, the sheath cells that are located in the apical cell layer form bizarre cytoplasmic shapes that extend into the cell-free space at the ventral side of the embryo (Fig. 2C, arrow). Double-stainings with a marker for cell nuclei and a dye that stains the actin cytoskeleton show that the nuclei of the secondary neural precursors are shifted basally similar to the primary invagination sites (Fig. 3A,A'; see also Fig. 1B,1D). The stained nuclei that surround the individual secondary invagination sites in the apical cell layer correspond to the sheath cells (Fig. 3A,A').
Figure 2 (A-C): Secondary invagination sites are surrounded by sheath cells. Electron micrographs of transverse sections through prosomal hemi-neuromeres. (A,B) Invagination sites (arrows) are surrounded by sheath cells (asterisks) that appear translucent in the electron microscope. The sheath cells extend processes (arrowhead) that enwrap the individual invagination sites. (C) Sheath cells that are located in the apical cell layer form bizarre shapes that extend into the cell free space at the ventral side of the embryo (arrowhead). The sheath cells are labeled with asterisks, the arrow points to an invagination site.
Figure 3 (A-B): The nuclei of cells within the secondary invagination sites are located basally. Confocal micrographs of flat preparations of embryos double-stained with phalloidin-rhodamine (red) and YOYO (green) (A,A') and single stained with phalloidin-rhodamine (B). (A,A') The apical optical section at 250 hours of development shows that the secondary invagination sites (arrow) are still attached to the apical surface. The nuclei of the secondary precursors are located basally, as revealed by the absence of nuclei staining in the apical cell layer. The asterisks in A' indicate the positions of the cell processes of the secondary invagination sites (compare to A). (B) The basal optical section shows the distinct morphology of the sheath cells (arrows) that subdivide the individual invagination sites.
Secondary invagination sites persist as epithelial vesicles
In contrast to the primary invaginating cell groups that are generated in four waves (see above), the secondary invagination sites appear almost at the same time (Fig. 4A). A detailed analysis of the ventral neuromeres of embryos stained with phalloidin-rhodamine, a dye that stains the actin cytoskeleton and accumulates in the constricted cell processes of the invaginating cells, revealed, that about 25 invagination sites are generated per hemi-segment. There is no clear dividing line between the formation of secondary invagination sites and invagination of the primary neural precurors. At 220 hours of development all secondary invagination sites are visible (compare Fig. 4A and 4B), while some of the primary precursor groups are still attached to the apical surface (Fig. 4A, arrowhead). However, at 240 hours all primary precursors have detached from the apical surface and dissociated (Fig. 4B; see also Fig. 1B). At about 250 hours, epidermal cells arise lateral and medial to the ventral neuromeres and overgrow the ventral nerve cord within 50 hours [2]. In Fig. 4C (280 hours of development) the border of the overgrowing epidermis is visible as a circle in the medial region of each hemi-neuromere. Although the secondary invaginating cell groups detach from the apical surface at this time, the individual cells of a group remain attached to each other and persist as epithelial vesicles (Fig. 4D; see also Fig. 1C). Due to morphogenetic movements at about 300 hours of development, the anterior-posterior extension of the individual hemi-segments is reduced leading to a rearrangement of the position of the epithelial vesicles (Fig. 4E,4F,4G,4H). After 350 hours 8 of the 25 invaginated cell groups are no longer visible indicating that the cells have detached from each other (Fig. 4F,4H). However, 10 cell groups are still visible at hatching (Fig. 4G,4I). Similar to the ventral neuromeres, groups of cells invaginate from the cephalic lobe neuroectoderm and persist as epithelial vesicles until larval stages (Fig. 4J). DiI labelling of cells within epithelial vesicles revealed that the cells of a group are attached to each other (Fig. 5A,5B,5C) and their short, thin cell processes run parallel to each other (Fig. 5B, arrow). They do not show any morphological features of differentiation, i.e. they do not grow long thin dendritic or axonal processes. These data show that 10 groups of neural precursors per hemi-segment do not differentiate during embryogenesis but give rise to neural cells that will be incorporated into the larval ganglia.
Figure 4 (A-j): Invagination of secondary neural precursors and formation of epithelial vesicles. (A-F) Confocal micrographs of flat preparations of embryos stained with phalloidin-rhodamine. (B-G) Flat preparations of the fourth prosomal hemi-segments. (A) At 220 hours about 25 secondary invagination sites form (arrow). There is no clear dividing line between the formation of secondary invagination sites (arrow) and invagination of primary neural precursors. Some primary invagination sites are still visible (arrowhead) The bars indicate the segment borders. (B) Apical optical section of the pattern of secondary invagination sites (arrow) at 240 hours of development. (C) Epidermal cells overgrow the ventral neuromeres between 250 and 300 hours (arrowheads) The arrow points to a secondary invagination group. (D) After invagination the individual cells of a groups remain attached to each other forming epithelial vesicles (arrow). (E) At 300 hours the anterior-posterior extension of the individual hemi-segments has been reduced leading to a rearrangement in the positions of the invaginated cell groups (arrow). (F) After 320 hours 8 of the 25 invaginated cell groups are no longer visible indicating that the cells have detached from each other. The arrow points to an invaginated cell group. (G) 10 cell groups are still visible at hatching (arrow). (H) Overview of the arrangement of epithelial vesicles (arrow) of the four prosomal hemi-segments corresponding to the four walking legs. The anterior-posterior reduction in size is clearly visible (compare to A). The bars indicate the segment borders. (I) Flat preparation of the prosoma at hatching. Epithelial vesicles are still visible (arrow). The bars indicate the segment borders, the arrowhead points to the midline. (J) Flat preparation of the brain at 350 hours. The arrow points to epithelial vesicles. ch, chelicera; l1 to l4, prosomal neuromeres corresponding to walking leg 1 to 4; leg 1, walking leg 1.p, pedipalp; ped, pedipalpal neuromere.
Figure 5 (A-C): DiI-labeling of cells within epithelial vesicles. Flat preparation of the fourth prosomal hemi-neuromere of an embryo labeled with DiI (red) and stained with phalloidin-rhodamine (green). (A-C) Invaginated cells in 40 segments of 10 embryos were labelled with DiI (red) and stained with phalloidin-FITC (green). The cells of a group (A, asterisks) are attached to each other (B,C large arrow head) and their short, thin cell processes run parallel to each other (B,C arrow). They do not show any morphological features of differentiation, i.e. they do not grow long thin dendritic or axonal processes. The small arrows (B,C) point to a cell of an adjacent invagination group.
Dissociation of epithelial vesicles is not associated with cell divisions
Analysis of the mitotic pattern during neurogenesis has revealed that the formation of the primary invagination sites is not connected with cell divisions [1]. In addition, mitotic activity seems to be restricted to the apical layer of the ventral neuroectoderm with the exception of a few cells indiciating that most of the invaginated primary neural precursors differentiate without further divisions. However, there are two waves of mitosis during the course of neurogenesis [1]. After formation of most of the primary invagination sites many cell divisions can be observed in groups of cells and single cells in the apical cell layer. The second wave arises when the primary precursors detach from the apical surface and the secondary invagination sites begin to form. Cell divisions are also restricted to the apical cell layer with the exception of a few cells [1]. These data indicate that the number of neuroectodermal cells is increased by cell proliferation prior to the recruitment of secondary neural precursors. A further analysis of the mitotic pattern during late embryogenesis with the mitotic marker anti-Phospho-Histon 3 and phalloidin-rhodamine revealed that only scattered mitotic cells are present in the ventral neuromeres. The pattern of cell divisions in the cephalic lobe and the prosomal segments (310 hours of development) shown in Fig. 6A is representative for the late embryonic stages. Since only a few mitotic cells are associated with dissociating epithelial vesicles (Fig. 6B,6C), it can be assumed that the secondary precursors differentiate without further divisions, similar to the primary neural precursors.
Figure 6 (A-C): Mitotic pattern in the ventral neuromeres after formation of the secondary invagination sites. Flat preparations of embryos stained with phalloidin-rhodamine (red) and anti-Phospho-Histon 3 (green). (A) Only scattered mitotic cells (arrowhead) are present in the ventral neuromeres after invagination of the secondary neural precursors (arrow). The pattern of cell divisions in the cephalic lobe and the prosomal segments at 310 hours of development is representative for the late embryonic stages. (B) Optical section through apical cell layers of the fourth prosomal hemi-neuromere. Only a few mitotic cells (arrowhead) are associated with epithelial vesicles. (C) A similar pattern is visible in basal cell layers of the same neuromere. The arrowhead points to a dividing cell, the arrow points to a dissociating epithelial vesicle. ch, cheliceral neuromere; cl, cephalic lobe; l1 to l2, prosomal hemi-neuromeres corresponding to walking legs 1 to 2; ped, pedipalpal hemineuromere.
achaete-scute homologues and neurogenic genes are re-expressed during formation of the secondary precursors
Two achaete-scute homologues have been identified in the spider [1]. CsASH1 is expressed like a proneural gene in the neurogenic regions prior to formation of the primary invagination sites and is necessary for the generation of neural precursors. CsASH2, in contrast, shows a pan-neural mode of expression: it is exclusively expressed in all invaginating neural precursors. Simlar to Drosophila melanogaster, the neurogenic genes Notch and Delta restrict the proportion of cells that adopt a neural fate at each wave of neural precursor formation [2].
During formation of the secondary invagination sites, the spider achaete-scute homologues and neurogenic genes [1,2] are re-expressed in the ventral neuromeres (Fig. 7). After invagination of the primary neural precursors, the expression of the achaete-scute homologues CsASH1 and CsASH2 and the neurogenic genes CsDelta1 and CsDelta2 is down-regulated, while CsNotch remains expressed at low levels in the ventral neuroectoderm (Fig. 7A,7B,7C,7D,7E). There is no clear dividing line between the invagination of the primary neural precursors and the formation of the secondary invagination sites (see above), which is also obvious by CsDelta1 staining: while CsDelta1 is down-regulated in the primary neural precursors (Fig. 7C, arrow), transcripts accumulate at high levels in the secondary invagination sites (Fig. 7C, arrowheads). Similarily, a transient stronger expression of CsNotch is visible in the secondary invaginating cell groups (Fig. 7E, arrow). Interestingly, CsASH1 is only expressed after formation of the secondary invagination sites, in single cells and groups of cells (Fig. 7F, arrow) while the gene shows a proneural mode of expression during formation of the primary precursors [1]. Like CsASH1, CsASH2 shows a pan-neural expression in the invaginating secondary precursors (Fig. 7G, arrow). CsDelta1 transcripts accumulate only in a subset of the secondary invaginating cell groups while CsDelta2 seems to be expressed in all of them (Fig. 7H,7I). CsNotch shows a ubiquituous expression in the ventral neuromeres (Fig. 7J).
Figure 7 (A-J): Proneural and neurogenic genes are re-expressed during formation of the secondary neural precursors. Flat preparations of the fourth and fifth prosomal hemi-segments after in situ hybridisation of whole embroys. (A-E) 220 hours of development, (F-J) 250 hours of development. Anterior is at the top, the midline to the left. (A) At 220 hours, CsASH1 expression has been down-regulated in all primary neural precursors (arrow) with the exception of one group (arrowhead). (B) At this time the pan-neural gene CsASH2 is still weakly expressed in the primary neural precursors (arrow). (C) CsDelta transcripts accumulate in the secondary invagination sites (arrow heads), while transcripts are down-regulated in the primary precursor groups. (D) A similar expression, although weaker, is visible after CsDelta2 in situ hybridisation. The arrow points to a region where CsDelta2 has been down-regulated, the arrowhead indicates expression in the secondary neural precursors. (E) CsNotch remains expressed at low levels in the ventral neuroectoderm. An up-regulation of CsNotch transcripts is visible in the secondary invagination groups (arrow). (F) At 250 hours CsASH1 expression can be detected in the secondary invagination sites (arrow), although it is not expressed in all of them. (G) CsASH2 seems to be expressed weakly in all secondary invaginating cells groups (arrow). (H) A high accumulation of CsDelta1 transcripts is visible in about 10 of the invagination sites (arrow), (I) while CsDelta2 seems to be xpressed in all invagination groups (arrow). (J) Cs Notch transcripts can be detected in all neuroectodermal cells at this time. l2 to l3, walking leg 2 to 3.
Discussion
Formation of epithelial vesicles – a conserved character in arthropod neurogenesis?
Analysis of the ventral neuromeres of spider embryos after invagination of the primary neural precursor groups revealed that secondary neural precursors arise during late embryogenesis that partially do not differentiate until larval stages. In contrast to the primary groups, the secondary invaginating cells do not detach from each other after invagination but maintain their epithelial character. In common with epithelial cells, they show a pronounced apico-basal polarity. The apical surface is covered with microvilli, while the lateral surfaces adhere to those of neighbouring cells of a group via specialized cell junctions, i.e. zonulae adhaerentes.
Although the formation of epithelial cell groups has not been observed in the ventral neuromeres of other arthropods, epithelial vesicles have been described during development of the stomatogastric nervous system and the brain in Drosophila melanogaster. After invagination of the individual neuroblasts that pioneer the frontal connective and recurrent nerve [14], three groups of cells invaginate from the stomatogastric nervous system primordium [15]. They loose contact with the surrounding stomodeal epithelium and form elongated, hollow epithelial vesicles, similar to the secondary neural precursors of the spider. Finally, they dissociate into apolar cells and are incorporated into different stomatogastric ganglia [15,16]. In a similar way, the vesicle forming the optic lobe invaginates from the posterior head region of Drosophila melanogaster embryos. In contrast to the stomatogastric vesicles, this cell group remains epithelial throughout embryogenesis and larval life [17].
It has been shown in Drosophila melanogaster that the Delta-Notch signaling pathway is involved in maintaining the epithelial character of the optic lobe and stomatogastric nervous system (SNS) precursors [16]. In Notch mutant Drosophila melanogaster embryos, cells with the identity of SNS and optic lobe precursors develop at approximately normal numbers, but they do not form epithelial vesicles. Instead, these cells appear as solid, irregular clusters of apolar cells [15-17]. In the spider, the function of CsNotch during development of the secondary neural precursors could not be analysed, because injection of ds CsNotch RNA leads to a premature differentiation of neural precursors due to an ealier function of CsNotch in lateral inhibition [2]. However, the up-regulation of CsNotch in the secondary invagination sites suggests a role in formation of the epithelial vesicles (see Fig. 7E).
Similar to Notch, the proneural genes achaete, scute and lethal of scute are continuously expressed in the SNS of Drosophila melanogaster [18]. Loss of proneural gene function leads to the absence of a subpopulation of SNS precursors and subsequently to an irregular invagination of the SNS placode. Furthermore, proneural genes seem to promote the dissociation of SNS precursors from the epithelial vesicles, since loss of proneural gene function results in a delay of this process. Similar to Drosophila melanogaster, both achaete-scute homologues of the spider are expressed in the epithelial vesicles that are formed by the secondary neural precursors. However, in contrast to its function in the recruitment of the primary neural precurors, the expression pattern of the spider proneural gene CsASH1 does not suggest a role in the establishment of the secondary neural fate. CsASH1 transcripts can only be detected in subsets of neural precursors after generation of the secondary invagination sites. A similar expression pattern can be observed for CsASH2, although the transcripts in the primary neural precursors are down-regulated later than the CsASH1 transcripts. The function of these two genes during generation of the secondary invagination sites and the formation of the epithelial vesicles could not be analysed. Due to their ealier role in the recruitment and differentiation of the primary precurors, injection of ds RNA of either gene leads to severe morphological defects in the ventral neuroectoderm [1].
The formation of epithelial vesicles leads to a delay in neural differentiation
As revealed by DiI-labeling, secondary neural precursors within epithelial vesicles do not show any morphological features of differentiation. Obviously, the formation of epithelial vesicles after invagination leads to a delay in the differentiation of the corresponding neural precursors. Although the epithelial vesicles are formed at about the same time, they dissociate from each other subsequently. About half of them are still visible at the end of embryogenesis indicating that they provide neural precursors for larval stages.
In insects a distinct mechanism has evolved for generating larval neural precursors during embryonic life. After a phase of cell cycle arrest from late embryogenesis to first larval instar, 'embryonic' neuroblasts proliferate again. [9,10]. Both in Drosophila melanogaster and in Manduca sexta, the larval progeny of these neuroblasts accumulate in groups of cells that are separated by glial cell processes and do not finish their differentiation until the onset of metamorphosis [10,19]. It has been shown that the secreted glycoprotein anachronism (ana) regulates release of central brain neuroblasts from cell cycle arrest [20]. Ana is expressed in glial cells that ensheath central brain and optic lobe neuroblasts. In ana mutant larvae, neuroblasts proliferate earlier than in normal development which in turn leads to a premature differentation of neurons in certain brain regions. This heterochronic defect has an impact on the axonal pattern: the ana mutant phenotype ranges from subtle missrouting of fiber tracts to massive disorganization that affects the entire optic lobe [20]. These data show that factors regulating the differentiation state of neural precursors can have an important influence on the organization of neural networks.
The distinct morphology of the sheath cells in the spider neuromeres, i.e. their translucent cytoplasm, the absence of microvilli and the extension of cell processes that enwrap the neural precursors suggests that these are glial cells. Further analysis will show if these cells express genes that can influence the epithelial organization, i.e. the differentiation state of the secondary neural precursors, comparable to the glial cells of Drosophila melanogaster.
Formation of epithelial vesicles – a basal mode of neurogenesis?
A recent study on neurogenesis in the onychophoran Euperipatoides kanangrensis shows that, rather than forming individual invagination groups, the whole medial regions of the hemi-segments invaginate into the embryo [21]. The invaginated cells remain attached to each other forming transitory epithelial vesicles. Although the phylogenetic position of Onychophora is still being debated, they are generally placed basally in the arthropodan clade [22-27]. Since onychophorans have retained many pleisiomorphic features, it can be assumed that they reflect a basal mode of CNS development [28-30]. This leads to the following model of changes in neural precursor formation during arthropod evolution: the basal mode of neurogenesis is the invagination of one large cluster of neural precursors from the central region of each hemi-neuromere. These clusters form transitory epithelial vesicles in the ventral neuromeres [31]. An advanced mode of neurogenesis is seen in chelicerates and myriapods: groups of cells that arise in several waves at stereotyped positions invaginate form the ventral neuroectoderm [1,3,4]. Interestingly, both chelicerate and myripod neurogenesis reflects some ancestral features. In the spider, epithelial vesicles are formed by secondary invaginating cell groups, while in myriapods the whole central regions of the hemi-neuromeres sink into the embryo after invagination of individual groups of neural precursors [3,32]. An even complexer mode of neurogenesis is seen in insects and crustaceans: individual neuroblasts are singled out from the ventral neuroectoderm that divide in sterotyped patterns to give rise to ganglion mother cells and finally neurons [33-42].
Conclusions
To summarize, the model suggests that the invagination of large groups of neuroepithelial cells that form transient epithelial vesicles represents the basal mode of neurogenesis. Subsequently, more parameters have been introduced to the process of neurogenesis during arthropod evolution, i.e. sequential invagination/delamination of neural precursors and connection between neural precursor formation and cell proliferation. It can be assumed that these additional parameters have contributed to the diversity of neural precursor populations. This diversity might have been used as an evolutionary tool to develop neural networks that are adapted to the specialized behaviour and morphologies of the individual arthropod groups.
Materials and Methods
Cupiennius salei stocks
Fertilized females of the Central American wandering spider Cupiennius salei Keyserling (Chelicerata, Arachnida, Araneae, Ctenidae) were obtained from Ernst-August Seyfarth, Frankfurt, Germany. Embryos were collected as described before [1].
Histology and stainings
Whole-mount in situ hybridisations were performed as described [1]. Phalloidin-rhodamine staining of spider embryos was performed as has been described for flies [43]. Anti-Phospho-Histone 3 immunocytochemestry has been performed as described [1].
DiI-labeling
After chemically removing the chorion, embryos were fixed in 4 % formaldehyde in PBS and 1 vol heptane. The vitelline membrane was removed with needles and the embryos stained with phalloidin-FITC. Flat preparations of these embryos were attached to a coverslip with a double-sticky tape and covered with PBS. 1,1'-dioctadecyl 3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) was dissolved in ethanol and applied with glass needles. A small droplet of DiI was injected into single or several cells of an invagination group using a 63 × water-immersion lens and a FITC filter on a Zeiss fixed stage microscope and a micromanipulator.
Acknowledgements
I thank Diethard Tautz for continued support, critical discussion and helpful comments. Many thanks to Pat Simpson, Volker Hartenstein and Diethard Tautz for critical reading of the manuscript. I am grateful to José A. Campos-Ortega for critical comments on the project and for providing access to the electron microscope and the histological equipment. I thank Michael Bate for critical comments on the project and access to the injection facility. Thanks to Ernst-August Seyfarth for providing the spiders. This work was supported by a grant from the Deutsche Forschungsgemeinschaft.
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| 15679931 | PMC544935 | CC BY | 2021-01-04 16:38:33 | no | Front Zool. 2004 Oct 25; 1:3 | utf-8 | Front Zool | 2,004 | 10.1186/1742-9994-1-3 | oa_comm |
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Front ZoolFrontiers in Zoology1742-9994BioMed Central London 1742-9994-1-41567992010.1186/1742-9994-1-4ReviewThe structure of biodiversity – insights from molecular phylogeography Hewitt Godfrey M [email protected] Biological Sciences, UEA, Norwich NR4 7TJ, UK2004 26 10 2004 1 4 4 18 10 2004 26 10 2004 Copyright © 2004 Hewitt; licensee BioMed Central Ltd.2004Hewitt; 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.
DNA techniques, analytical methods and palaeoclimatic studies are greatly advancing our knowledge of the global distribution of genetic diversity, and how it evolved. Such phylogeographic studies are reviewed from Arctic, Temperate and Tropical regions, seeking commonalities of cause in the resulting genetic patterns. The genetic diversity is differently patterned within and among regions and biomes, and is related to their histories of climatic changes. This has major implications for conservation science.
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Introduction
Phylogeography, named by Avise et al in 1987 [1], is a recent and rapidly developing field that concerns the geographical distribution of genealogical lineages. It grew from the newly acquired technical ability to obtain DNA sequence variation from individuals across a species range, and from this to reconstruct phylogenies. These are then plotted geographically to display their spatial relationships and deduce the evolutionary origins and history of populations, subspecies and species [2,3]. Genetic relationships between species based on polytene chromosome banding patterns had been used earlier to deduce the geographic history of colonization and speciation by Drosophila of the Hawaiian Islands [4], and allozyme variation may still complement DNA information, but the ready access to mitochondrial (mt) DNA sequences opened the door to most animal species and generated this new field [5].
DNA methods
Whilst mtDNA has lead the way in animal phylogeography, other DNA sequences are used, most commonly chloroplast (cp) in plants and non-coding nuclear (nc) regions in both animals and plants. MtDNA has a relatively fast rate of nucleotide divergence, well suited to examining events over the last few million years, but those of cpDNA and ncDNA are an order of magnitude lower and consequently less useful for such young divergences. More slowly evolving sequences are required for deeper phylogenetic history. For more recent events, like the last 10 thousand years, highly variable markers are needed, such as microsatellites and AFLPs, but whilst useful for population studies they suffer from homoplasy and produce equivocal genealogies [3].
Techniques for obtaining DNA sequence information are still advancing rapidly, with whole genome sequences being produced in a growing number of organisms. This allows sequences and markers to be identified and developed for many types of investigation, and some will be useful for phylogeographic studies. In particular, it is clear that genealogical data is required from several independent nuclear loci to provide a fuller and more reliable history of the species [6]. Single nucleotide polymorphisms (SNPs) are also becoming available across the genome, which will produce comprehensive measures of genetic diversity and allow the construction of better population histories [7].
Analytical approaches
The advance in DNA technology is producing a wealth of data for individuals, populations and species, and there are concomitant developments in analytical methods to divine demographic history and evolutionary relationships, and to test their significance. This progress in analysis is facilitated by access to increasingly powerful desktop computers on which the increasingly sophisticated software can be used. Haplotype sequences of a particular DNA region can be ordered into a genealogical tree or network, and hence produce their phylogeny. When combined with their population frequency and geographic distribution, this provides a strong basis for inferences on the evolutionary history of the populations and species. The usual phylogeographic approach is to build a phylogeny from haplotype sequences using distance, parsimony and maximum likelihood methods and then represent the lineages geographically. There are several approaches that are regularly used, such as DNA Distance Phylogeography, Nested Clade Analysis, Haplotype Networks, Sequence Mismatch Distribution and Genetic and Demographic Simulation, e.g. PAUP and GeoDis [8-10]. This last approach uses computers to explore broadly how DNA markers evolve in specified molecular, spatial and demographic conditions over history, and is being used increasingly [11]. Recent developments seek to use the genetic data to estimate the demographic history of a population, the dates of historical bottlenecks or expansions, the size of ancestral populations, the location of refugial areas, the dates of divergence, the extent of migration and gene flow, the extent of fragmentation, and the sequence of such events to produce the present geographic distribution of genotypes, e.g. [12-15]. We can expect further developments to provide even more discriminating analyses.
Each DNA sequence has its own genealogy and they may evolve at different rates. Furthermore, the various methods of analysis probe different aspects of the molecular and spatial history. Consequently, to reconstruct a species phylogeographic history one would ideally like to use a range of sequences (including nuclear, cytoplasmic, sex-linked, autosomal, conserved, neutral, high and low mutation rate) and apply a suite of pertinent analyses. This is not easy and often not possible with resources available. However, technological advances for molecules and computers have been explosive in the past decade, making much more detailed analysis possible today than only a few years ago, and this looks set to continue.
Paleoclimate and Paleobiology
The very different field of paleoclimatology is also experiencing great advances. The results of these are most pertinent to phylogeographic explanations, since they reveal the past environmental conditions and changes that have molded the evolutionary processes producing the present genetic structure. They provide a framework in which the phylogeny may be reconstructed. Past conditions can be deduced from carbon and oxygen isotopes, radiolarian skeletons, pollen grains and other residues from the sea bed, lake bottoms and ice sheets [16]. Novel information sources like insect exoskeletons, coral terraces and stalagmites are adding to this [17-20]. Such records show that earth's climate has been cooling for some 60 my with periodic (21, 41 and 100 kyr) global oscillations producing increasingly severe ice ages through the Quaternary (2.4 my). These involved greatly enlarged ice sheets and surrounding permafrost, and the lower temperatures and reduced water availability caused great changes in the distribution of species as demonstrated by the fossil record [16,21]. Nested within the major 100 kyr cycles are millennial scale oscillations, which can occur rapidly and are often severe [22,23]. Changes of 7–15°C may occur in decades and persist for centuries, as happened most recently in the Younger Dryas (11 kya), where fossils record shifts in the distributions of species.
These major changes in distributions of species occurred latitudinally as the ice sheets advanced and retreated, altitudinally in major mountain regions, and also longitudinally where new dispersal routes became available, as for example the Bering land bridge produced by the lowered sea level. The demographic fluctuations and adaptive challenges produced by such range changes would have had both stochastic and selective effects on the genetic variation and architecture, and the consequences of these can be studied by genetic and phylogeographic approaches. Thus the once distinct fields of paleobiology and phylogeography are now being combined, and have much to tell us about how present biodiversity was structured.
Fossil and Genetic Signals of Range Changes
With some effort it is now possible to obtain DNA data from specimens across the present range of a species, but fossil data is often more limited or absent. The most useful fossil data are for Europe and North America, which have extensive networks of pollen cores; a few span 3 ice ages (400 kyr), several reach the last interglacial (125 kyr), and a larger number cover back to the last glacial maximum (LGM 23-18 kyr) [21]. There are also some helpful detailed series of beetle exoskeletons [24]; animal bones and plant macrofossils tend to be localized and discontinuous, but are nonetheless useful markers of time and place. Reconstructions of paleovegetation have been made, e.g. [25], which are quite detailed from the LGM to the present, and when coupled with other fossil evidence indicate the extent and rapidity of changes in species distributions.
During the LGM the ice sheets and permafrost extended towards lower latitudes, so that generally species distributions were compressed toward the equator. Boreal species survived south of the ice in North America and Europe, but large areas of the north eastern Palearctic and Beringia remained ice free and some cold-hardy species appear to have survived here. Temperate species survived further south where habitats occurred to which they were each adapted. In Europe the disjunct southern peninsulas of Iberia, Italy and Balkans were particularly important, while in North America many temperate locations occurred around 40°N between the East and West coasts. Nearer the equator the pollen record is not extensive, but conditions were generally drier in the LGM and Tropical habitats were reduced while desert and savanna increased.
As a consequence, the habitats of many Boreal, Temperate and Tropical species were reduced and fragmented and they survived in refugia; but for some their habitats expanded, like those in the tundra and savanna. As the climate warmed after the LGM and the ice retreated, many Boreal and Temperate species were able to expand their ranges, as were some Tropical species. In some cases the refugial populations died out, but particularly in mountainous regions they could survive by ascending with the climate and their niche, as for example in the Alps, Andes, Appalachians and Arusha mountains. Such refugial regions allow the survival of species through several ice age cycles by ascending and descending to track their habitat, e.g. [26].
Such events modify the genetic content and structure of populations within species, and leave some traces for which we may search. Populations, races and subspecies that have been effectively separated for several glacial cycles will show divergence through the accumulation of neutral and possibly selected DNA changes. The extent of this divergence will be proportional to the time of separation. The haplotype tree or network of an evolving DNA sequence will reflect population expansions and contractions. Increasingly these effects can be analyzed, e.g. [27] and placed in some order of occurrence. When the geographic positions of haplotypes are included, a further range of deductions is possible. For example, recently derived populations will contain a sample of the same haplotypes as the parent populations, which combined with paleo-information allows colonization routes to be deduced [28,29]. The extent of distribution of younger haplotypes compared with that of older ones in the tree provides information on the past fragmentation of populations and processes involved in colonization [30]; this can also be combined with paleo-information to deduce the intraspecific phylogeographic history.
Higher Latitudes – the Arctic
Most phylogeography has concerned Temperate biota [2,26], but recently a number of species from higher latitudes have been analysed in sufficient detail across their range to provide some first genetic insights into their biogeographic history. These include mammals, birds, fish, crustaceans and plants adapted to such cold conditions [31,32]. Table 1 contains some major studies of Holarctic animal species complexes. During the LGM the greatly extended Arctic ice sheets forced such species south, as evidenced by fossil records in Europe and North America. At the same time, large areas of Northeast Asia and the NW corner of North America were covered in permafrost but not glaciated. Fossil evidence suggests that these also contained refugia, particularly Beringia [18,33,34] which with lowered sea level joined Asia and America across the Bering Straits. The different range changes involved would be expected to have various effects on the genetic diversity that may have left marks of their occurrence and extent.
Table 1 Animal species with Holarctic ranges showing distinct phylogeographic pattern, with some indication of their possible divergence times, glacial refugia and genetic signals of population history. CA = Circumarctic, HA = High Arctic. PA = Palearctic, NA = Nearctic, BE = Beringia, GL = Greenland, NT = North Temperate.
Species Range Phylogenetic Divergence (Myr) Likely Refugia
(fossil evidence *) Genetic signals of range changes Authors & Reference
Larus argentatus spp
Herring gull complex
CA HA
(+EurAsia Lakes) 9 clades
<0.5%
(0.1–0.3) Atlantic
Aralo-Caspian Allopatric fragmentation
Bimodal mismatch
Recent expansions
Hybridizations Liebers et al [96]
Rangifer tarandus
tundra reindeer
CA HA 7 clades
1–2%
(0.1–0.3) Beringia-Asia *
W EurAsia*
N America 150 kyr expand
15 kyr expand
Ragged mismatch Gravlund et al [97]
Flagstad & Roed [98]
Lemmus ssp
true lemmings HA PA
NA 4 clades
3.8–7.9%
(0.5–1.0) Beringia
E Asia
Siberia*
N America* Expand
Few haplotypes
Few haplotypes
Expand Fedorov et al [99]
Dichrostonyx ssp
collared lemmings HA
PA NA GL 6 clades
1–7%
(0.1–1.0) Beringia*
Arctic Islands
E Asia
C&W Siberia* Not to east
Expand
Low diversity
Low diversity Fedorov & Stenseth [100]
Microtus oeconomus
root/tundra vole
HA PA BE 4 clades
2.0–3.5%
(0.2–0.6) Beringia
S Urals*
Caucasus
C Europe* Not far
Few haplotypes
N expansion
N expansion Brunhoff et al [101]
Microtus agrestis
field vole
not HA, PA 3 clades
0.5–5.2%
(0.1–0.6) S Urals*
Carpathians*
Iberia Across Asia
N&W expansion
Not far Jaarola & Searle [102]
Lagopus mutus
rock ptarmigan
CA HA 7 clades
0.21–1.12%
(0.05–0.1) Multiple, eg Greenland – Beringia/Aleuts
Siberia Several recent Expansions
Only 4% diversity within lineages Holder et al [103, 104]
Calidris alpina
Dunlin (migrant)
CA 5 clades
1.1–3.3%
(0.1–0.3) W Africa
Arabia
SE Asia
C America W EurAsia
Siberia
Beringia
Canada Wennerberg [105]
Wenink et al [106]
Daphnia pulex
Waterflea (clonal)
CA HA 7 clades
0.5–3%
(0.2–1.5) Periglacial
Some more local clones Mixing, but some N Amer/Eurasian difference Weider et al [107]
Troglodytes troglodytes
Winter wren
NT not HA
EurAsia BE N Amer 6 clades
3.0–8.9%
(0.5–1.6) NW Amer
NE Amer
NE Asia
C Asia, S Europe Series of glacial vicariances
Recent expansions Drovetski et al [38]
Cleithrionomys rutilus/glareolus/gapperi
red-backed voles
HA PA NA BE 12 clades
1–10%
(0.1–1.8) C Europe*
E Asia*
Beringia
N Amer* Several colonizations of N Amer across Beringia from Asia Cook et al [37]
Distinct Parapatric Clades, Refugia and Range Changes
The phylogeographic structure, in terms of distinct regional DNA clades, is very marked in some species like the lemmings, voles and wren, moderate in the ptarmigan and dunlin, and less in the more mobile waterflea, reindeer and herring gull. The extent of DNA divergence between major clades in small mammals would suggest effective separation of up to 1 Myr, some 5–10 full glacial cycles, with further subdivision for shallower clades in more recent ice ages. In the gull, ptarmigan and reindeer the divergence among clades is low, indicating events occurring in the last or penultimate glacial cycles. Such recent structure would suggest that these species came from or were reduced to a small ancestral population in the late Pleistocene.
The deeper clades of the true and collared lemmings, the root and field voles, and to some extent the shallow ones of the ptarmigan and dunlin, are remarkably parapatric and many contacts between them coincide around major features like the Urals, Lena, Kolyma and MacKenzie Rivers (Fig 1) [32]. Regions where several subspecific and sister-specific boundaries coincide, called suture zones [35] have been recorded in North America and Europe, and are probably due to species having similar range changes and refugial areas [29]. Thus these regional parapatric genomes seem to have been diverging separately over a number of ice ages, with distinct refugia from which they colonized to fill their individual interglacial distributions. The pattern of range changes may not be exactly the same through each cycle, but there is no sign of genetic mixing among mtDNA clades, and the major boundary features and refugia are likely to have been similar over the last few ice ages. Other taxon contacts occur in these regions in groups like birds and butterflies, and it will be informative to investigate their phylogeographies to look for commonalties and causes.
Figure 1 A polar projection showing the general regions of contact between diverged DNA clades of six Holarctic species (see text and references for details and Latin binomials). Note the clustering near features like mountain ranges and major rivers. The Scandinavian cluster, which includes a number of other species, forms where the last remnants of the ice cap melted. Last glacial ice caps and sheets are in white, and tundra is darker grey (I am grateful to Richard Abbott for the basic map).
Each distinct regional clade would have had its own refugium, but the location of these remains to be determined. There are late glacial fossils of small mammals in Central Europe and also near the southern Urals, which were both parts of the extensive tundra and could have been refugia for European and west Asian clades. An additional refugium in east Asia would account for the clades meeting in the regions of the Lena and Kolyma Rivers, while fossils for a number of species in Beringia indicate that it was probably a significant refugium [32]. A recent detailed examination of the phylogeography of the tundra vole in Beringia [36] pinpoints the contact between the Central Asian and Beringian clades on the Omolon River in the Kolyma uplands, which formed a partially glaciated barrier during the last ice age. This would obviously affect other species and provide the western boundary to many Beringian distributions. The equivalent boundary of Beringia in the east was formed by the North American ice sheets which fringed Alaska from the MacKenzie River to the Aleutian Ranges.
The contractions, expansions and distant colonizations involved in these late Quaternary range changes would have influenced the genetic diversity, and should have left some signs. Low haplotype diversity and shallow clades are expected when populations have been severely contracted, and the age of the subsequent expansion may be gauged by mismatch analysis. The structure of haplotype networks and nested clades also provide indications of such events. Many of the populations and clades reported show these features (Table 1 and references). For example, tundra reindeer, herring gulls and rock ptarmigans have shallow clades diverging in the recent ice ages and populations with mismatch analyses indicating postglacial expansion and colonization of high Arctic regions. In those species like the lemmings and voles where DNA lineage divergence goes back several ice ages to perhaps the onset of more severe glaciations some 0.9 My, most clades are shallow with low diversity, particularly those for Central Asia, suggesting extensive colonization from a much reduced refugial source. This is an emerging feature of some significance. It does seem that even for cold adapted species life was hard in Central Asia during the ice age and it provided few refugia.
Beringia – an Arctic Refugium
Beringia, which spans Eastern Siberia and Alaska, was united and disjoined each Quaternary ice age by changes in the sea level of some 120 m. It was only partly glaciated and fossil evidence shows it supported a mixture of tundra habitats, which allowed it to act as a causeway for continental migration between Asia and America for various species, including humans. The history of Beringia is currently of interest and some of the lower parts now under the Bering Sea probably had conditions suitable for mesic tundra species even in colder times [18]. Several species have a distinct genetic clade across Beringia and some of these show higher diversity here than where previously glaciated regions were colonized, supporting its role as a glacial refugium [32]. A careful analysis of genetic diversity in the tundra, or root vole, Microtus oeconomus through Beringia indicates that the Beringian voles probably expanded from a population with low genetic diversity, which had colonized from Central Asia in the penultimate glacial cycle [36]. A broader phylogeographic study on related taxa of red-backed voles, Clethrionomys, demonstrated that North America has been colonized successfully from Asia at least twice, possibly three times in the mid-Pleistocene, and C. rutilus reached Alaska quite recently with the first Nearctic fossils in the Holocene [37]. The phylogenies of a number of other species, including Homo sapiens also indicate Beringia as a colonization corridor.
Analysis of the DNA differences among these Clethrionomys species reveals that they have been diverging from the Early Pleistocene, whilst the divergence within the other listed species is much younger (Table 1). An exception is the winter wren, where there are six clades that appear to have begun diverging some 1.6 Mya, suggesting possible cryptic species [38]. These distinct clades contain low diversity and signs of contraction and expansion. The species does not inhabit truly high Arctic habitats, reaching down to more Temperate latitudes. It would seem that its Holarctic range was progressively broken up by the increasingly severe Pleistocene glaciations, with connection across the oceans, Beringia and the centres of the continents becoming more difficult or impossible as its more Temperate range was forced south.
Mid Latitudes – Temperate Regions
So called Temperate species span a wide range of latitudes, since the climate determinants like insolation, oceans and altitude may produce suitable habitats between 20° and 60°. In Eurasia and North America those more cold hardy species generally have more northerly distributions than those better adapted to more southerly warmer climes, and this will also be reflected in altitudinal range differences. This would also be true of distributions in glacial periods, so that refugia for north Temperate species were nearer to the ice and permafrost than those of more southerly ones. Fossil evidence is very important in properly determining such Pleistocene range changes [39]. In the Arctic species considered (Table 1), the winter wren and field vole provide examples of low Arctic/north Temperate species in what is a progression of adaptations, niches and ranges on the cold to warm axis from pole to equator.
Refugia and Colonization in Europe and North America
Paleoclimate and phylogeography have been most researched in Temperate Europe and North America, generating many particular examples and several more general conclusions [2,26]. In particular, the similarity among DNA haplotypes across a species range allows the deduction of which northern populations came from which southern populations, and their likely postglacial colonization routes. The fossil record underpins these deductions, particularly for refugial areas and times of colonization [26,28]. Thus many species survived the LGM in southern Europe, the centre and north being covered in tundra and ice. Fossil and DNA evidence show that the peninsulas of Iberia, Italy, Greece and the Balkans were major refugia and contributed variously to the postglacial recolonization of the north. Whilst a few mobile species crossed from North Africa, the Mediterranean Sea appears to have been a major barrier throughout the Quaternary. For many species these southern refugial areas currently contain much genetic diversity for haplotypes, lineages and subspecific taxa. These southern parts of Europe are also mountainous, so that species may survive by ascending and descending with climate changes. The extent of genetic divergence among these peninsular populations clearly indicates that for many species they have been effectively separate for several to many ice ages. Importantly, this separate refugial survival is seen as a causative factor in divergence and speciation [3,29]. It is possible to deduce pathways of genetic divergence in a geographical framework that has produced the continent's diversity of populations, subspecies and species.
The fossil record, in particular that of pollen and beetles, shows that postglacial colonization was to a large extent a property of the individual species niche and the distribution of its habitat. Nonetheless, the patterns of DNA divergence within species have some common features, which argue for common colonization processes and routes [29]. Temperate species often show reduced haplotype diversity in the north, which is considered to be the result of rapid colonization with repeated founder events. This is seen in many species in Europe and North America, e.g. [28,40-46]. Furthermore the recolonized areas are often a broad patchwork of distinct genomes that have emanated from the different refugia, and which usually form hybrid zones where they make contact. These hybrid zones in different species often appear to be clustered together and so may be considered to belong to suture zones [29,35].
In Europe the Balkan haplotypes and genomes provided the main source for postglacial colonization for many species, while less came from Iberia and few came from Italy, probably hindered by the ice-capped Pyrenees and Alps. Species that exemplify these different patterns of colonization are the grasshopper, Chorthippus parallelus, the bear, Ursus arctos, and the hedgehog, Erinaceus europaeus/concolor [29]. Freshwater fishes like the chubb, Leuciscus cephalus, often show colonization by different haplotypes up the Danube and Dneiper Rivers from the Black Sea (Fig 2) [32]. Many European species phylogeographies are emerging and a considerable number broadly show these distribution patterns and probably followed similar colonization routes despite differences in their niche, mobility and life history. This apparent and remarkable commonality would seem to be a result of colonization following postglacial climate change in Europe's particular geography of southern peninsulas, transverse mountain ranges and northern plains. It demonstrates the explanatory power of combined phylogeography and paleoclimatology.
Figure 2 Likely postglacial colonization routes from refugial areas in Europe and North America for a distinctive sample of species that have been deduced from DNA haplotype relationships. Note that regions like central Scandinavia, Britain, the Pacific North West and central Canada contain a mixture of species whose genomes have come from different refugia (see text for discussion).
A particularly interesting consequence of these various colonization routes is that northern regions like Scandinavia and the Pacific NW of America have biotas that are mixtures of species whose genomes came from different refugia [26]. Thus in Central Scandinavia the grasshopper genome came from the Balkans, the bear from Iberia and Russia, the hedgehog from Italy and the chubb from the Black Sea. For the NW corner of America, where Alaska, the Yukon and British Columbia meet, DNA evidence suggests that it has been colonized sequentially from four directions; (1) initially from the south along the coast by the long-tailed vole, dusky shrew, ermine and marten, (2) then from the south by an inland route by all but the marten, (3) from Beringia by the ermine, and (4) from the Appalachians by the marten, and possibly bears and chickadees (Fig 2). Several plant species also colonized probably along the two southern routes [32]. Such mixed biotas carry a number of important implications. They mean that the component species from different refugia have not been evolving together during the previous glacial periods, so any close coadaptation must be either postglacial, or possibly survive from when their distant ancestors were sympatric. More general species-wide coadaptations may be maintained if the different species survive together in refugia. Where genomes from two or more refugia come together, genetic diversity will be increased by the presence of diverged lineages, as seen in hybrid and suture zones. Two populations in the same region living in similar habitats, but from different colonizing refugia will possess very different alleles and genomes, while conversely two very distant populations in distinct habitats may have the same refugial genome. This points to the importance of population history in the process of post glacial adaptation, and our understanding of it.
Mediterranean Latitudes – 30–40°N
The Mediterranean Sea, with Europe's refugial peninsulas and mountains in the north and North Africa to the south lies between roughly 30°N and 40°N, where at these latitudes in North America there are the Southern States across the Appalachians and Rockies to California. While in Europe the Scandinavian ice sheet came down to Warsaw about 52°N with extensive tundra to the south, the Laurentide ice sheet reached near 40°N. below the Great Lakes, with very little tundra and steppe. Such contrasts in geography have produced differences in the phylogeography of species, but there are also similarities. Species in these southern regions generally contain greater diversity for alleles, populations and subspecies, which in several form distinct geographic genomic patches. The divergence among southern lineages and patches is often deeper than further north, indicating a longer survival and probably in the same region. This southern divergence is often estimated to be over many ice age cycles from the Early Pleistocene or even the Pliocene for some species [26]. The best-studied regions in these latitudes are Iberia [47], South Eastern USA [2] and West Coast USA [48-51].
Molecular phylogeography began in the SE USA [1] and many terrestrial and aquatic species there have marked genetic substructure with concordant genomic boundaries [52]. A number of recent studies in Iberia, covering a range of organisms, e.g. beetles [53], lizards [54], salamanders [55], woodmice [45], rotifers [47], and several plants, also show this intraspecific diversity and substructure, with lineages from as early as the Pliocene and often in mountain regions. Likewise West Coast phylogeography for salamanders [56], woodrats [51], shrews [50], frogs [57] and other species from California and the Cascades [49] reveals geographically structured lineages diverging from the Pliocene and Pleistocene. This pattern and divergence would seem to be a product of the geological and climate changes that have occurred, involving major mountain building and Quaternary ice ages. Lineages and populations from the northern parts of such southern regions, like the southern Appalachians, west and central Iberia and the northern Cascades, appear to have provided the main source for northward postglacial colonization, while genomes to the south survived with altitudinal shifts in broadly the same regions [32].
Species with distributions north and south of the Mediterranean Sea must have managed to cross this now major barrier to terrestrial organisms at some stage before or since the opening of the Gibraltar Straits some 5.3 Mya after the Messinian crisis [58]. If this was before 5.3 My, they are likely to have diverged to sister species if there has been little gene flow. There are now phylogeographic studies on a few species both terrestrial and volant. In terrestrial species of salamanders Salamandra spp and scorpions Buthus spp [59,60] the DNA data shows that the N African/European divergence is old, before the opening of the Gibraltar Straits. While in the woodmouse Apodemus sylvaticus [45] the N African haplotypes appear recently derived from southern Iberia, possibly transferred by humans. Interestingly an old divergence in holm oak, Quercus ilex, may also perhaps be due to humans [61]. Five flying species have been examined, of which the chaffinch, bearded vulture and barbastelle bat [62-64] have DNA phylogenies that indicate the Gibraltar Strait has not been a major barrier. In dragonflies Calopteryx spp [65] the North African genotypes are related to the Italian ones, and in honey bees [66] some African mtDNA haplotypes are found in south Iberia and Sicily, but nuclear markers indicate little migration. These latter two species and the bearded vulture appear to have crossed the Sicilian Channel, which was also narrow during the lowered sea level of the pleniglacials.
Lower Latitudes – Tropics and Savannah
Much of Africa, South America, South East Asia and North Australia lie in the Tropics. They are rich in species diversity, but with a few notable exceptions little is known of their phylogeography or their paleobiology [26]. During the recent ice ages the climate was colder and drier in the Tropics, with increased deserts and savannah and reduced rain forests. The pollen record is unfortunately poor, but it seems that forest species descended the mountains (~6°C lower LGM), while lowland forest species may have survived in many local wet places and gullies [16,67,68]. It would seem clear that even tropical biotas have undergone repeated changes as a result of climatic oscillations through the Quaternary [26,69].
Wet Tropics
Several phylogeographic studies from American and Australian rainforests and a few from Africa and Asia indicate that there is great genetic diversity produced by a complex history often diverging in the Pliocene [26,70]. A nice example of this has been studied in the montane forests of the central Divide of Costa Rica, where a North American salamander Bolitoglossa has radiated into tropical Middle America [71]. There are strong allozyme and mtDNA differences between several populations only a few kilometres apart (Dnei 0.18, cytb 4%), and 2 putative species within 10 km (Dnei 0.45, cytb 9%). Such genetic distances indicate divergence from the late Pliocene through the Pleistocene. This has involved several adaptations to elevation zones that would have been amplified by local topographic isolation and climatic oscillations. These amphibians may have peculiar attributes, but phylogeographies of birds and freshwater fish in Middle America are also complex with many lineages [72]. There are few studies yet, but it may be that the phylogeographic status of Bolitoglossa is not so unusual. Recently over 100 species of rhacophorine tree frog were described in Sri Lanka using mtDNA in combination with exophenotypic measures, when only 18 were previously known, and despite recent extinctions by Man's activities [73]. This suggests that tropical biotas are not only amazingly diverse but highly structured genetically, both above and below the species level.
Genetic studies in tropical rainforests of SW Amazonia and NE Australia show phylogeographic divergence that is geographically concordant across a number of taxa and also originating in the Pliocene. The first set concern some 35 species of small mammals sampled along the Jurua River, and where for the majority there is a deep phylogenetic divide coincident with the Iquitos Arch. This formed as a bulge in front of the uplifting Andes in the Pliocene creating two basins that filled with sediment. The depth of mtDNA divergence between clades in these two basins places their separation at this time [74]. The Iquitos Arch is also implicated in the phylogeographic structure of the dart-poison frog Epipedobates femoralis, which was also sampled along the Jurua River traversing this ancient ridge, and which also has coincident mtDNA divergence (cytb 12%) dating to the Pliocene [75]. Interestingly, the collection sites differed markedly for their haplotypes, particularly in the headwaters region, again suggesting considerable local genetic structure. There has been a multitude of hypotheses proffered to explain the structure of Amazonian diversity, and such molecular phylogeographic approaches are beginning to distinguish amongst them.
The second set of phylogeographic studies involves several birds, reptiles and frogs from the remnant strip of tropical forest in NE Queensland [76]. This wet forest has undergone contraction and fragmentation during the drier colder stages and expanded in the interglacials. These show concordant mtDNA divergence that possibly dates back to the Late Pliocene. This is coincident with the Black Mountain Corridor, a narrow region from which rainforest disappeared in the Pleistocene ice ages producing main north and south refugia. The north and south clusters of haplotypes have various structures, some of which show low diversity probably due to population contractions during the ice age, while others have retained more haplotype diversity possibly by survival in local patches of forest. The rainforest snail Gnarosophia bellendenkerensis also shows these main phylogeographic features, and with some finer subdivisions. Its distribution from the LGM to the present has been modeled using current climate envelopes for this snail species mapped onto reconstructed paleoclimate distributions [77]. There is good agreement between the changes in the modeled snail distribution from the LGM to the Holocene and the signals from mtDNA data of refugial locations and expansions. Such an approach provides support for the deductions of both paleoclimatic modeling and phylogeography. Furthermore, the relationship between particular paleoclimatic changes and genetic structure is sharpened by studies on other species from this region that are not adapted to rainforest, like grasshoppers and frogs [78,79]. These species show genetic subdivision that is coincident with other physical features that would provide refugia and barriers commensurate with their lifestyle and climatic history, such as the coastal humidity transition or Burdekin Gap.
The phylogeographic pattern demonstrated by amphibians, reptiles and small mammals, is also found in bird species from tropical Africa and South America, which show old Pliocene lineages in the lowland forest and mixed old and recently diverged clusters in the mountains. This has lead to the proposal that such mountains provided a relatively stable environment through the ice ages and rising mountains, in which older lineages survived and new ones were created [80]. DNA divergence in spinetails from the Andes [81] and greenbuls from East Africa [82] provide evidence of montane speciation through the Pleistocene. Such mountain ranges appear to act as generators and reservoirs of lineages and species, and this probably is a function of their low latitude and topographic variety, which provide warm wet habitats through climatic and altitudinal range changes. The repeated small shifts in distribution driven by climate, along with continued uplifting of these mountains would provide conditions for contraction, selection, expansion and speciation.
Dry Tropics
There have been a number of recent DNA studies of several larger mammals from Africa that provide some interesting insights into how Pleistocene climatic changes modified their ranges and hence their genetic structure and divergence (Table 2). Whilst sampling such species across Africa is a major task, the threat posed by reductions in their numbers means that considerable efforts are being made to assess their genetic makeup for management and conservation, and many have a useful fossil record. They are not inhabitants of the wet forests, which were reduced during the colder drier glacial periods, but are found largely in the savannah grasslands and woodlands that had a different pattern of contraction and expansion. These habitats increased with the onset of the Pleistocene and its increasing glacial activity (3-2 Mya), with periods of dominance recorded around 1.7 and 1.2 Mya. They show increasing prevalence from 0.6 Mya through the Late Pleistocene, and fossils record the emergence of the associated mammal species and their subspecies since then (see references in Table 2). The DNA data, although not an accurate measure over this time scale, also indicates that these are recent events with most divergences in the last 0.4 My.
Table 2 Larger mammal species from across African savannah grasslands and woodlands showing phylogeographic pattern, with some indication of their phylogenetic structure and possible divergence times, refugia in west W, east E, and south S, and genetic signals of colonization and population history. LP = Late Pleistocene, MP = Mid Pleistocene, EP = Early Pleistocene, → = colonization/expansion. MS = mismatch expansion. All studies used d-loop mtDNA; also elephant used cytb and microsats, impala cytb, warthog and dog microsats, and buffalo Y.
Species Phylogenetic structure Likely Refugia (fossil evidence *) Genetic signals of range changes Reference
Hartebeest
Alcelaphus buselaphus 3 step clades
W→S→E, W, E subdiv, S,
*0.7 My> S→, E→,
some shallow clades Arctander et al [83]
Topi
Damaliscus lunatus 3 clades
S→E (+ S) (W), E, S,
*0.7 My> S→, 2 clades, MS
E→, shallow clade, MS Arctander et al [83]
Wildebeest
Connochaetes taurinus 2 clades, S→E,
S structured E, S,
*1.5 My> S→E, MS in E
LP Arctander et al [83]
Kob (& Puku)
Kobus kobus sl 3 clades,
W→E + S W, E, S
* EP> W↔E several times,
lineage mixing, MP LP Birungi & Arctander [108]
Greater Kudu
Tragelaphus strepsiceros 3 clades,
E, SW→S→E E, S,
SW isolate
*widespread S→E LP
shallow clades
diversity S>E Nersting & Arctander [84]
Impala
Aepyceros melampus 2 clades,
SW, S→E (E), S
SW isolate
*widespread S→E LP
network cluster E
diversity S>E Nersting & Arctander [84]
Wild dog
Lycaon pictus 2 shallow clades,
E, S,
few haplotypes (W), E, S W→E&S?, 340 ky>
each clade 70 ky>
mobile – mixing Girman et al [89]
Buffalo
Syncerus caffer 2 shallow clusters
W→E+S
S nested in E W, E, S,
* LP W→E <180 ky
E→S twice <130 ky
MS LP Van Hooft et al [85]
Elephant
Loxodonta africana 3–5 clades
W→E+S→W W, E, S,
*EP
*LP→ W→S&E, EP
W→S&E, MP
E→W, LP Nyakaana et al [109]
Eggert et al [88]
Warthog
Phacochoerus africanus 3 distinct clades
W→S→E W, E, S,
*0.78 My>
*0.4 My→ 3 clades isolated MP by dry climate
contract/expand LP Muwanika et al [87]
Most phylogeographies show some 3 major clades that are associated with 3 main areas of Tropical Africa, the west, east and south, indicating that these have been major refugial areas for the development of this divergence through climatic cycles in the Late Pleistocene (Fig 3). Many have shallow clades, mismatch analyses and star-like networks that are the expected result of contractions and expansions of these populations, and their phylogeographies indicate various colonizations between these major regions. In three species, the wildebeest Connochaetes taurinus, greater kudu Tragelaphus strepsiceros and the impala Aepyceros melampus, the data support colonization northwards to the east from refugia in the south of Africa [83,84]. The greater kudu and impala have distinct SW clades, which suggests isolation and survival there, as well as central South Africa, where many species appear to have had a refugium. Interestingly the first wildebeest fossils are from east Africa, so that the species seems to have disappeared from this region and been recolonized recently from the south.
Figure 3 Africa with major vegetation and mountain areas. Reduced Tropical rainforest at the LGM is shown. The Savannah species often show West, East and South clades (see Table 2 for details) and the general areas of these are indicated. The genetic data also indicates colonisations between these possibly refugial areas in the middle and late Quaternary Period.
On the other hand, the Cape buffalo Synercus caffer caffer has younger haplotypes in the south, which along with mismatch analyses suggest one or two recent colonizations from populations in eastern Africa. The DNA divergence of these from central African buffalo subspecies is perhaps only 180-130 kyr, and roughly coincident with the Cape buffalo's genetic expansion and evidence from fossils [85]. The hartebeest Alcelaphus buselaphus and the topi Damaliscus lunatus probably survived in a few places in southern and eastern Africa from which they expanded with better conditions [83,86]. The warthog Phacochoerus africanus is now relatively widespread, but has 3 distinct clades equivalent to subspecies, and with low divergence within each one [87]. This points to strong isolation through the last few ice ages with considerable recent population reduction followed by population expansion.
The mtDNA phylogeny of the African elephant is more complexly structured, with haplotypes of the putative species of forest Loxodonta cyclotis and savannah L. africanus elephants mixed together in several clades [88]. It suggests successive production of clades from the early Pleistocene, which involved colonization from the centre to the south and east with increasing savannah habitats, loss of competitors and punctuated by climatic cycles. Such admixture of clades in regions and taxa is probably a reflection of several such colonizations, and a recent invasion of western Africa from central regions is also indicated by the haplotype distribution. The African wild dog is very mobile and populations over the middle part of its current eastern through southern range show a mixture of haplotypes. There are 2 shallow but distinct mtDNA clades that would have diverged perhaps 340 kya, with each coalescing about 70 kya [89]. The cause of this divergence is not clear, but restriction in habitat by climatic oscillations, and separate colonization of east and south from western Africa are possible.
Whilst there are common features within and distinguishing ones between the wet and dry examples reviewed from the Tropics, there are individualities to each species phylogeography; these reflect differences in biology and history that have produced differences in genetic structure. The richness and diversity seen at the species level is multiplied by that within species, and more studies are needed on biotas from such habitats to properly describe and understand them. Little is known from the species rich Tropics of SE Asia, or from the plains of S America and the large areas of Temperate Asia. There are a number of individual studies on a range of species, but one needs several representatives from each community to look for generalities. Given the species diversity in these areas, such genetic and phylogeographic knowledge is particularly important to inform sensible decisions on management and conservation of these resources.
Lessons for Conservation from Phylogeography
Man is in the unique position to know and predict the consequences for the environment and its biota of his innate will to survive and reproduce. Our actions are greatly modifying both of these, so we face the challenge of managing this sensibly. But we must also think of these natural and induced biotic changes in the light of future major changes in the climate. It is clear that global oscillations, producing great climatic changes, have occurred and will continue; in particular the increasingly severe Quaternary ice ages, which are now well researched and clearly demonstrated. Biotas have changed greatly due to these, and will do so again. We are currently well into an interglacial, the Holocene, and there is debate about how soon it will end and how quick this will be. If the North Atlantic conveyor is turned off, and Man may assist in this, colder conditions may return very quickly. On the other hand, in the shorter term global warming may continue, and Man may assist this! What should we do about biodiversity, and how does phylogeography inform us for this?
The distribution of biodiversity across the world is largely measured as species diversity – their numbers, proportions and distinctness. But within a species there are often several geographic subspecies, and genetic studies have added greatly to knowledge of subspecific diversity, with some regions possessing more lineages and older divergence. Mountain ranges in warm Temperate and Tropical regions are seen to be important because they harbour much diversity at species, lineage and allelic levels. Phylogeographic studies reveal that this is likely a product of species surviving through climatic oscillations by tracking their habitat altitudinally and locally in a varied topography in regions not so affected by the extremes of climate change, e.g. [26]. The southern mountains of Europe, the southern Appalachians and western mountains of USA have clearly been important as refugia and provided most colonists for the vast north Temperate regions today. DNA divergence argues that this has happened repeatedly and so will probably happen again. To date there is little information on the patterns from Asia or S America, and it is needed. Such regions of Temperate refugial genetic diversity have accumulated lineages and alleles through several ice ages and deserve particular research and conservation.
The mountain regions in the wet Tropics of Africa and South America are very rich in species; while phylogeographic studies reveal that these contain divergences often into the Pliocene with subsequent diversification through the Pleistocene. This retention of older diversity through millions of years along with younger lineages argues that they are both generators and reservoirs of divergence and species [26,80]. Less is known about the Tropical regions than Temperate ones, but the phylogeographic evidence does suggest that they can contain greater diversity and divergence in an area, as for example in the wet forests of Costa Rica or Queensland [71,90]. One wonders just what genetic diversity the species in the mountains of China and SE Asia contain. However, tropical Asia is even less studied than Africa and Central America, and with the rapid anthropogenic changes there studies on their phylogeography is urgent. With such information the genetic value of particular regions will be clearer, however their conservation and management involve complex and difficult political matters.
Besides these more general issues, there are a number of more particular lessons and questions. For example, it has recently been noted in several butterflies that the lower genetic diversity produced by postglacial colonization of northern Europe is correlated with their recent decline, as evidenced in national records. Moreover, different deduced postglacial colonization patterns show the same correlation of low genetic diversity and population decline [91]. This suggests that the phylogeography of a species may be used as a predictor of demographic threat and loss. Clearly similar evidence from more species and groups is required to substantiate this.
Another particular example is the genetic diversity created by the formation of a hybrid zone as two genomes meet with postglacial colonization, which is multiplied when several species zones coincide as a suture zone, e.g. [26]. It has been argued that such regions are important because of their genetic diversity [92]. They may well allow the generation of occasional hybrid species [93,94] and possibly reinforcement [95], but except perhaps for climatically very stable locations in the wet Tropics they are transient, and will disappear with each major climatic reversal. They may reform in roughly the same place each cycle, but fossil evidence suggests that this is not necessarily the case [29]. Furthermore, for most zones the diversity they contain is accumulated in their refugia over several cycles, and hence these regions have greater long term value.
The demonstration that the extent of divergence among lineages within and between sister species generally increases from the High Arctic to the wet Tropics reflects their evolutionary age. It can be argued that the richer Tropical biotas are more valuable than the poorer temperate ones, both in terms of their present allelic, lineage and species diversity, and their long term survival. However, this overlooks the particular adaptations of Temperate species and the vast highly productive biotas they produce. The agriculture of the Temperate regions also supports much of the world's population. The consideration of regional diversity and adaptation raises a number of related questions. How well coadapted are recently assembled Temperate ecosystems? Are north Temperate and Arctic species particularly selected for colonization by repeated range change? Are genetically richer genomes more able to adapt to change? Do putative refugial regions contain genetic variation that may be useful in agriculture? And there are many more such considerations.
Conclusion
The frequent major climatic oscillations in the last 2 My caused repeated changes in the ranges of surviving taxa, with extensive extinction and recolonization in higher latitudes and altitudinal shifts and complex refugia nearer the tropics. As a result of these past dynamics, the genetic diversity within species is highly structured spatially, with a patchwork of genomes divided by often coincident hybrid zones.
The extent of divergence among lineages within and between sister species generally increases from the High Arctic to the wet Tropics and reflects their evolutionary age. Holarctic animal species show shallow but clear phylogeographic structure from the last or recent glaciations. Clades of several species are parapatric near major geographic features like rivers and mountains, suggesting they had similar range changes and refugial areas.
In Temperate regions like Europe and North America there is much more diversity in the south, where it has accumulated in refugia over many ice ages, and much less in the north, where it was lost during postglacial colonization. These northern places have been colonized by species from different southern refugia, and have had little time to become closely coadapted. Furthermore, this loss of diversity in the north is implicated in the present reduction of population abundance in some species. Mammals from the Dry Tropics of Africa often show major clades in the west, east and south indicating major refugial areas for recent divergence through climatic cycles in the Late Pleistocene.
Mountain ranges in warm Temperate and Tropical regions would seem to be important for the survival of lineages through climatic changes, and hence for genome divergence and speciation. Such understanding of the distribution of biodiversity carries serious implications for the theory and practice of conservation.
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| 15679920 | PMC544936 | CC BY | 2021-01-04 16:38:33 | no | Front Zool. 2004 Oct 26; 1:4 | utf-8 | Front Zool | 2,004 | 10.1186/1742-9994-1-4 | oa_comm |
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Front ZoolFrontiers in Zoology1742-9994BioMed Central London 1742-9994-1-51567991710.1186/1742-9994-1-5ResearchReticulate sympatric speciation in Cameroonian crater lake cichlids Schliewen Ulrich K [email protected] Barbara [email protected] Department of Ichthyology, Bavarian State Collection of Zoology (ZSM), Münchhausenstr. 21, 81247 Munich, Germany2004 26 10 2004 1 5 5 19 10 2004 26 10 2004 Copyright © 2004 Schliewen and Klee; licensee BioMed Central Ltd.2004Schliewen and Klee; 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
Traditionally the rapid origin of megadiverse species flocks of extremely closely related species is explained by the combinatory action of three factors: Disruptive natural selection, disruptive sexual selection and partial isolation by distance. However, recent empirical data and theoretical advances suggest that the diversity of complex species assemblages is based at least partially on the hybridization of numerous ancestral allopatric lineages that formed hybrids upon invasion of new environments. That reticulate speciation within species flocks may occur under sympatric conditions after the primary formation of species has been proposed but not been tested critically.
Results
We reconstructed the phylogeny of a complex cichlid species flock confined to the tiny Cameroonian crater lake Barombi Mbo using both mitochondrial and nuclear (AFLP) data. The nuclear phylogeny confirms previous findings which suggested the monophyly and sympatric origin of the flock. However, discordant intra-flock phylogenies reconstructed from mitochondrial and nuclear data suggest strongly that secondary hybridization among lineages that primarily diverged under sympatric conditions had occurred. Using canonical phylogenetic ordination and tree-based tests we infer that hybridization of two ancient lineages resulted in the formation of a new and ecologically highly distinct species, Pungu maclareni.
Conclusions
Our findings show that sympatric hybrid speciation is able to contribute significantly to the evolution of complex species assemblages even without the prior formation of hybrids derived from allopatrically differentiated lineages.
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Background
Recent empirical data and theoretical advances suggest that the diversity of complex species assemblages is based at least partially on the hybridization of numerous ancestral allopatric lineages that formed hybrids upon invasion of new environments [1-5]. A growing amount of studies show that cytoplasmatic (mitochondrial or chloroplast) gene phylogenies of recent diverse species radiations often conflict with phylogenies based on numerous nuclear genes [3,5-9]. Theoretical arguments as well as empirical evidence from hybrid zones predict that in newly colonized habitats the effect of transgressive segregation, i.e. the generation of extreme traits in hybrid populations, may lead to a drastically increased phenotypic variation. This effect may in turn serve as a substrate for evolution of novel adaptive traits [2,10-12]. These arguments in combination with an increasing body of evidence showing that species resulting from interspecific hybridization are common in plants [13] and highly probable in animals [5,7,9-14] gave rise to hypotheses about a prominent role of hybridization for the evolution of adaptive radiations [6]. Both, the initial formation of hybrid swarms of originally allopatric populations meeting in a newly colonized habitat ("hybrid swarm origin hypothesis") as well as secondary hybridization of in situ diverged lineages ("syngameon hypothesis") could possibly explain the rapid formation of megadiverse species flocks. The scenario may either involve secondary localized hybridization, i.e. hybridization of parapatric ("microallopatric") lineages within the geographical range of the primary radiation, or alternatively hybridization of sympatrically diverged lineages. Distinguishing between these two alternatives is central for the understanding of the processes that lead to the evolution of megadiversity. The first alternative predicts that increased species richness due to hybridization is dependent primarily on the spatial scale and the accompanied possibility to establish localized metapopulations. The second alternative predicts that hybridisation can aid the build-up of diversity even under fully sympatric conditions. Several studies, some published, some in preparation support the hybrid swarm origin hypothesis for some species assemblages endemic to comparatively large areas [3-5,8] but a critical evaluation of the syngameon hypotheses rests on the ability to test for sympatric hybrid speciation.
However, despite increasing evidence for sympatric speciation, uncontested examples remain rare and rarely go beyond the formation of single species-pairs [15,16]. In addition, evidence for sympatric speciation of complex species-assemblages is often based on mitochondrial phylogenies of limited taxon-sampling. This is problematic as mitochondrial phylogenies or those based on few nuclear loci may obscure true species phylogenies either due to introgressive hybridization among already established species or due to incomplete lineage sorting during rapid speciation. Hence it is not surprising, that studies applying several nuclear markers occasionally yield phylogenetic hypotheses about the origin and pattern of sympatric species assemblages which contrast with mitochondrial hypotheses. As a consequence of the uncertainty about phylogenetic relationships among members of complex species flocks questions about the processes that contribute to sympatric speciation remain difficult to test due to the lack of appropriate model systems.
Until recently the phylogeny of mitochondrial lineages of the cichlid species flock of crater lake Barombi Mbo (Cameroon) [17] was considered as one of the best examples for sympatric speciation [15]. According to this phylogeny which was based on haplotypes of single specimens, the monophyly of the 11 endemic species suggested strongly that they had formed after a single colonisation by a riverine founder species, Sarotherodon galilaeus. Because the lake's conical basin is only 2.15 km in diameter, because there are no migration-barriers along the shore, and because the lake is isolated from nearby river systems by cataracts of its outflow, allopatric scenarios for the origin and diversification of the flock were ruled out. However, in the light of the aforementioned methodological drawbacks of mitochondrial phylogenies, both the monophyly-hypothesis for the Barombi flock and the relationships among its 11 endemic species are worth to be reevaluated.
The ability to score thousands of amplified fragment-length polymorphisms (AFLPs) has created a powerful possibility for the phylogenetic reconstruction of rapidly originated species flocks. It has been successfully applied to a limited number of taxa belonging to the Lake Malawi and Lake Victoria haplochromine species flocks and revealed previously undetectable phylogenetic patterns including those supporting the hybrid swarm origin hypothesis [4,18,19]. In this study, we tested hypotheses about sympatric speciation with a focus on hybridization by applying a combination of mitochondrial DNA-sequencing and AFLP-genotyping as well as a set of recently proposed analytical tools [20] to the phylogenetic analysis of a complete and complex species flock.
Results
Mitochondrial phylogenetic inferences
We obtained a DNA sequence-alignment with 2553 bp including two complete mitochondrial genes, NADH dehydrogenase subunit 2 (ND2) and cytochrome b (cytb), partial proline tRNA as well as from part of the control region from all Barombi species (two samples per species) and relevant S. galilaeus populations (one to two samples per population). 2191 sites of the alignment were constant, 198 variable characters were parsimony-uninformative and the number of parsimony-informative characters was 164. Empirical base frequencies in this data set were A = 0.2721; C = 0.3271; G = 0.1268; T = 0.2740. Bootstrapped Maximum Parsimony (MP), Maximum Likelihood (ML) and Neighbour Joining (NJ) trees all recovered identical 50%-majority rule consensus-trees (figure 1). As the sistergroup to the monophyletic Barombi flock a Sarotherodon galilaeus clade was recovered which includes all west African populations except S. galilaeus sanagaensis which emerged as the sistergroup to all other ingroup taxa. Within the Barombi Mbo flock four lineages were recovered with high bootstrap support, one containing the predators of genus Stomatepia, one combining the fine-particel feeders of the genus Sarotherodon, one consisting only of the dwarf zooplanctivore Myaka and one containing the macro-invertebrate or eggfeeding sistertaxa of the genus Konia plus the highly specialized spongivore Pungu. For taxa represented by more than one sample, all conspecific samples grouped together except those of the morphologically merely distinguishable S. caroli and S. linnellii. A rough time estimate as deduced from the ultrametric tree (chronogram) [21] derived from non parametric rate smoothening (NPRS) of bootstrapped ML-distances suggests that all four lineages almost simultaneously came into existence, which must have taken place approx. 1 myr years ago. Soon after this primary radiation, the divergence of the Pungu haplotypes from Konia took place, while all other clades radiated into several species much later. A 94 sample data set with only cytochrome b and partial proline tRNA sequences (3 to 7 samples for the Barombi taxa, and 1 to 7 for all other; 1212 bp with 1003 constant characters, 59 parsimony-uninformative and 150 parsimony-informative characters) confirmed the previous findings for the Barombi flock and suggests that lineage sorting between the four large clades is complete (for a Neighbour Joining Tree see Additional File 1). However, between species within the clades lineage sorting was only complete for a subset of taxa (Pungu, Myaka, Konia ssp, S. lohbergeri and S. steinbachi), but not within Stomatepia ssp., S. caroli and S. linnellii.
AFLP based phylogenetic inferences
The phylogenetic reconstruction based on the same individuals and using 22 restrictive primer combinations with 3489 AFLP size fragments (3004 variable) confirmed the monophyletic origin of the Barombi Mbo flock and the monophyly of the Stomatepia and Konia clades (figure 1). However, several other phylogenetic groupings were recovered with high bootstrap support, which contrasted conspicuously with the mitochondrial phylogenetic hypothesis. S. gal. multifasciatus and S. gal. "Niger" were recovered as sistergroups to the clade containing all other ingroups including S. gal. sanagaensis. Within the Barombi flock Pungu is now sistergroup to a Sarotherodon subclade (S. steinbachi and S. lohbergeri), Myaka is sistergroup to the other Sarotherodon subclade consisting of the pelagic species S. caroli and S. linnellii. The Konia clade is resolved as the sistergroup to the rest of the flock. In contrast to the unresolved basal topology in the mitochondrial tree, the two species from Lake Ejagham are resolved as a monophylum which is the sistergroup to the geographically closest S. galilaeus population from the river Cross. An additional data set with 3 selective amplifications but 80 samples recovered only three supraspecific nodes with moderately high bootstrap support, the monophyly of the Barombi flock, Konia and the node containing the rest of the Barombi flock without Pungu. The latter was placed intermediate between the two well supported nodes.
Testing for sympatric reticulate speciation
Both the Shimodaira-Hasegawa and Templeton's test confirmed significantly the difference for the alternative tree topologies for each sequence and AFLP data sets, respectively (figure 1). These discordant phylogenies suggested strongly that hybridization among previously evolved lineages had taken place and that at least one taxon of the Barombi Mbo flock, Pungu maclareni, is the result of speciation by hybridization. By identifying the clades which contain taxa with discordant phylogenies we hypothesized that traces of three ancient hybridization events are still detectable in the multilocus AFLP data. To test for the presence of the respective phylogenetic signal for these three hypothetical ancient syngameons in the large AFLP data set, we used the recently developed method of Canonical Phylogenetic Ordination (CPO) [20]. In addition, this method is useful for differentiating between contributions to variation in the observed AFLP character pattern that were generated by the segregation of ancestral polymorphisms inherited from a common ancestor due to incomplete lineage sorting rather than by the contribution of derived characters of hybridizing lineages. This, as the contribution to the variation that is assignable to the phylogenetic group uniting the common ancestor of the hybridizing lineages (coded as phylogenetic variables) is partialled out in the CPO separately from the contribution of the phylogenetic groups characterizing the derived lineages that may have hybridized (see also Methods section).
All phylogenetic groups detected either by the mitochondrial or the AFLP data set, or formulated according to the three hypothetical syngameons were coded as phylogenetic variables and their explanatory value for the variance in the AFLP data set was tested (table 1). As a result, almost all supraspecific clades which were not conflicting among the two data sets were recovered as contributing significantly to the variation in the data set. Conflicting clades, which were either found in the mitochondrial or AFLP phylogenetic hypothesis, yielded non-significant results except for the AFLP based nodes uniting (1) all S. galilaeus and lake taxa without S. gal multifasciatus and S. gal "Niger" and (2) the node of the AFLP based hypothesis of the monophyly of the Lake Ejagham sister pair. Of the three hypothetical syngameons, the one uniting Pungu with its potential ancestor clades represented now by the Konia ssp and S. steinbachi and S. lohbergeri contributed significantly to the variation in the AFLP data set, whereas the other two did not.
As a final test for the putative hybrid origin of Pungu, a tree-based method as outlined in Seehausen [6] was performed in order to test for homoplasy excess introduced by this potential hybrid taxon in the AFLP-data set (figure 2). Only the removal of Pungu resulted in far outlying higher support values for the respective nodes, whereas all other removals resulted in much lower values (Fig. 2). In addition, the other nodes tested were not affected by the removal of Pungu. Interestingly, the removal of several other taxa produced far outlier values in other nodes. However, these removals concerned part of the two hypothetical syngameons that were not supported significantly in the CPO, which were therefore not explicitly tested. The removal of Konia dikume resulted in an increased bootstrap support for the clade uniting Konia and Pungu in the analysis of the 530 loci data set and an accompanying though weaker signal in the S. caroli/S.linnellii clade. The removal of S. caroli resulted in a distinct although weak increase of the bootstrap support for the Myaka/S. linnellii node, the exclusion of both Myaka and Konia eisentrauti increased the bootstrap support for the S. linnellii/S. caroli node. Finally, the removal of S. mariae increased the support for the S. pindu/S. mongo split strongly. Among the riverine populations of S. galilaeus, the removal of S. gal. "Meme" increased strongly the value for the clade uniting the S. gal. "Cross" specimen with the two endemic species from Lake Ejagham (Details in Additional File 2). These additional findings suggest that the comparative phylogenetic analysis of mtDNA and AFLP data alone was not sufficient to uncover all possible hybridization events.
Figure 2 Box-plots of the distribution of %-bootstrap support values for the nodes uniting the two Konia species (Figs. 2a) or Sarotherodon lohbergeri and S. steinbachi (Figs. 2b) after iterative removal of single species or taxon groups. Values are based on 2000 bootstrap replicates in the AFLP-based tree reconstruction using the Link et al algorithm [48]; they are based on either the 32-sample/2355 loci dataset for the S. lohbergeri/S. steinbachi split or on the 80 sample / 530 loci dataset for the Konia split. Outside (*) and far outside values (°) are plotted as asterisks and circles, respectively. Arrows denote far outside values resulting in a distinctly higher bootstrap support for the two clades after exclusion of Pungu maclareni. n refers to the number out of 18 maximum possible removal experiments. Removal of Pungu did not result in outside or far outside bootstrap support values for any other node out of 30 nodes tested (Additional information concerning far outside values yielded for other nodes see Additional File 2).
Discussion
Our results demonstrate that the sympatric origin of a diverse and complex species flock was aided substantially by reticulate evolution among lineages that emerged in a much smaller primary radiation. Our data suggest that at least one out of 11 taxa of the species flock in Lake Barombi Mbo, Pungu maclareni, is the result of speciation by hybridization. On the other hand, conflicts among mitochondrial and nuclear data sets as well as results of the homoplasy excess tests suggest that in the course of the evolution of the flock hybridization must have taken place among several additional Barombi taxa, too.
According to the ultrametric time-calibrated tree ("chronogram") based on the well supported phylogeny of mitochondrial haplotypes in the lake, the primary radiation in Barombi Mbo resulted into the almost instantaneous split into four distinct lineages approximately one million years ago. The accumulation of numerous apomorphic characters that support these mitochondrial lineages suggests strongly that they represented reproductively isolated species at that time. Only in an advanced stage of species flock formation and after considerable time had elapsed, their cohesion was broken partially by hybridization events between these lineages. However, according to the chronogram the ancestral mitochondrial clades that contributed, for example, to the hybrid origin of Pungu continued to accumulate apomorphic characters well after the origin of Pungu. This suggests that the species status of the ancient hybridizing lineages in terms of sufficient reproductive isolation must have allowed for their ongoing genetic cohesion and accompanied coalescence of haplotypes before additional speciation events took place.
Traditionally the rapid origin of megadiverse species flocks of extremely closely related species was explained by the combinatory action of three factors: Disruptive natural selection, disruptive sexual selection and partial isolation by distance [22-24]. Although introgression among species is known for many fish species [7,9,25-27] and although reticulate evolution and hybrid origins of species are well documented in plants [14], it is only of recent that hybridization has been proposed to play a major role in generating diversity in animals in general [27-30] and in "explosive" speciation in species flocks in particular [6]. Especially in newly colonized habitats with increased ecological opportunities, secondary hybridization of primarily diverged lineages may provide rapidly sources of heritable advantageous variation by producing additional adaptive diversity through recombination of functional genotypes. Interestingly, the species with the most likely hybrid origin in Lake Barombi Mbo, Pungu maclareni, represents an ecologically highly specialized ecotype. Both its peculiar dentition and the accompanying hypertrophic jaw-muscles are unique not only in Barombi Mbo but in cichlids in general [31]. Accordingly, one putative second species with a hybrid genome in the lake, Konia dikume, ranks among the most unusual cichlids as it is the single species which is able to exploit chironimid larvae in the almost oxygen-free deep water due to its extremely high haemoglobin concentration in its blood [32]. In the light of our findings we hypothesize that hybridization produced these extreme phenotypes by transgressive segregation which allowed the exploitation of extreme niches. This supports the notion that speciation by hybridization is not only able to produce additional random variation but may significantly increase the ecological complexity in a rapidly evolving species community by providing extraordinary genetic opportunities. If indeed transgressive segregation of hybrid genotypes plays a major role in the evolution of evolutionary novelties, members of complex species-assemblages with unusual ecological adaptations should predictably turn out to be of hybrid origin more often than species with common adaptations.
Methods
Taxon sampling, collection of samples and deposition of vouchers
We obtained genetic data from relevant Sarotherodon galilaeus populations and from all species endemic to crater lakes Barombi Mbo and Ejagham, which are related to S. galilaeus. Oreochromis niloticus and Sarotherodon melanotheron were used as outgroups based on published information and pilot-study data confirming their outgroup status with respect to S. galilaeus and the investigated species flocks.
Adult specimens from Lake Barombi Mbo were collected by UKS during field visits to the lake in 2001 and 2002 and from Lake Ejagham in 1994. Identification to species is according to Trewavas et al. [33] and was straightforward except for Sarotherodon caroli and S. linnellii. Black adult breeding males of the S. caroli/S. linnellii-phenotype from the deepwater were identified as S. caroli, whereas golden males from the shallow inshore area were identified as S. linnellii. Fin-clips were taken from the right pectoral fin in the field directly after collection and preserved in 96% Ethanol p.A.. Samples from additional specimens belonging to different populations of Sarotherodon galilaeus and outgroups were either collected by UKS in the field in Cameroon or donated by others. Vouchers are deposited in the ichthyological collection of Bavarian State Collection of Zoology (ZSM). Of few S. galilaeus-samples, only photographs of the specimens are available and are deposited in the ZSM, too. Informations about specimens and their species identifications, geographic origin, accession numbers and information about which specimens were sequenced and AFLP typed in different data sets are provided in Additional file 3.
Molecular Methods
DNA Preparation
DNA samples were isolated from approx 10 mm2 fin tissue with the DNeasy™ Tissue Kit (Qiagen). DNA-quality was visually inspected under UV-light on a 0.8% agarose-gel stained with ethidium bromide. For subsequent AFLP-analysis only samples with a clearly visible high-molecular band were used. DNA-concentration was determined using the VersaFluor-Fluorometer-System (BioRad) using the stain Picogreen® dsDNA Quantitation Kit (Molecular Probes). All samples were adjusted to 60 ng/μl.
Mitochondrial DNA Amplification and Sequencing
Two different data sets were assembled, one long data set with approx. 2550 bp including the complete NADH dehydrogenase subunit 2 (ND2), the complete cytochrome b (cytb) gene and part of the proline tRNA, and finally, one part of the control region. For this long data set only two samples per species were used, but a short data set with more individuals but only the cytochrome b and the partial proline tRNA genes was generated, too. ND2 was PCR-amplified using the primers "ND2Met" 5'-CATACCCCAAACATGTTGGT-3'"ND2Trp" 5'-GTSGSTTTTCACTCCCGCTTA-3'; a second fragment containing the cytb, proline and threonine tRNAs and the 5'-end of the control region was amplified with the primers "L14725" 5'-TGACTTGAAAAACCATCGTTG and "H16498" 5'-CCTGAAGTAGGAACCAGATG [34] ; internal sequencing primers were the newly designed "cytL640" 5'-CACGAAACCGGATCAAAC-3' for cytochrome b and "L71" 5'-TACCCCTAGCTCCCAAAGCT-3'7 for the 5'-end of the control region. PCR was performed by using a PTC 220 DYAD thermocycler (MJ Research) in a 25 μl reaction volume using the Expand PCR system (Roche Diagnostics) with 25 pmol of each primer, 20 pmol of dNTPs, 12.5 pmol MgCl2 and 0.88 units of Taq polymerase. PCR parameters were 94°C for 4 min, 35 cycles with 94°C for 1.5 min, 55°C for 1 min, 72°C for 1.5 min, followed by a final elongation at 72°C for 3 min. PCR products were cleaned by using MinElute PCR purification kit (QIAGEN) and their DNA-concentration adjusted to 100 ng/μl. PCR-Products were then used as templates for cycle-sequencing reaction using the "Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit" (Applied Biosystems) with each of the PCR primers or internal primers. Cycle parameters were the following: 94°C, 2 min; 25 cycles of 94°C, 20 s; 52°C, 10 s; 60°C, 4 min. The sequenced product was filtered through Sephadex-G50 fine (Fluka) packed spin columns (Amersham) to remove unincorporated dye terminators, primers, and salts, and finally dried in a speed-vac. These products were resuspended, electrophoresed and analysed with an ABI PRISM™ 377XL-96 automated sequencer using a 4.25 % polyacrylamid gel (BioRad). Electrophoretic information was transcribed to sequence data using the program Genescan (PE Applied Biosystems).
Individual sequence files were edited and contigs assembled using Sequence Navigator™ (PE Applied Biosystems). Homologous protein-coding regions (ND2, cytb) were aligned manually and confirmed by translating DNA data into amino acid sequences in BioEdit [35]. The short fragment of the control-region was first aligned with default settings in Clustal W as implemented in Sequence Navigator™. No indels larger than 1 basepair (bp) were detected and alignment therefore was straightforward. All sequences were tested for an anti-G bias characteristic of the mitochondrial DNA to confirm that we have collected genuine mitochondrial DNA data [36]. Sequence data have been deposited in GenBank (for accession numbers se Additional File 3).
Amplified Fragment-Length Polymorphisms (AFLPs)
We followed the original protocol of the AFLP-method [37] using the AFLP™ Plant Mapping Kit (Applied Biosystems) with slight modifications of the accompanied protocol: Restriction and ligation were carried out in a single step under standardized conditions in a thermocycler (2 h at 37°C and 8 h at 16°C). 1,5 μl of the preselective amplification product were used in only 10 μl total reaction volume of the selective amplifications. The restriction enzymes used were EcoRI and MseI. Primer sequences for preselective PCR were GACTGCGTACCAATTCA and GATGAGTCCTGAGTAAC. An additional two bases were added to the 3' end for selective PCR. Analogous to the mitochondrial data set we assembled two datasets. For the long one in total 22 primer pairs were used in the following combinations and fluorescent dye-labelling (MseI-primer/ EcoRIDYE): TC-CAFAM, AT-CAFAM, AC-CCNED, TA-CAFAM, AA-CTFAM; AA-GGJOE; AC-CAFAM; AA-CAFAM; AG-CAFAM; AA-CGJOE; AC-CTFAM; AG-CTFAM; AT-CTFAM; AG-GGJOE; TC-CTFAM; TA-GGJOE; AG-ACNED; AT-ACNED; AG-CCNED; AC-ACNED; AT-CCNED; TA-CGNED. For the short data only the five primer pairs TC-CAFAM, TA-GGJOE, AC-CAFAM, AC-CCNED, AT-CAFAM were used but typed for 80 individuals (see Additional File 3).
Selectively amplified fragments were separated on 6% LongRanger polyacrylamid gels (FMC BioProducts) with an ABI PRISM™ 377XL-96 sequencer. Fluorescent signals were detected using the GENESCAN software (Applied Biosystems) with internal size standard (GS-500 ROX; Applied Biosystems). The fluorescent threshold was set to 50 units and the correct identification of ROX-marker bands by GENESCAN was checked for all electropherograms.
Bands between 100.5 bp and 499.5 bp were scored in a first step for presence or absence using the software Binthere (developed by N. Garnhart and available through the T. Kocher laboratory . The program generates aligned spreadsheets from GENESCAN-sized AFLP-data by assigning each sized fragment to a size-class of user-defined distance to the next size-class. Using a spreadsheet routine, fragments were inferred in a second step to be homologous if they differed by no more than 1.00 bp, and if the scoring procedure identified the same size-classes whether scored from small to large size-fragments (forward) or vice versa (backward). Size-classes with inconsistent allocation of fragments according to forward and backward scoring were excluded, as well as adjacent size-classes differing by less than 0.35 bp, which corresponds to the double standard deviation of 0.15 bp of the sequencer [38]. As a result of this procedure a final 0/1 data-matrix for all scored individuals was prepared. All samples of the same primer combination were run on same gel for the 33 specimen dataset and on two gels for the 80 taxon dataset. Single unsuccessful amplifications were repeated and fitted to the data matrix using the size-class assignment criteria as outlined above.
Phylogenetic Analyses
Mitochondrial Sequence Data
MrModeltest 1.1b [39], a simplified version of David Posada's "Modeltest 3.06" [40] was used to perform hierarchical likelihood ratio tests (HLRT) and to calculate approximate Akaike Information criteria (AIC) to determine the optimal nucleotide substitution models for the dataset. If the two tests did not select the same model, we chose AIC over HLRT, as AIC is a useful measure that rewards models for good fit but imposes a penalty for unnecessary parameters [41,42], which may cause erroneous phylogenetic conclusions especially in Bayesean phylogenetic analyses [43]. For the 33 sequence dataset the HLRT selected the HK+I+Γ model (α = 0.2781; proportion of invariable sites = 0) a transition/transversion (Ti/tv) ratio of 8.5662 was calculated. The AIC selected the HKY+I+ Γ model (α = 0.9468; proportion of invariable sites = 0.4264) with a transition/transversion ratio of 8.6616. For the 94 sequence dataset, the HLRT selected the GTR+ Γ model (α = 0.4014; proportion of invariable sites = 0), whereas AIC selected the GTR+I model (proportion of invariable sites = 0.5817). Empirical base frequencies in the data 2553/1212 data sets were A = 0.2443/0.2440; C = 0.3264/0.3261; G = 0.1443/0.1446; T = 0.2581/0.2852.
The AIC settings were subsequently used for Maximum Likelihood (ML) analyses and to estimate ML distances for minimum evolution (ME) analyses in the program PAUP* 4.0b1.0 (PPC/Altivec) [44] and for the 94 sequence dataset in Treefinder [45]. Maximum Parsimony (MP) analyses were conducted with heuristic searches (TBR branch swapping and MULTREES option effective; 10 random stepwise additions of taxa for 33 sequence set and simple addition for the 94 sequence set; gaps in the control region treated as a 5th character). Non-parametric bootstrapping with 1000 (ME or MP analyses) or 100 (ML) pseudoreplicates was used for testing the robustness of the inferred trees. Tree topologies using the HLRT settings under ME were not different from topologies gained with AIC settings (data not shown).
A LRT [46] as implemented in PAUP* was performed with the respective 33 sequence ME tree under the AIC model assumptions with (-ln L = 6770.47061) and without (-ln L = 7120.92833) molecular clock enforced. Overall constancy of rates of evolution was rejected (chi2 = 701.0154, df = 32, p = 0.001). To date cladogenetic events in the absence of rate constancy, the nonparametric rate smoothing (NPRS) method [21] as implemented in Treefinder [45] was used to construct an ultrametric tree ("chronogram") using the bootstrapped 33 sequence ML derived tree-topology and associated bootstrapped branch lengths as input.
AFLP Data
We used PAUP* [44] to calculate the skewness parameter g1 [47] to test for adequacy of phylogenetic signal in the 0/1-data set. g1 calculated from 1000000 random trees revealed significant non-random structure under the parsimony optimality criterion in the 22 restrictive amplifications for the complete data set: g1 -0.805, 33 samples, 3004 variable sites out of 3489 scored); g1 values were lower in the 3 restrictive amplifications data set used for some homoplasy excess tests (see below): g1 -0.298, 80 samples, 717 variable sites out of 859 scored. 2355 and 530 loci respectively were parsimony informative within Sarotherodon galilaeus sensu lato (excluding Oreochromis niloticus and Sarotherodon melanotheron). "Pruned" data matrices using only those parsimony informative sites were constructed for Principal Canonical Ordination and homoplasy excess tests (see below) in order to account for noise in the data potentially introduced by the distant outgroup. Pairwise genetic distances were calculated from the binary data-matrix with two different algorithms: One developed by Link et al. [48] as implemented in TREECON v.1.3b [49], which is based on shared and unique characters and ignores shared absence. This algorithm is adequate for AFLP data, since noise in the data may often be created by weak signal intensities and hence absence of band-detection despite a possible weak presence of signal. Alternatively, we calculated pair-wise distance matrices with the restriction-site program RESTDIST within the PHYLIP 3.6A2 package [50]. Trees were constructed from the Link et al.-distances with the neighbour joining (NJ) algorithm as implemented within TREECON or from the RESTDIST-distances using the Fitch-and-Margoliash-algorithm [51] with unconstrained branch length as implemented in the program FITCH within the PHYLIP 3.6A2 package [50]. Non-parametric bootstrapping was performed with 100 bootstrapped data sets analyzed 10 times with random input orders, and with local and global optimization.
Hypothesis testing
Alternative phylogenetic hypotheses produced as described by tree topologies based on mtDNA and AFLP data were compared with each other and statistically evaluated using either the Shimodaira-Hasegawa LRT [52] for mitochondrial data or the Templeton's Wilcoxon signed-rank test [53] for AFLP-data (both as implemented in PAUP*).
In order to test for the presence of a phylogenetic signal that possibly reflects reticulate events in the AFLP-data, two methods were applied.
First, a canonical correspondence analysis (CCA) was performed using CANOCO 4.0 [54]. This method has previously been used successfully for testing the effect of tree-like hydrogeographic data and supplementary ecological data on microsatellite allele-frequencies in freshwater fishes [55], as well as an alternative to traditional phylogenetic comparative methods [20]. A presence/absence matrix with the 2355 AFLP-characters which were parsimony-informative within the Sarotherodon galilaeus-clade (S. galilaeus sensu lato and lake endemics) provided the data-matrix to be tested. Phylogenetic hypotheses derived from mtDNA- and AFLP-analyses as well as hypothetical syngameons as derived from the conflict between the two data-sets were translated into a phylogenetic matrix by assigning binary indicator variables, each coding for the membership of investigated samples to phylogenetic groups (e.g. nodes in phylogenetic trees or hypothetical syngameons) [20,54]. 9999 full model Monte Carlo (MC) permutations were used to test whether a given phylogenetic group as coded by the indicator variables and identified by automatic forward selection of variables was significantly related to the AFLP-data pattern.
Second, a tree-based method as outlined in Seehausen [6] was performed in order to test for homoplasy excess introduced by potential hybrid taxa in the AFLP-data as suggested by the mtDNA-AFLP-phylogeny conflict and the CCA. Theoretically, hybrid taxa are overall intermediate to the parental taxa because they carry a mosaic of parental characters. Consequently, the inclusion of a hybrid taxon into a multilocus based phylogeny estimate introduces an excess of homoplasies and therefore conflict in the subset of clades that contributed to hybridization. Removal of the putative hybrid taxon should therefore decrease the amount of homoplasies and hence increase support for those nodes that unite descendants from taxa which gave rise to a hybrid taxon. In contrast, removal of a non-hybrid taxon should not affect support for the respective nodes. We computed Link et al bootstrap-supports (2000 replicates) for the nodes uniting Sarotherodon steinbachi and S. lohbergeri in the 2355 loci data with n = 16 experiments (each taxon removed once). Analogous support values for the node uniting the Konia eisentrauti and K. dikume were computed with the 530 loci data set with n = 14 experiments, because bootstrap support in the larger data set was always larger than 98.85% and identical runs yielded values differing by more than 1.15 % (data not shown). By reducing the number of loci but increasing the number of samples we obtained a meaningful distribution of bootstrap support values for that node.
Authors' contributions
UKS designed the study, carried out the field work, part of the lab work, the major part of the data analysis and wrote the manuscript. BK carried out the major part of the lab work and carried out parts of the data analysis. Both authors read and approved the final manuscript.
Table 1 Results of canonical phylogenetic ordination of AFLP data
Variation explained¶
Node ¥ Marginal effects† λ1 Conditional effects‡ λa P-Value#
Phylogenetic groups according to mtDNA-based phylogeny
S. galilaeus sensu lato incl. Barombi taxa * 1 0.15 0.10 0.000
S. galilaeus sensu lato w/o S. g. sanagaensis incl. Barombi taxa 2 0.12 0.05 n.s.
S. galilaeus w/o S. g. sanagaensis excluding Barombi taxa 3 0.15 - n.s.
S. g. multifasciatus * 4 0.18 0.13 0.000
S. galilaeus w/o S. g. sanagaensis and S. g. multifasciatus 5 0.12 - n.s.
S. galilaeus "Meme" * 6 0.10 0.08 0.000
"Cross-clade" + S. g. "Niger" 7 0.10 - n.s.
"Cross-clade" * 8 0.09 0.07 0.001
"Barombi Mbo clade" * 9 0.18 0.11 0.000
Stomatepia ssp.* 10 0.08 0.06 0.000
St. mongo * 11 0.07 0.05 0.054
St. mariae * 12 0.06 0.05 0.008
St. pindu * 13 0.05 - n.s.
St. mariae – St. pindu 14 0.06 - n.s.
Pungu maclareni – Konia ssp. 15 0.07 0.05 0.025
Konia ssp. * 16 0.06 0.06 0.001
Pungu maclareni * 17 0.06 - n.s.
Konia eisentrauti * 18 0.05 0.04 n.s.
Konia dikume * 19 0.05 - n.s.
Myaka myaka * 20 0.05 0.04 n.s.
Sarotherodon lohbergeri * 21 0.06 0.04 n.s.
Sarotherodon steinbachi * 22 0.05 - n.s.
"Barombi Sarotherodon clade" 23 0.08 - n.s.
Phylogenetic groups according to AFLP-phylogeny
Myaka + S. caroli/S.linnellii 27 0.07 - n.s.
Pungu + S. lohbergeri/S. steinbachi 28 0.07 - n.s.
St. mongo + St. pindu 29 0.07 - n.s.
S. galilaeus s. l. incl. Barombi taxa w/o S.g.multifasciatus and S.g. "Niger" 30 0.21 0.21 0.000
S. lohbergeri + S. steinbachi 31 0.06 - n.s.
S. linnellii + S. caroli 32 0.06 - n.s.
S. sp. "mudfeeder" + S. sp. "bighead" 33 0.08 0.05 0.013
Hypothetical ancient syngameons according to conflict between mtDNA-based and AFLP-based phylogenetic hypotheses groups ¥¥
P. maclareni + Konia ssp. + S. lohbergeri + S. steinbachi red (24) 0.08 0.06 0.002
M. myaka + S. caroli + S. linnellii + S. lohbergeri + S. steinbachi green (25) 0.09 - n.s.
S. g. sanagaensis + S. galilaeus w/o S. g. multifasciatus. S. g. "Niger" blue (26) 0.15 - n.s.
Each phylogenetic group as identified by nodes in phylogenetic analyses of mtDNA-, or AFLP-data or by the conflict between the data sets (hypothetical syngameons) was tested for their explanatory value in explaining variance in the AFLP data set.
¥ Node numbers correspond to encircled node numbers for mtDNA-clades and AFLP-clades supported in Fig. 1.
¥¥ Groups refer to hypothetical syngameons as indicated by coloured blocks in Fig. 1a and b.
¶ "Variation explained" refers to eigenvalues of each phylogenetic group that explain part of the variation in the pruned AFLP-data (2355 loci / 33 samples identical to those in Fig. 1a. Sum of all unconstrained eigenvalues was 1.667, sum of all canonical eigenvalues 1.179.
† Marginal effects refer to eigenvalues ("fit") of each phylogenetic group if taken singly as the only variable on the pruned AFLP data set.
‡ Conditional effects refer to the increase in eigenvalues ("additional fit") of each phylogenetic group as selected by automatic forward selection.
# P-values refer to the significance level of the conditional effects as obtained with a Monte Carlo permutation test under the full model with 9999 random permutations.
* mtDNA-clades as indicated in Fig. 1b that are compatible with clades as identified by the AFLP phylogenetic tree in Fig. 1a.
Figure 1 Phylogenetic tree (a) based on AFLP-data and chronogram (b) based mtDNA data of all Barombi Mbo cichlids (inside blue box), reference specimens of the closest riverine ancestor of the Barombi flock, Sarotherodon galilaeus, and two undescribed Sarotherodon from Lake Ejagham (S. sp. "bighead" and S. sp. "mudfeeder"). Photographs refer to the taxon to the left, different populations of S. galilaeus are depicted by two identical photographs only. (a) Numbers at nodes in the AFLP-tree are bootstrap-values (%) of tree reconstructions using the pruned (above) and unpruned (below) AFLP data set. Topologies were identical except for the position of S. g. "Niger", which was sistergroup to S. g. multifasciatus when using the unpruned data (bootstrap value: 86). Long terminal branches in the original phylogram were cut to identical lengths for graphical reasons; interior branch lengths are as in the original phylogram. (b) Numbers at nodes in the mtDNA-chronogram refer to bootstrap-values of the ML (above) and MP (below) tree reconstructions. The absolute time scale above the tree is based on the maximum age of the Barombi Mbo crater formation of 1.0 mya [56]. Encircled numbers mark nodes referring to phylogenetic groups tested with PCO (see Table 1). Red, blue and green shaded boxes unite hypothetical ancient syngameons as deduced from the conflict between mtDNA- and AFLP-based phylogenetic hypotheses. Further details of tree reconstruction see Methods.
Supplementary Material
Additional File 1
Neighbour Joining phylogram of 94 specimens Neighbour. Joining phylogram calculated from complete sequences of the mitochondrial cytochrome b gene (1141 bp) and the partial proline tRNA gene (71 bp) sequences for 94 specimens based on the GTR+ Γ model and rooted with Sarotherodon melanotheron (not shown)
Click here for file
Additional File 2
Additional box-plots of bootstrap supports in taxon removal experiments. Box-plots of the distribution of %-bootstrap support values for nodes that yielded additional higher outside or far outside values in taxon removal experiments (methodology compare with legend of Fig. 2 of the main text). a. The removal of Konia dikume resulted in an increased bootstrap support for the clade uniting Konia and Pungu in the analysis of the 530 loci data; set. b. The removal of S. caroli resulted in increased outside values for the bootstrap support of the b. the Myaka/S. linnellii node and the c. the clade that unites all Barombi taxa with the exclusion of Konia; d. the exclusion of both Myaka and K. eisentrauti increased the bootstrap support for the S. linnellii/S. caroli node; e. Finally, the removal of S. mariae increased the support for the S. pindu/S. mongo split strongly. f. Among the riverine populations of S. galilaeus, the removal of S. gal. "Meme" increased strongly the value for the clade uniting the S. gal. "Cross" specimen with the two endemic species from Lake Ejagham ("Cross clade"); in addition, the exclusion of S. lohbergeri increased the bootstrap value for the same node, albeit weakly.
Click here for file
Additional File 3
Specimens genotyped, Genbank Accession Numbers and vouchers. Specimens included in the study with information on DNA-reference-numbers, voucher deposition in the Zoological State Collection Munich (ZSM), Genbank-Accession numbers and representation of specimens in two AFLP-data sets.
Click here for file
Acknowledgements
We thank the Barombi people, who are the traditional owners of Lake Barombi Mbo, for their permission and support; the Ministry of Science of the Republic of Cameroon for the research permit (n° 31/MINREST/B00/D00/D10/D12); WWF Cameroon (L. Usongo) for logistic support; F. Herder, S. Mbakwa and R. Schliewen assisted in the field, M. Miller and D. Neumann in the lab. F. Herder, A. Nolte, R. Schelly, D. Tautz and especially O. Seehausen contributed helpful suggestions to the manuscript. This study was financed by a grant to UKS of the Deutsche Forschungsgemeinschaft DFG (SCHL 567/1).
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| 15679917 | PMC544937 | CC BY | 2021-01-04 16:38:33 | no | Front Zool. 2004 Oct 26; 1:5 | utf-8 | Front Zool | 2,004 | 10.1186/1742-9994-1-5 | oa_comm |
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Front ZoolFrontiers in Zoology1742-9994BioMed Central London 1742-9994-1-61567992710.1186/1742-9994-1-6ResearchHomologs of wingless and decapentaplegic display a complex and dynamic expression profile during appendage development in the millipede Glomeris marginata (Myriapoda: Diplopoda) Prpic Nikola-Michael [email protected] Department for Evolutionary Genetics, Institute for Genetics, University of Cologne, Weyertal 121, 50931 Köln, Germany2004 24 11 2004 1 6 6 27 10 2004 24 11 2004 Copyright © 2004 Prpic; licensee BioMed Central Ltd.2004Prpic; 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 Drosophila genes wingless (wg) and decapentaplegic (dpp) comprise the top level of a hierarchical gene cascade involved in proximal-distal (PD) patterning of the legs. It remains unclear, whether this cascade is common to the appendages of all arthropods. Here, wg and dpp are studied in the millipede Glomeris marginata, a representative of the Myriapoda.
Results
Glomeris wg (Gm-wg) is expressed along the ventral side of the appendages compatible with functioning during the patterning of both the PD and dorsal-ventral (DV) axes. Gm-wg may also be involved in sensory organ formation in the gnathal appendages by inducing the expression of Distal-less (Dll) and H15 in the organ primordia. Expression of Glomeris dpp (Gm-dpp) is found at the tip of the trunk legs as well as weakly along the dorsal side of the legs in early stages. Taking data from other arthropods into account, these results may be interpreted in favor of a conserved mode of WG/DPP signaling. Apart from the main PD axis, many arthropod appendages have additional branches (e.g. endites). It is debated whether these extra branches develop their PD axis via the same mechanism as the main PD axis, or whether branch-specific mechanisms exist. Gene expression in possible endite homologs in Glomeris argues for the latter alternative.
Conclusion
All available data argue in favor of a conserved role of WG/DPP morphogen gradients in guiding the development of the main PD axis. Additional branches in multibranched (multiramous) appendage types apparently do not utilize the WG/DPP signaling system for their PD development. This further supports recent work on crustaceans and insects, that lead to similar conclusions.
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Background
The genes wingless (wg) and decapentaplegic (dpp) are important factors for the normal development of the Drosophila legs. Both genes encode secreted morphogens that generate combinatorial gradients across the developing imaginal leg discs (e.g. [1]). These gradients form the top level in a PD axis patterning cascade and they control expression of the genes at the next level of the cascade, the leg-gap genes (e.g. Distal-less (Dll), dachshund (dac)) (e.g. [2,3]). Thus, wg and dpp are key factors involved in the early events of PD axis formation.
In recent years several comparative studies in other arthropod species have suggested that the action of the leg-gap genes in PD patterning is evolutionarily conserved [4-9]. Tus, the question arose as to whether the regulation of the leg-gap genes by the WG/DPP morphogen gradient is also conserved. The currently available data provide no clear answer. Initially, the expression patterns did not support the conservation of this top level of the PD axis patterning cascade [9,10]. Other authors, however, have argued in favor of a conservation of WG/DPP morphogen signaling in PD axis formation [6]. Furthermore, many arthropods have appendages with more than one PD axis. It is currently debated whether these multiple axes are all patterned by a cascade involving wg and dpp at the top level, or whether the different branches are patterned through branch-specific mechanisms.
The comparative analyses of wg and dpp expression during appendage formation to date mainly focus on insects (e.g. Tribolium, Gryllus, Schistocerca, Athalia [9-12]). Only a few representatives of the crustaceans and chelicerates have been studied from other arthropod classes [6,13]. Here, I report on results concerning wg and dpp expression in the appendages of a representative of the fourth extant arthropod class, the myriapod Glomeris marginata. The wg gene of Glomeris is expressed on the ventral side of the appendages compatible with a conserved role in PD axis development. Additionally, Glomeris wg may induce expression of the genes Dll and H15 in the sensory organs of the mouthparts. The results with the Glomeris dpp gene are more ambiguous. Although the data can be interpreted in favor of a conservation of the WG/DPP morphogen gradients, clearly more work on the subject is necessary to clarify the evolution of PD axis patterning in arthropod appendages. In particular, it will be necessary to elucidate the mechanisms through which the additional PD axes in multibranched appendages are patterned.
Results
Cloning of Gm-dpp cDNA fragments
A fragment of a Glomeris gene that shows sequence similarity to members of the TGF-beta gene family was isolated. In order to establish the orthology of this fragment, I performed a phylogenetic analysis incorporating a selection of TGF-beta proteins from Drosophila, other arthropods and a variety of deuterostome taxa. The resulting phylogenetic tree distinguishes two groups of proteins. One group comprises the arthropod dpp genes and their deuterostome homologs, the BMP2/4 genes. The second group consists of the Drosophila TGF-beta genes screw (scw) and glass bottom boat (gbb), and the remaining deuterostome BMP genes with TGF-beta homology, including zebrafish anti dorsalizing morphogenetic protein (ADMP). The Glomeris fragment resides in the dpp/BMP2/4 group and is therefore designated as Gm-dpp (Fig. 1). The resolution within the dpp/BMP2/4 group is low, with many nodes lacking statistical support. The Glomeris fragment forms a group together with dpp from the two-spotted cricket (Gryllus bimaculatus) and BMP2/4 from the yellow acorn worm (Ptychodera flava). However, support for this grouping is not statistically significant (reliability value = 27). A higher resolution of the dpp/BMP2/4 group may be achieved in the future by the aquisition of additional sequence information.
Figure 1 Phylogenetic analysis of the Glomeris dpp fragment. The analysis included TGF-beta genes from mouse (Mm), zebrafish (Dr), lancet (Branchiostoma floridae; Bf), acorn worm (Ptychodera flava; Pf), the sea urchins Strongylocentrotus purpuratus (Sp) and Lytechinus variegatus (Lv), fruit fly (Dm), flour beetle (Tribolium castaneum; Tc), sawfly (Athalia rosae; Ar), buckeye butterfly (Junonia coenia; Jc), the grasshoppers Schistocerca americana (Sa) and S. gregaria (Sg), cricket (Gryllus bimaculatus; Gb), pill millipede (Glomeris marginata; Gm), and the spiders Achaearanea tepidariorum (At) and Cupiennius salei (Cs). Shown is the unrooted Puzzle tree computed from 1000 intermediate trees produced with the Quartet Puzzling method [41]. The numbers at the edges denote the reliability values.
Expression of Gm-dpp during embryogenesis
The expression of a number of developmental genes has been studied in the pill millipede Glomeris marginata [7,14-17]. Of all genes studied so far the expression of Gm-dpp is the weakest. A specific in situ hybridization signal is observed after approximately six hours of staining, whereas the normal staining interval of other genes ranges between 15 and 30 minutes. This extended staining time is responsible for the intense artificial background that is visible in the preparations displayed in this paper (see Figs. 2, 3).
Figure 2 Expression of Gm-dpp in G. marginata embryos. (A) stage 2. The arrow points to expression in the dorsal portion of the neuroectoderm. (B) stage 3. The arrows point to the dorsal and ventral (middle) portion of the neuroectoderm, respectively. (C) stage 3. Aspect of the head. The arrow points to expression in the brain. (D) stage 4. Arrow: expression in the optic lobe. Asterisk: expression in the antennal neuromere. Arrowheads: expression in the heart. (E) stage 5. Arrow: expression in the optic lobe. Arrowheads: expression in the heart. (F) stage 6.1. The arrowheads denote expression in the dorsal portion of the germ band that is probably correlated with heart formation. A-E are in ventral aspect. F is in lateral aspect. Abbreviations: md, mandible; mx, maxilla; an, antenna; t1, t2, t3, first three trunk legs.
Figure 3 Expression of Gm-dpp during appendage development. (A, E, I) trunk legs. (B, F, J) maxilla. (C, G, K) mandible. (D, H, L) antenna). The arrows in A, E, I, D, H, L point to the distal expression domain in the trunk legs and the antenna, and denote the border of this domain against the ventral side of the appendage, where no Gm-dpp expression is detected. The arrows in B, F, G, K point to a ventral expression domain in the gnathal appendages. In all panels arrowheads denote expression along the dorsal side. The asterisk in K denotes expression in the external lobe. The asterisk in D denotes expression in the antennal neuromere at the base of the antenna. The asterisk in L denotes expression within the base of the antenna. The square in L is located next to a weak ring of expression in the antenna. Stages are as indicated in the top right corner the panels. Abbreviations: max, maxilla; mdb, mandible; ant, antenna.
In younger stages, a specific staining is seen in the forming appendage buds and along the external, i.e. dorsal, rim of the neuroectoderm (Fig. 2A; arrow). It is known that the neuroectoderm of each hemisegment is divided into a dorsal, medial and ventral portion [16]. Judging from its expression, it is possible that Gm-dpp has a role in the development of the dorsal portion of the neuroectoderm. A role in the developing ventral portion is also possible, since Gm-dpp is transiently expressed along the ventral midline (Fig. 2B; arrows). A further expression domain in the central nervous system is seen in the area of the developing optic centers of the brain (Fig. 2C,2D,2E; arrows).
Starting with stage 4, Gm-dpp is expressed along the external rim of the germband in tissue that will later form the heart (Fig. 2D,2E; arrowheads). Later on, segmentally repeated patches of weak Gm-dpp expression appear on the dorsal side of the embryos (Fig. 2F; arrowheads). These patches are presumably also correlated with the developing heart of the embryos. Finally, expression of Gm-dpp is found in the stomodaeum, and very weakly in the proctodaeum.
Expression profile of Gm-dpp during appendage development
The appendages buds show weak expression of Gm-dpp at the very beginning of their formation (Fig. 2A,2B). Later on, different appendages display appendage-specific expression patterns. In the trunk legs, the strongest expression is seen at the leg tips. In early developmental stages the expression fills almost the entire tip, and the border against the ventral portion of the legs (which is devoid of expression) is rather distinct (Fig. 3A). There is also expression of Gm-dpp along the dorsal side of the trunk legs, but this is visibly weaker than the expression in the leg tips. The expression at the leg tips is clearly confined to the dorsal side of the tip in legs of stage 5 embryos (Fig. 3E), while the expression along the dorsal side persists, but becomes weaker and diffuse. Finally, expression of Gm-dpp in the legs vanishes almost completely at stage 6 (Fig. 3I). The dorsal expression is virtually undetectable, and only a few cells express Gm-dpp at the tip.
In the maxilla there are two expression domains of Gm-dpp, a dorsal and a ventral one (Fig. 3B). The ventral domain is located on the internal side at the base of the maxilla. This domain slowly vanishes during development (Fig. 3F) and finally disappears around stage 6 (Fig. 3J). The dorsal expression domain runs along the dorsal edge of the base of the maxilla (Fig. 3B,3F). This domain also gradually disappears during development, and at stage 6.1 only a faint dorsal expression is detectable (Fig. 3J).
In the mandible, a dorsal expression domain that runs along the entire dorsal rim of the appendage is visible (Fig. 3C). Later on, however, this expression is restricted to the basal portion of the mandible and has a distinct border against the external lobe (Fig. 3G). Additional expression domains are detectable at later stages within the external lobe (Fig. 3K; asterisk) in addition to the internal side of the internal lobe (Fig. 3G,3K; arrow).
In the antenna, Gm-dpp is expressed in the dorsal half of the appendage with a distinct border against the non-expressing ventral half (Fig. 3D). In addition, a patch of weaker Gm-dpp expression is located on the ventral side of the antenna (Fig. 3D; square). Another patch of Gm-dpp expression is visible at the transition between the antennal base and the neuroectoderm of the antennal neuromere (Fig. 3D; asterisk). The latter two patches of expression disappear during the further course of development. By stage 5 the ventral spot has disappeared completely, and the patch at the antennal base is virtually gone as well (Fig. 3H). Similar to the other appendages at stage 6.1, the level of Gm-dpp expression has also significantly decreased, though one can discern three specific expression domains at this stage. There are two groups of cells (at the tip and at the base of the antenna) weakly expressing Gm-dpp (Fig. 3L; arrow and asterisk, respectively), and a ring of cells at the distal third of the antenna, where Gm-dpp expression is even weaker (Fig. 3L; square).
Expression profile of Gm-wg during appendage development
The expression of Gm-wg during germ band segmentation, neurogenesis, and the development of the digestive system has already been described [14]. Here, I focus on Gm-wg expression during the development of the appendages. Before the onset of limb development, Gm-wg is expressed in a stripe in each hemisegment. This stripe is located approximately in the middle of the segment and runs across the neuroectoderm and the presumptive appendage tissue. Comparison to the expression of engrailed (Gm-en) has shown that the stripe of Gm-wg expression abuts the parasegment border and, thus, is located in cells of the anterior segmental compartment [14]. The buds of the appendages form from the tissue at the external ends of the Gm-wg stripes. Preparations of complete hemisegments of stage 3 embryos show, that expression of Gm-wg extends more or less contiguously across the neuroectoderm into the forming limb buds in all four different appendage types (Fig. 4A,4B,4C,4D). The extent to which the expression reaches into the limb buds varies depending on the appendage type. In the antennal bud the expression of Gm-wg is restricted to the ventral side (Fig. 4D). In the maxillary and mandibulary buds expression includes larger areas, approximately two thirds of the buds (Fig. 4B,4C). Finally, expression is most extensive in the buds of the trunk legs: almost the entire buds express Gm-wg (Fig. 4A).
Figure 4 Expression of Gm-wg during appendage development. (A, E, I, M, Q) trunk leg. (B, F, J, N, R) maxilla. (C, G, K, O, S) mandible. (D, H, L, P, T) antenna. The arrowheads in A-D, and the arrows in E, I, M, Q and H, L, P, T point to the transition of ventral to dorsal tissue in the appendages. The arrows in A-D point to expression in the neuroectoderm of the respective body segment. The arrows in F, J, N, R denote the expression surrounding the maxillary sensory organs. The squares and asterisks in G, K, O, S denote expression of Gm-wg in the internal and external lobe, respectively. Stages are as indicated in the top right corner of the panels. Abbreviations see Fig. 3.
In the trunk legs expression of Gm-wg is restricted to the ventral side during the further course of development (Fig. 4E,4I,4M,4Q). The expression is contiguous from the base of the legs to the tips, but the level of expression is somewhat heterogeneous. The strongest expression is seen near the base and in the distal part of the legs, while expression is visibly weaker between these parts. A similar phenomenon is present in the antenna (Fig. 4H,4L,4P,4T), where expression is restricted to the ventral side of the antenna and the level of expression at the distal end is much stronger than in more proximal parts. However, unlike the pattern in the legs, the intensity of expression at the base of the antenna is not increased.
The maxilla displays a rather dynamic expression profile of Gm-wg. Beginning at stage 4 the gene is expressed along the ventral edge of the maxilla (Fig. 4F). Three domains can be distinguished that are not completely separated. The innermost domain is more diffuse than the other two domains and at stage 5 separates into two separate patches of expression (Fig. 4J,4N,4R; two-headed arrow). The two other domains remain separate during the development of the maxillary appendage and are reminiscent of the expression pattern of Gm-Dll (see below).
In the mandible, a similar fragmentation of the initial mostly homogeneous expression pattern takes place. In the external lobe the expression is strong throughout and is separated from the expression domain in the internal lobe by an area of very weak expression (Fig. 4G,4K,4O,4S). The expression domain in the internal lobe splits (Fig. 4K), then retracts from the tip of the lobe (Fig. 4O) and decreases in expression strength (Fig. 4S).
Expression of Gm-wg and Gm-Dll in the gnathal sensory organs
As mentioned above, the expression pattern of Gm-wg in the maxilla is reminiscent of the pattern described for Gm-Dll [7], and at first glance both patterns appear virtually identical. The Gm-Dll gene is expressed in the primordia of the maxillary sensory organs. Expression of the Gm-wg gene, however, is at least partially complementary to the pattern of Gm-Dll. In older stages, strong expression of Gm-wg is not detected within the primordia of the sensory organs, but rather it surrounds the primordia (Fig. 4R). In preparations simultaneously labeled with probes against Gm-wg and Gm-Dll the composite expression pattern stains the entire internal side of the maxilla (Fig. 5A,5C), indicating that both patterns complement each other. However, the maxillary expression of Gm-wg in three (later four) domains is more extensive than the more restricted expression pattern of Gm-Dll in two (later three) well-defined stripes (see [7]). Therefore, the expression patterns are certainly not mutually exclusive. The presumed overlap of the two expression patterns, however, cannot be detected with the double labeling technique used here.
Figure 5 The relation between the expression of Gm-wg and Gm-Dll. Preparations of maxillae (A, C) and mandibles (B, D) simultaneously labeled with a mixture of probes against Gm-wg and Gm-Dll. In the maxilla the patterns complement each other to stain the entire ventral edge of the appendage, whereas in the mandible no significant difference is observed compared to Gm-wg expression detected alone. Compare to Fig. 4. Stages are as indicated in the top left corner of the panels. Abbreviations see Fig. 3.
In addition, a complex and dynamic pattern of Gm-Dll has been described in the mandible [7]. In contrast to the maxilla, the patterns of Gm-wg and Gm-Dll appear to overlap completely in the mandible. In preparations of mandibles labeled with a cocktail of probes against both genes no significant difference to the pattern of Gm-wg alone is observed (Fig. 5B,5D), indicating that the Gm-Dll pattern is entirely included in the Gm-wg pattern.
Discussion
Establishment of the primary PD axis
In Drosophila dpp is expressed in a narrow dorsal sector in the leg imaginal discs, whereas wg is expressed in a similar sector on the ventral side (e.g. [1]). Together these two genes generate morphogen gradients in the developing leg imaginal discs. These gradients are utilized by several genes to guide the development of the PD axis of the leg imaginal discs. Evolutionary developmental studies have shown that the expression of wg homologs along the ventral side of the appendages is highly conserved in the arthropods (e.g. [6,9,10,13]). In contrast, dpp expression differs from the expression pattern found in Drosophila in all arthropods studied thus far (e.g. [6,9-12,18,19]). At early stages expression of arthropod dpp homologs is restricted to the leg tip, while at later stages expression rings of unclear significance appear in some species. Despite these differences in expression, it has been argued that the combined action of the WG and DPP morphogen gradients is conserved, and that the differences in expression of dpp are correlated with the differences in the mode of leg development between Drosophila (via imaginal discs) and most other arthropods (normal leg outgrowth) [6].
The data from Glomeris presented here may be interpreted in favor of this hypothesis. The Gm-wg gene is expressed along the ventral side in the legs and Gm-dpp is expressed most strongly in the leg tips. Taking these expression loci as the sources of Gm-WG and Gm-DPP protein, the resulting hypothetical protein gradients would facilitate PD patterning events similar to the ones in the Drosophila leg discs (see also Fig. 11 in [6]). However, Gm-dpp is weakly expressed along the dorsal leg side. This is similar to the Drosophila situation, but is contrary to the predictions of the above hypothesis since Glomeris does not develop the legs via flat imaginal discs and therefore should show a dpp expression pattern typical of directly developing legs rather than a pattern similar to Drosophila. The fact that Gm-dpp is also weakly expressed along the dorsal side of the legs may be explained by several possibilities. It may be argued that the dorsal expression is so weak that it has no significant influence on the shape of the Gm-DPP protein gradient, which would therefore mainly be dependent on the morphogen source at the tip. It is also possible that the dorsal expression is unrelated to PD axis formation and instead functions during DV axis formation (see below).
In any case, the picture emerging from the available data on dpp expression in arthropods is that the dorsal sector in Drosophila seems to be an exception rather than the rule. The hypothesis proposed by Prpic et al. [6] attempts to explain this by the differences in leg architecture between Drosophila and most other arthropod species. However, according to their hypothesis, the presence of combinatorial protein gradients is conserved. It should be pointed out in this context that the existence of a DPP gradient (or a WG gradient for that matter) has yet to be demonstrated in an arthropod other than Drosophila. Thus, although the expression data may be interpreted as the PD axis patterning using WG/DPP signaling being conserved among arthropods, it is obvious that comparative expression analyses alone cannot answer the question satisfactorily. It must now be considered whether experiments capable of demonstrating WG/DPP signaling during leg development in arthropods other than Drosophila may be conceived.
Establishment of secondary PD axes
Aside from the primary PD axis, many arthropods have limbs with additional branches (rami). It has been proposed that these additional rami are patterned in the same way as the main branch, simply by duplications of the WG/DPP signaling system [20]. Recent results from the study of insect mouthparts argue against this notion [11]. The insect labium and maxilla have ventral branches (endites) that apparently do not utilize a combinatorial WG/DPP gradient system to guide their outgrowth. A similar conclusion has been reached by a study of the development of crustacean multibranched appendages [13].
The presence of endites in the mouthparts of myriapods is unclear, mainly because of the modified morphology of the adult gnathalia. Certain elements of the centipede mandible and first maxilla are probably derived from endites (e.g. [21,22]) and there are attempts to assign parts of the diplopod mandible as homologous to crustacean or insect endites (e.g. [21,23]). Indeed, the embryonic mandible and maxilla in Glomeris develop ventral lobes that are very reminiscent of the endite lobes of the embryonic mouthparts in insects. The exclusive ventral origin of these lobes is further corroborated by the lack of expression of the dorsal marker optomotor-blind [17]. Furthermore, the Glomeris lobes possess Dll-positive sensory organs, which is typical of arthropod endites [7,24-26]. Thus, although the interpretation of the millipede mouthparts is disputed (see e.g. [27]), these ventral lobes are likely homologous to the endites present in insect mouthparts.
The embryonic Glomeris mandible develops two lobes, the internal and external mandibular lobe. Both lobes express Gm-dpp, but not at a position suggestive of a role in PD axis formation (Fig. 6C). In addition, the expression domain in the external mandibular lobe appears after the lobe has already grown and is therefore unlikely to be involved in PD outgrowth. The development of the maxillae is more complex. They start out as separate appendages, but around stage 6 the left and the right maxilla fuse along their internal sides (Fig. 6A,6B). Each maxilla has a single lobe, containing the primordia of three sensory organs that can also be visualized by Gm-Dll expression [7]. The two external sensory organ primordia form the lobus medius and the lobus exterior in the adult (see [27] for a detailed description of Glomeris maxillary morphology). These two sensory structures grow from the internal side of the stipes (Fig. 6A). The internal sensory organ primordium is different from the other two in the sense that it will not end up on the stipes, but will form the lobus interior that grows from the lamella lingualis (Fig. 6A). The lamellae linguales of the right and left maxilla fuse around stage 6 to form the intermaxillary plate (Fig. 6B). The single lobe in the Glomeris embryonic maxilla therefore has a rather complex fate in the adult mouthpart (the gnathochilarium [27]). The portion of the maxillary lobe that will give rise to the stipes expresses Gm-dpp at only later stages when the expression along the dorsal side of the maxillary base is extending weakly into the stipes. The portion forming the lamella lingualis also expresses Gm-dpp, but at its internal edge, a location hardly suggesting a role in PD outgrowth of the maxillary lobe.
Figure 6 Possible endite homologs in the mouthparts of Glomeris. Schematic representations of the mouthparts of Glomeris (A-C). An insect mouthpart (maxilla of Schistocerca) is shown for comparison (D). (A) Glomeris maxilla. (B) Left and right maxilla of Glomeris already fused forming the gnathochilarium. (C) Glomeris mandible. The palp is proposed to be lost in Glomeris gnathalia ([7]; grey hatched line). The black hatched line indicates where the appendage inserts on the segment. Expression of dpp is shown in grey (hatched area in the maxilla: very weak expression). Please note that the figure shows all observed expression domains in a single drawing although really some domains appear at different time points (see text for a description of the temporal expression profile). None of the possible endite homologs in Glomeris mouthparts (lli, st, iml, and eml) expresses Gm-dpp in a fashion suggestive of a role in PD axis patterning. See text for details. Expression of dpp in Schistocerca is after [11]. Abbreviations: bs, base; ca, cardo; eml, external mandibular lobe; ga, galea; iml, internal mandibular lobe; lc, lacinia; le, lobus exterior; li, lobus interior; lli, lamella lingualis; lm, lobus medius; plp, palp; st, stipes.
In summary, none of the maxillary and mandibulary lobes in Glomeris appear to utilize conventional WG/DPP signaling to organize PD growth. Similar results have been obtained recently for the endites in the grasshopper Schistocerca and the beetle Tribolium [11]. In Schistocerca at least one endite (the galea) grows without dpp expression (Fig. 6D), and in Tribolium both maxillary endites lack detectable dpp expression [11]. This indicates that the development of the PD axis of the endites does not generally require the WG/DPP morphogen system.
Relation of wingless and dpp expression to DV axis formation
A second role of wg and dpp in Drosophila is the activation of some factors involved in DV axis formation in the legs [28,29]. wg, being expressed along the ventral side, is an instructor of ventral fate, whereas dpp is expressed on the dorsal side and establishes dorsal fates. The primary factors controlled by wg and dpp are H15 on the ventral side and omb on the dorsal side. These factors have been recently studied in Glomeris and in a spider (Cupiennius salei) [6,17]. The expression patterns suggest that the role of omb as dorsal instructor is evolutionarily conserved, but H15 does not seem to be a general ventralizing factor in all arthropods. Thus, the dorsal, but not the ventral developmental mechanisms seem to be conserved. It is interesting that the expression data of wg and dpp suggest that the opposite is true. The wg expression on the ventral side is highly conserved among the arthropods, but the dpp patterns differ between species and in most part expression is not localized to the entire dorsal side. This paradox clearly demonstrates the limited understanding of the evolution of DV axis formation in arthropod appendages.
Patterning of appendicular sensory organs
The maxilla of Glomeris has several sensory organs. Recent studies have identified the genes Dll, dac and H15, which show a restricted expression pattern in the primordia of the maxillary sensory organs [7,17]. Two of these genes, Dll and H15, are known from Drosophila to be activated upon signaling through the wingless pathway [29,30]. It is interesting to note that expression of Gm-wg surrounds the sensory primordia in the Glomeris maxilla. It may therefore be the case that cells expressing Gm-wg in the surrounding of the primordia signal to their neighbors within the primordia and stimulate them to activate Gm-Dll and Gm-H15. Minimally the activation of Dll appears to be a general feature of appendicular sensory organs in arthropods since Dll expression has been observed in appendicular sensory organs in chelicerates, crustaceans, myriapods and insects (e.g. [7,24,25,31]). Moreover, data from Drosophila suggest that Dll expression is critically required for sensory organ formation, as mutants lacking Dll fail to develop Keilin's organs (the sensory structures of the embryonic leg anlagen) [32,33].
Conclusions
The expression of Gm-wg and Gm-dpp during appendage development indicates a role for both genes in guiding this process. Involvement of wg and dpp in appendage development appears to be conserved among all extant arthropod classes including myriapods. The data from Glomeris and other arthropods suggest that the WG/DPP morphogen signaling system as it is known from Drosophila leg discs is present in all arthropods. However, this morphogen system apparently functions in only the main branch of the appendages, the so-called telopodite [34]. Limb types with additional branches (e.g. endites) obviously use additional, yet unidentified mechanisms to organize proximal-distal growth of the extra branches. Gene expression in potential endite homologs present in Glomeris mouthparts supports this notion. Aside from the role in PD axis formation, the expression profile of Gm-wg suggests an additional role for this gene in patterning appendicular sensory organs.
Methods
Animal stocks
Animals were collected during Spring 2003 in beech forests in the vicinity of Cologne, Germany and near Kranenburg, Germany. They have been treated as described before [6,14]. The animals were released after the end of the breeding season (Summer '03).
Molecular cloning
The cloning assays were based on cDNA transcribed from polyA-RNA extracted from selected Glomeris embryos of all developmental stages up to stage 6.1 (see [14,35] for a description of embryonic stages) and were performed in duplicate. For the amplification of dpp-like gene fragments, the primers dpp-fw-1 (GAY GTN GGN TGG GAY GAY TGG) and dpp-bw-1 (CKR CAN CCR CAN CCN CAN AC) were used in the initial PCR, and the primers dpp-fw-2 (GGN TAY GAY GCN TAY TAY TG) and dpp-bw-1 were used in the nested PCR. Additional sequence information was gained by RACE PCR. No full-length fragment could be obtained and several artificial clones were encountered, probably representing chimeric products resulting from jumping PCR between different TGF-beta-like cDNAs. Using species specific primers, artificial and genuine fragments were identified. A confirmed genuine fragment of almost 360 bp was isolated and cloned. This fragment was used for sequence analysis and probe synthesis. The isolation of Gm-wg has been previously reported [14]. The GenBank accession numbers are as follows: Gm-wg (AJ616907); Gm-dpp (AJ843875).
Alignments and sequence analysis
Pairwise alignments of aminoacid sequences were performed by searching GenBank [36] using the Gapped BLAST program [37]. The alignments were calculated based on the BLOSUM 62 matrix [38] (gap costs: 11 for opening, 1 for extension). Multiple sequence alignments were calculated based on the GONNET matrix [39] (gap costs: 10 for opening, 0.2 for extension) implemented in CLUSTAL_X [40]. The resulting alignments were subjected to maximum likelihood analysis using the Quartet Puzzling method [41] as implemented in PAUP* 4.0b10 [42].
In situ hybridizations, specimen preparation and microscopy
In situ hybridization with digoxigenin-labeled RNA probes has been performed as previously described [7]. Whole-mount embryos were photographed in PBST under a Leica dissection microscope. Appendages were dissected with fine insect needles and photographed in 50% glycerol under a Zeiss Axioplan microscope. All images were corrected for color values, brightness and contrast using Adobe Photoshop 5.5 for Apple Macintosh. The image processing software has also been used to enhance image backgrounds by retouching dirt or yolk remains, and to group together single pictures into multipanel figures.
Acknowledgements
I thank Diethard Tautz for his encouragement and advice during all phases of my work. The clone of Gm-wg has been a gift from Ralf Janssen. The Glomeris RACE template has been a gift from Hilary Dove. I also thank Wim Damen for comments on the manuscript. I thank John Baines for his help with the English language. This work has been funded by a grant from the Deutsche Forschungsgemeinschaft (grant number TA99/19-2).
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Emerg Themes EpidemiolEmerging Themes in Epidemiology1742-7622BioMed Central London 1742-7622-1-11567990810.1186/1742-7622-1-1EditorialThe birth of Emerging Themes in Epidemiology: a tale of Valerie, causality and epidemiology Tam Clarence C [email protected] on behalf of the editorial board of Emerging Themes in Epidemiology2 Deputy Editor, Emerging Themes in Epidemiology3 Infectious Disease Epidemiology Unit, Department of Infectious & Tropical Diseases London School of Hygiene & Tropical Medicine, London, United Kingdom2004 6 10 2004 1 1 1 10 6 2004 6 10 2004 Copyright © 2004 Tam; licensee BioMed Central Ltd.2004Tam; 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.
Emerging Themes in Epidemiology (ETE) is a new, online, Open Access peer-reviewed journal. The Journal is unique in that it was conceived and is managed by research degree students in epidemiology and related public health fields. The Journal's management is overseen by its Editor-in-Chief and Associate Faculty Editors, all of whom are senior members of faculty. ETE aims to encourage debate and discussion on the theoretical, methodological and practical aspects of epidemiologic research and practice. In addition, ETE is dedicated to the promotion of Open Access publication and the training of research students in the scientific publishing process. This editorial, to coincide with the launch of ETE, sets out the Journal's philosophy and aims. Epidemiology is a rich and innovative science that has much to gain from broader discussion of the causal frameworks that underpin it. ETE aims to be a major forum for such discussion.
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"Causality. There is no escape from it, we are forever slaves to it. Our only hope, our only peace is to understand it, to understand the 'why'[1]."
- The Merovingian
As far as we know, the Wachowski brothers' Merovingian was not an epidemiologist, but his sentiments should resonate well with those in the field. Probably more than those in any other profession, epidemiologists are slaves to causality. Our professional lives are dedicated to the pursuit of pumphandles, spiderwebs and causal pies [2,3]. Adding to these colourful metaphors are ones proposing frameworks for epidemiologic research that involve Chinese boxes, computer-generated fractals and prison breaks [2,4,5]. But what is the student of epidemiology to make of all these curious abstractions? Lost in a sea of metaphors, they might very likely throw their arms up in the air and, in thorough confusion, decide to take a long and much-needed coffee break [6-8].
The epidemiologic literature on causality certainly makes for stimulating reading, but it would be interesting to know how many of us have causal pies and fractals on our minds as we reach for that red folder labelled "Logistic regression 101". Discursive articles on the usefulness of such metaphors are widely regarded as philosophical flights of fancy that we might eventually get around to reading after clearing that backlog of papers waiting to be written in the next two months. Yet what are our alternatives? The newly-qualified epidemiologist leaves their degree with a solid grasp of error, bias, confounding and statistical methodology, but with perhaps a single lecture on Koch-Henlé postulates and Bradford-Hill 'criteria' as the extent of their training into causal thinking. It is interesting to note that Last's Dictionary of Epidemiology does not include the term 'cause', opting instead to give a definition of 'causality' that involves a brief discussion of necessity and sufficiency [9]. Any reasoned assessment will quickly lead to the conclusion that guidelines for determining causal pathways as commonly taught in epidemiology courses are woefully inadequate, regardless of whether one decides to take an inductionist, refutationist, or hypothetico-deductivist view [10,11], or admits to not having a clue what any of these terms mean. The challenge for the modern epidemiologist is to put those newly-learned methods to use from within a causal limbo, with Robert and Austin as their guides and John Snow as spiritual counsellor.
The anatomy of a cause
James Wong's 2000 teen horror movie Final Destination [12] is unlikely to go down in history as a cinematic classic, but it is memorable for its clever, if rather gruesome, depictions of causal processes leading invariably to the death of a number of its unfortunate characters. The movie's motto is that "you can't cheat death's plan". Having narrowly avoided a fatal plane accident thanks to the protagonist's chilling premonition, a French teacher and five of her students are destined to fall victims to the Grim Reaper one by one- in the order they would have died had they been on the ill-fated plane with the rest of their class- unless they can find a way to break the cycle and cheat death. In the most elaborate death scene, Valerie, the French teacher, alone in her house and still visibly shaken by the loss of her colleague and students, becomes unnerved by noises outside. With John Denver's Rocky Mountain High ironically playing in the background, she tries to calm herself by making some tea. Moving to the kitchen sink, she fills the kettle with water, spilling some down the side. She wipes the kettle, turns towards the gas stove and tosses the towel carelessly behind her, which catches onto a knife block. With the kettle whistling, she picks up a school coffee mug, drops two tea bags inside and fills it with boiling water. Picking up the mug, she suddenly recognizes the school logo and, in shock, reflexively throws the mug's contents into the sink. Opting now for something stronger to calm her nerves, she takes some ice cubes from the freezer, drops them into the still-warm mug and re-fills it, this time with vodka. Oblivious to the crack that has appeared in the mug, she walks towards the living room, leaving a trail of vodka behind. As she stands by her computer monitor, vodka drips into the circuitry. An electrical surge creates a spark that ignites the alcohol, causing the monitor to explode and sending out flying shards of glass that slash her throat. Shocked and bleeding, she stumbles towards the kitchen sink, chased by a trail of burning alcohol. Reaching the stove, the trail of flames ignites the gas burners, lighting up her clothes and hair. Falling to the ground and still bleeding, she rolls around violently trying to put out the flames. In an act of desperation, she reaches up and grabs the dangling towel, tilting the knife block and sending a half dozen blades cascading into her stomach while flames catch the curtains and set the house on fire.
Suppose you were the investigator arriving on the scene. There is a half burnt-down house, a blown-up computer, a broken mug and a corpse with third-degree burns, stab wounds and a cut throat, but no signs of struggle. What would you determine was the cause of this tragedy? The severe burns, the protruding knives and the neck wound would be pretty obvious choices. But perhaps it is more complicated than that. Perhaps there were extenuating circumstances without which this tragic outcome might not have occurred. The exploding computer maybe, or what about the vodka that burned leaving no trace, or the cracked mug? No, maybe it was the towel, complicitly catching onto the knife block. Or maybe we should blame the teacher's drinking habit.
The point of this rather unsavoury story is that without the benefit of such extrinsic observation, detailed reconstructions of causal processes are unattainable. A reasoned observer might conclude that all of the above factors were in some way responsible. They all contributed to the process in their own small way, and had any one of them not been involved things may not have turned out the way they did. They were all what one might call 'component causes'. But is this enough to convince us of what the real cause was? Clearly not.
Suppose now that you were an audience member and were somehow able to communicate directly with the characters in the movie. You might wish to warn Valerie of her impending ill fate. At which point in the whole sequence would you alert her so that her death could be prevented? Clearly you would not deny her a mug of tea and you would most likely have no way of knowing that the towel would land on the kitchen block with dire consequences just a few moments later. You might, of course, realize this at the last moment and warn her against reaching up and grabbing the towel, but she might still have died from her neck wound. You might have shouted for her to grab a fire extinguisher and put out the trail of flames, but it is unlikely she would have listened to you as she tried hopelessly to stop the bleeding from her neck. You might, with better foresight, have pointed out to her that vodka was dripping from her mug. Or perhaps with hindsight, you might have recognized that the best thing would have been to provide some moral support and consolation in her time of grief, with which the whole sorry incident might have been avoided altogether.
One thing is now clear. There are steps within causal processes on which we can act to try and alter their course- the trail of flames might have been extinguished, and the consequences of the dripping vodka might have been avoided. There are other steps on which we can have no influence, eg. the tossed towel landing on the knife block. Another important thing is also apparent: causal processes have hierarchies. Depending on what happens at one stage, a number of alternative events may result at the next. Had Valerie been able to stop the trail of flames, she might have reached the kitchen sink, realized she could not stop the bleeding and called an ambulance. Or she might have run out of the house shouting for help and her neighbour, trained in first aid, might have saved her life. Saving victims of horror movies, however, is not an easy job. Knowing when best to step in is not necessarily that simple, as we have seen with Valerie. In some cases we may be given a number of opportunities and intervening at any of them might lead to a positive outcome. But in other cases, once certain factors are in place there will be an air of inevitability in everything that follows, and all our attempts to intervene thereafter may prove to be little more than an exercise in futility.
Perhaps it is now time to admit that I have extended this fanciful analogy far beyond what is appropriate. I make no apology if in so doing I have in some way managed to convey the idea that epidemiology thrives on causal processes, on elucidating their complexities and identifying the most effective points for intervention no matter at what level of their elaborate hierarchies. If this is a worthwhile venture, then the discussion of how we conceptualize and study these causal processes surely is so too.
Emerging Themes in Epidemiology (ETE) was born out of this ideal- that contrary to common belief, epidemiology is not merely a collection of standardized tools to be applied at will to any health-related situation, but that it is a rich and innovative science that aims to describe reality in all its complexity, spanning the molecular to the global, with the ultimate goal of improving the health of individuals and populations. And that in order for this to be achieved, we need to improve our understanding of how factors, at any level of biological or social organization, eventually lead to ill health.
ETE is founded on three core principles:
• That epidemiology and epidemiologists have much to gain from a broader and more fundamental discussion of the concepts and theoretical frameworks that underpin the practice of epidemiologic research
• That Open Access publication has a crucial role to play in reducing the current inequities in access to scientific information, which should be a universal and freely-available resource for the benefit of the whole of society
• That students of epidemiology and related fields can make substantial contributions to the introduction of new concepts and ideas into mainstream epidemiologic research, not only through writing, but also through having a direct influence over what is published
In keeping with this philosophy, we recognize that epidemiology has much to gain from developments in other fields and we welcome contributions from all public health professionals. We will consider articles that comment critically on current epidemiologic theory and practice, either generally or within a specific specialty, including articles from other fields that have implications for the conduct of epidemiologic research. ETE will not generally publish research reports, although exceptions may be made in cases where the results can be placed within a broader public health context to present a new concept or theoretical framework.
By making all of this material freely available online under the auspices of the Open Access publisher, BioMed Central, we aim to make Emerging Themes in Epidemiology a global forum for the discussion of new developments in epidemiologic thinking and practice that will benefit the global public health community. In doing so, we recognize that there is much that the scientific community can do to support Open Access publication. The health consequences of inequitable access to scientific information remain largely ignored, yet for years we have adhered to a system of publication that is restrictive and largely subsidized by institutions and libraries at great expense. Open Access is an important step towards making the publication process, and its associated costs, more transparent. We thus call on individuals, institutions, funding bodies and governments to engage in the Open Access movement by promoting and supporting Open Access publication. This will involve a major shift not just in publishing costs, but also in thinking. Our current measures for assessing the impact of academic research are inherently intertwined with our inequitable publication tradition. Developing new ways of assessing the quality of scientific research that are independent of journals' perceived 'impact' are imperative for the wider recognition of Open Access.
In promoting the role of students in the publication process, we intend for ETE to be a training ground for postgraduate students, providing them with an opportunity to be involved at every stage of the publication process, including commissioning, reviewing and writing articles. Our editorial board is formed principally of research students, who operate the Journal with support from an international group of associate faculty. Our collaborations extend to a growing number of students from diverse institutions serving as article referees. We welcome suggestions for extending these collaborations in the future.
The third millennium has brought with it exciting challenges for epidemiologists. An explosion in the availability of genetic and molecular information, the development of bioinformatics, the increasing application of sophisticated statistical analyses to model complex systems and the gradual incorporation of sociological approaches to understanding health inequalities have arrived together with sobering statistics on the state of global public health. Knowing when and how to apply these and other developments at a time of rapid political, social and biological change while maintaining clear sight of our public health goals will be the major challenge for current and future generations. It is our hope that Emerging Themes in Epidemiology will be a tool for epidemiologists confronting that challenge.
Acknowledgements
My thanks to Ben Lopman, Dina Handan, Sue Lee and various members of the Editorial Board for their comments on the manuscript.
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Emerg Themes EpidemiolEmerging Themes in Epidemiology1742-7622BioMed Central London 1742-7622-1-21567990310.1186/1742-7622-1-2EditorialEmerging Themes in Epidemiology: Form and function Lopman Ben A [email protected] Deputy Editor, Emerging Themes in Epidemiology2 on behalf of the editorial board of Emerging Themes in Epidemiology3 Department of Infectious Disease Epidemiology, Imperial College London, London, United Kingdom2004 6 10 2004 1 2 2 7 7 2004 6 10 2004 Copyright © 2004 Lopman; licensee BioMed Central Ltd.2004Lopman; 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.
Emerging Themes in Epidemiology is a new, online Open Access journal. This editorial – which coincides with the Journal's launch – describes its unique review and publication model. The editorial board and review process of ETE will be managed by research degree students and will therefore be a training ground for students (though final editorial control rests with senior faculty Associate Editors). With our mix of Open Access publishing and the strong involvement of students in editing and reviewing, we believe that Emerging Themes in Epidemiology will be a progressive medium for promoting new ideas in epidemiology.
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The idea that has given birth to Emerging Themes in Epidemiology (ETE) was conceived late on a Friday afternoon. As the rest of the academic and professional work week was coming to an end, the creators of this journal were just beginning to think straight. Clearly the work of students. However, it was not just clandestine meetings at unsociable hours that brought this diverse group of epidemiology PhD students together to launch and direct a new journal.
Through training and various research projects PhD students become versed in the technical, methodological and practical aspects of epidemiology. While some will stay in academia and some will use their skills in other sectors of public health, our time as PhD students is seen as a time to learn and a time to be a part of the academic community. However, aside from the occasional correspondence with editorial boards, research students receive little training or involvement in the process of scientific publication. We think that this is an important process for us to learn about since, as researchers, our careers are dependent on publication. So we came together to form ETE, which will be a training ground for PhD students.
This Open Access journal will also be our contribution to the field of epidemiology. Students will manage the editorial board and they will be a part of the review committee for each paper. Through this process we hope to learn. But, we also hope to facilitate the publication of new ideas and to raise issues that concern us as students of epidemiology. In the other editorial in this launch 'issue' of Emerging Themes in Epidemiology, the philosophical underpinnings of the Journal have been illustrated. Now, some explanation is required of how we will achieve our vision of ETE.
The Editorial Board
The editorial board of ETE is made up of doctoral students and faculty. The research students are from various academic and public health institutes in London (though we are open to the prospect of overseas expansion). The student members are responsible for the running of the Journal, for managing the review process and communicating with authors and readers. The board is headed by our Editor-in-Chief, Professor Peter Smith, and is overseen by an international panel of Associate Editors. The role of these senior faculty is to consider the major decisions of the Board and to preside over final publication judgements. And, because PhD studentship is a transient state (of varying length), these Associate Editors will maintain the continuity of ETE by recruiting new student editors as the current board graduates.
The review process and student reviewers
At present, there is a lively debate as to whether peer-review should be 'open' or 'closed'. Recognizing the forceful arguments on both sides, we believe that the scientific community is best served by a system that encourages a constructive dialogue between colleagues. Authors and reviewers will know each others' name and affiliation. However, we recognize that some highly specialized fields of research are small and competitive. In reality, this means that there are often repercussions for giving both criticism and praise to a colleague's work. So at the reviewers' request, we will withhold their names from the authors.
When a paper is submitted to ETE, it is assigned to one of our two Deputy Editors. A committee of five research students then screens the manuscript to decide whether it fits within the scope of the Journal. If they decide it does, the paper is sent for external peer review. All papers are seen by a minimum of two reviewers. As part of our dedication to involving research students in the editorial process and facilitating the publication of new ideas, at least one PhD student and one expert in the appropriate field will be asked to review each paper. How will PhD students perform as reviewers? There is evidence that young reviewers (under 40 years old) trained in epidemiology or statistics, and those who can dedicate about 3 hours provide better quality reviews [1]. Clearly, these are characteristics of most of our student reviewers, so we are optimistic about our review process. Furthermore, in accord with our objective to make the Journal a training resource, all student reviewers will be informed of the Editorial Board's decision. They will see how their review influenced that decision and how it complemented or contradicted with the reviews of more experienced reviewers (the 'experts').
Final decisions to accept or reject will always be done with the endorsement of one of the Associate Editors, who are senior researchers in various epidemiologic fields. The involvement of students in the review process is admittedly novel, but we believe there are sufficient checks and balances in our review system to ensure quality. Testament to this is the decision by PubMed and PubMed Central to index ETE. Authors can therefore be confident that their papers will have the highest visibility and will be available in PubMed's archives.
Conflict of interest in a journal dedicated to discourse
The issue of conflict of interest is anathema to many scientists. We are trained to pursue objectivity; the suggestion that one's methods, results or conclusions are in any way affected by funding sources or other affiliations can be seen as a direct challenge to credibility. It shouldn't be. In the complex funding and political environments in which we work, we can no longer deny that these issues must be faced. Evidence has mounted that funding is associated with research outcomes (in tobacco and food) and, recently, high-profile cases related to vaccine safety and nutritional supplements have furthered the case for explicit statements of potential conflicts [2-5]. Once again, ETE will encourage openness from authors as well as reviewers. An individual's financial or professional associations do not necessarily constitute conflicts of interest, but failing to declare them can be highly misleading to the reader and the scientific community [6].
ETE is a journal dedicated to active discourse in epidemiology. We will ask contributors to disclose their source(s) of funding – as it could be seen to influence opinions or research agenda. We will also ask authors to disclose links to other associations that are not explicit from their institutional affiliation.
Open Access and financing of ETE
Perhaps there is no discipline more appropriate for open-access publication than epidemiologic research and the allied population health sciences. Our work has the aim of revealing the determinants of disease in human populations, with the ultimate goal, through knowledge empowerment, intervention and policy, to reduce suffering from ill health. With such an ethos, how can we justify keeping research findings locked away in expensive medical journals, inaccessible to those who may need it most?
The open-access publishing model has the potential to revolutionize the way authors use scientific literature and the manner in which it is received by other scientists and the community at large. The advent of open-access publishing means "it is now possible to make our treasury of scientific information available to a much wider audience, including millions of students, teachers, physicians, scientists, and other potential readers, who do not have access to a research library that can afford to pay for journal subscriptions" [7].
Currently, the most common economic model for sustaining Open Access journals is one where the author pays for the costs incurred in the editorial and publishing process. BioMed Central, our publisher, charges authors per accepted publication (currently $525), but fees are waived for authors from institutions with membership to BioMed Central or authors with financial hardship. Our hope is that a long-term solution will in future be developed by the scientific community as a whole, perhaps through the provision of grant schemes for Open Access publication. Nonetheless, recognizing the difficulties with the current 'author pays' model, we aim to cover the article processing fees for at least the next few years for all submissions not already covered under institutional memberships or other grants or subsidies.
Special issues and online publishing
From time to time ETE will call for papers on a particular subject pertinent to the theory and practice of epidemiology. The series of articles will be built around a framework, designed by the editorial board and a guest editor. The series of articles will be published in tandem, and will be highlighted on a special page on the website. The flexibility of online publishing will allow for articles submitted after the initial publication of the issue and for commentary on the original articles to also be included. Guest editors for special issues will work with the Editorial Board to develop an issue, commission appropriate articles and write a leading editorial. Anyone interested in collaborating with ETE to curate a special issue is encouraged to contact the Editorial Board.
The birth of ETE
Epidemiology is 'young' science, rapidly evolving in its theory, methods, and subject matter. With our mix of Open Access publishing and the strong involvement of students in editing and reviewing, we believe that Emerging Themes in Epidemiology will be a progressive medium for promoting new ideas that will contribute to the development of the field.
Acknowledgements
Thanks to Clarence Tam, Sue Lee, Victor Schoenbach, Peter Smith, Michel Coleman and the rest of the members of the Editorial Board for their comments on this manuscript.
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Horton R The lessons of MMR. Lancet 2004 363 747 749 15016482 10.1016/S0140-6736(04)15714-0
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| 15679903 | PMC544940 | CC BY | 2021-01-04 16:38:26 | no | Emerg Themes Epidemiol. 2004 Oct 6; 1:2 | utf-8 | Emerg Themes Epidemiol | 2,004 | 10.1186/1742-7622-1-2 | oa_comm |
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Emerg Themes EpidemiolEmerging Themes in Epidemiology1742-7622BioMed Central London 1742-7622-1-31567990910.1186/1742-7622-1-3Analytic PerspectiveAssessing the impact of humanitarian assistance in the health sector Roberts Les [email protected] Charles-Antoine [email protected] Humanitarian Policy Group, Overseas Development Institute, London, United Kingdom2004 7 10 2004 1 3 3 5 10 2004 7 10 2004 Copyright © 2004 Roberts and Hofmann; licensee BioMed Central Ltd.2004Roberts and Hofmann; 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 have been significant improvements in the design and management of humanitarian aid responses in the last decade. In particular, a significant body of knowledge has been accumulated about public health interventions in emergencies, following calls for developing the evidence base of humanitarian health interventions. Several factors have prompted this, such as the increased volume of humanitarian assistance with subsequent higher levels of scrutiny on aid spending, and greater pressure for improving humanitarian aid quality and performance. However, documentation of the ability of humanitarian interventions to alleviate suffering and curb mortality remains limited. This paper argues that epidemiological studies can potentially be a useful tool for measuring the impact of health interventions in humanitarian crises. Survey methods or surveillance systems are mainly used for early warning or needs assessment and their potential for assessing the impact of aid programmes is underutilised.
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Introduction
In the last decade, humanitarian aid agencies have put a great deal of effort into improving the design and management of humanitarian responses in conflict situations: humanitarian agencies have agreed on international standards, numerous technical guidelines have been produced, training programmes are offered on different aspects of humanitarian programming, and aid programmes are evaluated more systematically. Initiatives as distinct as the Code of Conduct, Sphere, Active Learning Network for Accountability and Performance in Humanitarian Action, the Humanitarian Accountability Partnership International, the Quality Project from the French group Urgence, réhabilitation, développement and People in Aid all have in common a concern for the quality, performance and impact of humanitarian assistance [1]. In particular, a substantial body of knowledge has been accumulated regarding public health interventions in emergencies, in response to calls for developing the evidence base of humanitarian health interventions [2-4]. Most of these developments have been stimulated by the findings of the system-wide Rwanda evaluation, which highlighted serious questions about the performance of some humanitarian organisations and, more importantly, emphasised the responsibility of the international community: humanitarian aid alone cannot substitute for political action [5,6].
Despite the technical progress in the last decade, knowledge of the impact of humanitarian interventions in alleviating suffering and ultimately reducing mortality in the health and other sectors remains limited. There is a real question whether all the developments in accountability and implementation actually improve the overall performance of humanitarian assistance [7,8]. Whereas there is a significant evidence base on the effectiveness of interventions in acute emergencies, especially in refugee settings, the evidence base is much weaker for situations of protracted conflict with longer term programmes in less controlled settings. It is also difficult to determine the relative contributions of humanitarian aid programmes as distinguished from local coping mechanisms and/or the interventions of national governments. This lack of knowledge exists at the programmatic level and more generally at the sector-wide level.
This paper examines the current practice of humanitarian agencies for measuring the impact of health interventions. It is based on a review of the literature as well as on the authors' own field experience. The question explored is whether field epidemiology provides a useful set of tools and methods to determine more accurately the impact of health interventions.
The impact of humanitarian assistance
The issue of impact is particularly high on the current humanitarian agenda. The increasing interest in impact analysis arises from a number of interlinked developments: the rapid increase in the overall volume of humanitarian assistance in the last decade- from 2 billion in 1990 to $5.5 billion by 2000 [9]- and the resulting scrutiny on how money is spent, the new public management agenda within the public sector and the adoption of results-based management systems by donors and some aid agencies.
There is no definition of impact in humanitarian assistance. The most commonly used definition of impact in international aid is the one provided by the OECD/DAC which defines impact very widely as ' [t]he positive and negative, primary and secondary, long-term effects produced by a development intervention, directly or indirectly, intended or unintended' [10]. This paper concentrates on the intended effects of aid interventions, e.g. whether the original objectives of a programme have been met. The question of negative, unintended impact, which is undoubtedly important, falls out of the scope of this paper.
A central question in an impact assessment is 'what would have happened in the absence of the aid programme?' In theory, there are two approaches to answering this question [11]:
1. To compare the impact with a control group that did not receive aid – a "with/without" comparison.
2. To do a "before/after" comparison for the beneficiaries of an intervention.
These two approaches pose different sets of issues. The "with/without" comparison with the use of control groups creates ethical problems: it is difficult to deliberately exclude a group from access to potentially life-saving relief. It may happen that some particular groups do not receive relief, due to problems with access or lack of resources, but, as Hallam warns, comparisons between people who received assistance and those who did not need to be used very carefully [12]. As a result, very few experimental trials with randomised allocations of services have ever been conducted in emergency or refugee situations [13,14].
The "before/after" comparison generates different problems. In order to be valid, the comparison implies that no other factor influences the impact, so that it can be fully attributed to the intervention. In reality, a multiplicity of other factors have an influence on the impact, such as the presence of other aid programmes, local coping mechanisms, or changes in the social and economic environment. For example, it is very difficult to attribute a reduction of conflict-related excess mortality to humanitarian aid only: variations in the baseline mortality (due to seasonal trends, disease epidemics, HIV/AIDS etc.) can be significant. There is an increasing recognition of the importance of these other factors, and that humanitarian aid, however vital, is only one element of the picture [15-18].
A theoretical model for measuring impact
There are three main considerations that are necessary for measuring the impact of health programmes: the strength of the evidence suggesting causation between the intervention and the change in health status, the validity of a baseline of comparison, and the validity of the indicator employed. These conditions also apply to other types of humanitarian assistance.
Criteria of causation
Over time, a great deal of debate has arisen over what epidemiological evidence constitutes proof that some exposure or input produced an effect versus what evidence simply implied an association. This distinction can be more than academic, as was seen over three decades of debate regarding the effects of smoking on health. Bradford-Hill put forward criteria for attempting to ascribe causation between an exposure and a health outcome [19]. These criteria are so widely utilised that some introductory text books simply refer to them as the epidemiological criteria of causation [20]. While his main motive was to attribute causation of a disease due to exposure to a chemical or biological agent, the logic of these criteria also applies to assessing the positive effects of favourable exposures, such as health programmes.
Bradford-Hill said that all of the following conditions can contribute to the argument that an exposure induces a health consequence:
1. The greater the strength of the association, the more likely that it is causative
2. There is a dose-response relationship between the exposure and the health outcome
3. Exposure consistently induces the health consequence in different settings at different times
4. The exposure occurs before the health outcome
5. There is a biologically plausible explanation for the exposure resulting in the health outcome
6. There are not more plausible explanations for the health outcome
7. Experimental results add particular weight to the evidence
For virtually all cause and effect health relationships, some of these criteria will not apply. For a programme to be shown to have an impact, criteria 4, 5 and 6 should always be met. Of particular concern to programme evaluation is the issue of biological plausibility and the amount of service provided. Programmes need to be evaluated with particular regard to the likelihood that the level of inputs provided could plausibly result in the outcome reported. That is, the number of clinic visits, or the amount of food provided per child etc. need to be sufficient to induce the health effects observed. Criteria of causation are important for interventions and settings where project impacts are usually not, or cannot be, measured.
These criteria of causation can be applied to populations and programmes as readily as Bradford-Hill applied them to specific disease agents. For interventions with a vast literature documenting the attributable benefits (e.g. measles vaccination or Vitamin A supplements), the need to show "proof" that the intervention produced a health benefit may be small, but for many other emergency interventions (e.g. HIV prevention through educational efforts or health benefits from shelter) there may be little or no evidence that such programmes produce any health benefits, making the importance of documenting any benefits great. Most humanitarian programmatic efforts fall somewhere in between, employing types of programmes that have produced documented benefits in some settings, but have failed in others, and may or may not be producing benefits in the setting at hand. Table 1 provides an example of how Bradford-Hill's criteria can be used.
Table 1 Application of Bradford-Hill criteria: Katana, Democratic Republic of Congo
Starting in December of 2000, the International Rescue Committee (IRC) began a general health programme to support existing government services in Katana Health Zone, Democratic Republic of Congo (DRC). The IRC conducted population-based mortality surveys in this area with 345,000 mostly rural residents. The programme consisted of the provision of drugs, supplies, training and medical oversight in the clinics, a water provision and hygiene education programme in villages with the highest rates of cholera in 2000, a measles immunisation and vitamin A provision campaign, and support to the local health committees which included the donation of vouchers for the most indigent community members. Figure 1 below shows the crude mortality rate (CMR) over the period covered by 5 surveys conducted between 1999 and 2002. IRC claims to have reduced the excess CMR by 60% (from 4.9 to 2.8 deaths per 1000 per month where the baseline is assumed to be 1.5) during the period from 6 to 12 months after implementation and by 70% (from 2.8 to 1.9 deaths per 1000 per month) over the period from 12 to 24 months after implementation. In support of the results in figure 1 being a consequence of the health programme, IRC reported that:
• attendance at the clinic rose by 147% between 1999 (~7400 visits per month) and 2001 (~18,300 visits per month average)
• 70% of treatments were for malaria and diarrhoea, the main reported causes of death in the 1999 and 2000 surveys, and decreased as a cause of death in 2001 & 2002
• CMR in the five eastern provinces of DRC was estimated by IRC to have increased slightly in 2001 compared to 2000
• A survey in November of 2001 found that 60% of residents that had experienced fever in the preceding two weeks had sought treatment at a clinic
Employing Bradford-Hill's criteria, this example shows that: 1) there was a considerable drop in CMR associated with the establishment of the intervention, 2) there was no dose-response effect, 3) the fact that IRC's two other areas of health programmes had similar (but somewhat less dramatic) reductions implies repeatability, 4) the benefit occurred after implementation, 5) the findings are biologically plausible (although 1 visit per resident per year seems low), 6) alternative explanations for the reductions cannot be ruled out given the variance over time and the dramatic changes in violent conflict, although IRC reports that the violence did not dramatically subside until 2002, 7) these are not experimental data. Finally, the fact that the CMR was measured by an apparently valid survey method implies that IRC probably did contribute to a reduction in mortality in Katana [26].
Validity of the baseline of evaluation
Attempts to analyse the impact of humanitarian interventions are often handicapped by a lack of baseline data and a lack of knowledge about regular seasonal variations in key indicators of impact. For instance, baseline mortality rates are often not known. Countrywide figures are either unreliable, out of date, or not appropriate as they do not capture the regions where the conflict is occurring. A related problem that is continuously faced by humanitarian agencies is the lack of reliable population statistics [21].
Figure 1 Mortality in Katana, 1998 – 2002
When there are no baseline data, established norms can be used instead. For example, when people are arriving in a new location or are returning home, it is often impossible to determine the baseline before their arrival. In those cases, norms can be applied as an assumed baseline or as a threshold above or below which the indicator should not fall. Programmes lacking baseline data which keep mortality "low" or keep water and food provision high may be successful in terms of meeting their objectives but still not be able to quantify the impact of the intervention.
Validity of the indicator employed
The identification and use of relevant indicators is a crucial part of determining the impact of an intervention. Although the terminology varies, the literature generally distinguishes between performance (or process) indicators and impact (or outcome) indicators.
• Performance (or process) indicators concerns both the outputs of a programme (number of latrines built, number of training conducted, the quantities of food delivered) and the process of implementation (coverage of a programme, equity of distribution, targeting)
• Impact (or outcome) indicators are measures of the actual achievements intended by a programme. Mortality and malnutrition rates are the most commonly used impact indicators for humanitarian programmes, although interventions that aim to support livelihoods as well as save lives might require a broader set of indicators
For example, in a measles immunisation campaign, the immunisation coverage within a certain age group is a performance indicator, and the incidence rate of measles cases within that group is an impact indicator [22].
The Standardised Monitoring and Assessment of Relief and Transition (SMART) inter-agency initiative is an attempt to systematise the collection of impact indicators. SMART has recently stipulated that two measures (Crude Mortality Rate and nutritional status of children under five) are the most basic essential indicators for assessing the severity of population stress and for monitoring the overall effort of the humanitarian community. Those involved in the SMART initiative are currently developing standardised survey methodologies with associated reporting formats and software to address some of the challenges to effective evaluation. Part of the purpose of SMART is to enable global comparisons about the extent of humanitarian need in order to enable resources to be focused where they are most needed. A better understanding of needs based on nutrition and mortality might also enable better analysis of impact. Of course, malnutrition and mortality rates will not necessarily enable impact to be attributed to particular projects or agencies and may only be able to demonstrate the extent to which a relief system as a whole is meeting the needs of a population [23].
There is a tendency for humanitarian agencies to collect performance rather than impact indicators. This is due to various reasons, such as donor requirements that tend to favour the collection of performance indicators or the belief that impact indicators (such as mortality or morbidity) are sometimes difficult to collect. Arguably, it is easier for humanitarian agencies to monitor their own activities than to go out and monitor or assess the effect these activities have on the population they are assisting. Performance indicators may in some cases provide sufficient evidence about the likely impact of interventions and hence be used as a proxy for impact. However, when the validity of the proxy measure is unclear, there are risks in using performance indicators as a measure of success. For example, there is strong evidence that immunising children against measles has a direct effect on reducing mortality from measles; therefore, immunisation coverage can be used as a proxy for impact on mortality [24,25]. Other types of health interventions, such as reproductive health care, require more research on the link between the intervention and the health outcome before performance indicators can be used as a proxy for health outcomes [24]. Table 2 illustrates some commonly used indicators with regard to their strength of association to health outcomes, and the ease with which they can be monitored.
Table 2 Characteristics of indicators commonly used to justify health programmes.
Established validity as measure of health impact Indicator General ease of acquiring data to show health effects
Highest • Crude Mortality, <5 mortality Difficult in rural/diffuse settings, easier in camps
• Case fatality rate
High • Nutritional status of children Easy at the clinic data level, difficult but more valid with population surveys
• Disease rates
• Immunisation status of children
• Patient-specific mental health evaluations Logistically easy, requires skill on part of evaluator
• Safety of blood supply
Moderate • Food-basket evaluations Easy in camps, more difficult in more diffuse populations
• Water and sanitation availability
• Reduction in measles, mumps and rubella through reproductive health services Very difficult to measure even though benefits are likely to be occurring
• Improved patient outcomes via referrals
• Impregnated bednets distributed
• Comprehensive, timely health information system Nearly impossible. These are difficult to measure, and all require a series of events to induce a health benefit
• Good coordination between sectors
• Knowledge & attitudes about services available
• Population practices
Low • People given seeds and tools, shelter, or other materials Easy to measure. Links to health are likely to be mediated via many steps.
• Drainage, fly control activities or tasks
• Number of clinic visits
• Distance to facilities, health workers per capita
• Trainings conducted, numbers trained Easy to measure. May produce no effects on health.
• Change in knowledge without documented change in behaviour
• Messages/curricula developed
Current practice of humanitarian agencies in the health sector
In the humanitarian sector, assessment of impact has most often been seen as a sub-set of evaluation. Impact is one of the Organisation for Economic Co-operation and Development / Development Assistance Committee (OECD/DAC) evaluation criteria [4,27]. However, current evaluation practice rarely provides sufficient time for proper impact assessments. Most evaluation reports reviewed for this paper do not go beyond making statements about the impact of interventions.
Whereas the question of impact is unarguably important in evaluation practice, a detailed analysis of impact requires a different form of investigation. This may be done either through ongoing monitoring of project implementation, or as a separate research exercise (through surveys, operational research, reviews etc.). The tools developed in field epidemiology provide a set of potentially useful methods for measuring the impact of interventions. Existing approaches such as survey methods or surveillance systems, although seldom used by humanitarian agencies, can provide significant insight about the impact of humanitarian aid. So far, these tools remain mainly used in early warning systems or needs assessment [21]. The two most common approaches are survey methods and surveillance systems.
Survey methods
Most surveys are an attempt to actively go out and survey a representative sample of the population, although they may take different forms. WHO and others have produced manuals to guide health workers to conduct specific kinds of surveys, with nutritional anthropometry and Expanded Program on Childhood Immunizations (EPI) coverage methodologies being among the most succinctly described. Aid workers often do not have sufficient skills to take a valid sample and analyse the results of a survey. This is why many initiatives to improve the quality of relief programmes have emphasised the importance of training relief workers in survey methodologies. Some organisational headquarters and some groups such as Epicentre have specialists who can be deployed to assist with the conducting of surveys.
Uncertainty about population figures creates particular difficulties in constructing sampling frames for use in surveys. Census data may be many years old while the crisis may have had a dramatic impact on demographics and population numbers due to migration and high mortality. Although cluster surveys are a compromise measure, in many situations (especially in conflict situations or where terrain is very difficult) it may prove difficult to gain access to the 30 clusters proscribed. Nomadic groups may also prove difficult to sample [23,28,29]. Despite these difficulties, recent experiences have shown that surveillance methods can be successfully carried out in volatile environments [26].
Surveillance Systems
Surveillance is the systematic collection of information over time for decision making. Surveillance systems are often part of general monitoring systems and have been used for analysing impact in both health and nutrition programmes.
Aid agencies sometimes evaluate health programs by establishing a surveillance system at the beginning of a funding cycle and contrasting the rate of health events at the beginning and the end. This is valid if either: a) all of the events of interest are captured by the surveillance network, or b) the data from within the system are representative of the health conditions of the entire population and remain consistently so over the course of the project. Neither of these conditions is commonly met for clinic-based surveillance systems in rural and urban areas, although both of these conditions are often met in well-defined settings like refugee camps. If the majority of a population does not have access to formal health care then a clinic or hospital-based surveillance system will be able to tell very little about the health conditions of the broader population. Of course, not all surveillance systems are linked to utilisation of formal health services. Sentinel site surveillance systems for nutrition monitoring, for instance, involve the monitoring of purposively selected communities in order to detect changes in context, programme and outcome variables. Surveillance systems can be less costly than surveys and may reveal more in-depth information on the causes of malnutrition.
Problems with the lack of skills
A recurrent problem with the use of epidemiological methods such as surveys and monitoring is the lack of appropriate skills for conducting good quality assessments. Reviewers from the Centers for Disease Control and Prevention (CDC) evaluated the monitoring of projects and the measurement of nutritional status and mortality in Somalia from the period 1991–93 [30]. They developed a set of criteria for evaluating different kinds of information (surveillance and surveys) and systematically reviewed available reports. They found that the range of methodologies employed and outcomes measured were so variable and of such poor quality that they prevented widespread comparisons, and that, regardless of consistency, much of the data were simply not credible due to poor collection methods. Spiegel et al. from CDC reviewed 125 nutritional surveys conducted in Ethiopia in 1999 and 2000 during a time of famine but relative peace and stability [31]. The surveys were carried out by 14 organisations with a wide range of survey expertise. Only 67 of the 125 surveys attempted to conduct a sample that represented the population served. Only 9 of those 67 surveys assigned clusters to the population in a manner that was proportional to the sub-units of the population and only 6 of those possessed the minimum number of clusters (30) and children (900) suggested by most nutritional manuals. Spiegel concluded that non-governmental organisation (NGO) workers were woefully unprepared to conduct quantitative assessments of this kind, and that most of the surveys were of such poor quality as to be unhelpful toward making sound relief policy decisions [31].
The measurement of anthropometry is relatively standardised compared to many other health outcomes such as mortality and mental health status. For example, a mortality survey in Kabare Health Zone in the Eastern Democratic Republic of Congo (DRC) in 1999 was conducted simultaneously with an EPI coverage survey and only included households with a child under 5 years of age. The resulting estimate of crude mortality (1.9 per 1000 per month) was far lower than in a later repeat survey (finding 2.7 per 1000 per month) and, in fact, the initial survey missed most of the excess mortality [32]. For some project objectives, such as the prevention of HIV transmission, there is not even a widely agreed upon outcome to be measured that serves as a proxy for HIV incidence. The difficulty of assessing outcomes such as mortality is a principal reason for the use of process indicators in place of health outcomes.
Thus, without improved staff skills and capacity and a significant change in attitude among donors, it is likely that humanitarian agencies will continue to rely heavily on process indicators and not be expected to prove that programmes influenced the health of the targeted beneficiaries.
Review of 15 reports of health-related programmes
All final reports of health-related programmes funded by the US Department of State, Bureau of Population, Migration, and Refugees (BPRM) and submitted in 2003 were reviewed for the Humanitarian Policy Group study. Proposals that contained objectives of health-related activities (e.g. shelter provision, food transport) but that did not specifically say they would influence health status were excluded. The remaining 15 final reports were evaluated against the following five criteria:
• Was there a health-related objective?
• Was the baseline rate measured or a comparison group identified?
• Was the health-related outcome measured and reported?
• Was the societal level of the evaluation appropriate given the intervention?
• Were there any major issues supporting or raising concerns about the reported outcome data?
The societal level of a health project and evaluation was categorised as being either on the patient level, the household level or the community level. The expectation was that programmes that intervened on a specific level should be evaluated on that level. For instance, a curative health programme might have benefits at the individual level but it may not be possible to evaluate its impact at a wider level.
Six of the 15 reports did not attempt to measure or report any health-related rates or status. Proposals corresponding to five of these six reports only contained process indicators as the initial objectives, and thus the lack of documented health benefits was assured before the projects began. An additional three of the 15 reports contained health data-based objectives but did not present any health-status data, instead reporting process indicators such as the numbers of clinics supported, consultations given, or tons of food distributed.
Only four of 15 final reports could demonstrate a health benefit, and three others were likely to have produced a population-based benefit although this was not documented. These four were the only projects to measure baseline rates. Nine of the 15 did not have objectives and measures that matched to societal level. The results of this analysis confirm the general conclusion reached at the July 2002 SMART Monitoring and Evaluation Workshop, that while NGO's and agencies often want to monitor health outcomes, they usually monitor process indicators. Problems with process indicators seen in the BPRM review include:
• The cited activity may be related to the health outcome, but the significance of this effort depends on the activities being done well and in sufficient numbers (e.g. Eritrea and Sierra Leone wanted to reduce mortality and reported numbers of clinic-based activities)
• The health-related objective is only distantly related to the health outcome (e.g. a programme in Uganda wanted to induce "food self-sufficiency" but reported tons of food distributed)
• In some cases, the link between the process indicator and the outcome was simply implausible (e.g. a Balkans programme wanted to reduce dependency on aid of chronically "Extremely Vulnerable Individuals" and reported doing this for some by distributing school books)
Interestingly, a mental health programme in Guinea, with perhaps the most difficult-to-measure outcomes, had the most rigorous documentation, which included pre-intervention and post-intervention patient evaluations and the use of non-patient controls. Representatives for the other three programmes which documented impacts felt that very little of the project budget (perhaps <2%) was spent on documenting the impacts.
Over 20 NGOs were providing general health services in the eastern DRC in 2000 and 2001 with funding from either Office of U.S. Foreign Disaster Assistance (OFDA) or European Commission's Humanitarian Aid Office. According to OFDA, only two of those agencies could show health benefits associated with their programmes [33]. This seemed plausible at the time given the violent and chaotic circumstances within which the NGOs operated. The short funding cycles and volatile nature of emergencies often prohibit a systematic and rigorous evaluation of either the impact or the monitoring of multiple agencies in the same setting.
Wider level of impact analysis
There is an increasing interest in impact analysis at higher levels and a 'system-wide approach to performance' [7]. Several initiatives and mechanisms, from donors and humanitarian agencies, are attempting to move beyond the project level and consider sectoral, multi-sectoral, or system-wide impacts. For example, one of the objectives of the SMART initiative is to enable judgements about the overall impact of the humanitarian effort. Another example is the Inter-agency Health Evaluations in Humanitarian Crises Initiative that proposes to establish inter-agency health programme reviews in order to find new ways of looking at health programme performance and its impact on the health of affected populations [34].
A number of issues must be considered with wider levels of impact assessment. First, there is no reason to think that the constraints encountered when measuring the impact of particular interventions are erased when looking at a wider level. A particular difficulty is that of aggregation. The wider the level is, the more aggregation impact data require. Clearly, a donor or an aid agency looking at the overall effectiveness of its aid over a number of years needs far more aggregation than the evaluation of the impact of a single project conducted by a single agency. Finally, wider levels of impact assessment also generate new problems such as that of responsibility. Who is responsible for the collective impact of a number of individual humanitarian projects? Projects may have a positive impact taken individually, but the overall humanitarian effort may be insufficient compared with the level of needs. Who will account for the overall success or failure (if that is in fact possible to measure) of the humanitarian enterprise? This is a typical question that came out of the system-wide Rwanda evaluation. There is also a need for consensus in the relief community about the fundamental objective of health programmes.
Conclusion
Despite existing efforts to improve the quality, accountability and performance of humanitarian aid in the health sector and more broadly, this paper has shown that there is limited knowledge about the health impact of humanitarian aid. The epidemiological tools potentially useful for analysing the impact of aid programmes are seldom used. As a result, humanitarian efforts rest on a limited evidence base. This is in large part due to lack of epidemiological skills found within NGOs working on the ground. Addressing this skills deficit will be essential if the rigour of routine assessments is to be improved.
In the current practice, the health impact of programmes is too often assumed rather than demonstrated. This is largely due to the use of performance or process indicators as proxy for impact, without the necessary evidence that the intervention is robustly linked with a health outcome. There needs to be a consensus regarding which types of intervention (measles immunisation, assuring that people have enough food and water) are linked to good health and the levels of service that are sufficient, in order to document that aid money is well spent. Further research on the links between particular interventions and health outcomes is required to build up this evidence base.
Efforts to document project impact should be woven into monitoring and surveillance activities, not only to reduce costs, but as a tool to improve program quality. The absence of systematic monitoring and surveillance in the humanitarian sector is a serious obstacle to assessing the impact of humanitarian aid. All too often an assessment of the impact is considered as a separate activity that takes place at the end of a project. The question of impact must be included throughout the project cycle, from the formulation of objectives to the final evaluation.
For health impacts to be more widely documented there needs to be adequately trained, experienced, and motivated staff present at the design and evaluation phases of projects. Part of the solution is increased funding for training and retaining staff who can act as a resource, but there must be also an increased collaboration between donors and relief workers that develops a culture rewarding the documentation of programme failures as well as successes as learning opportunities. While initiatives such as SMART provide a potentially useful platform for analysing the global impact of humanitarian aid, there is a risk that the efforts will focus exclusively on technical discussions regardless of the wider political dimension of humanitarian aid. Some agencies also fear that these mechanisms will reinforce the donor control over humanitarian agencies, instead of solely aiming to increase the quality and performance of humanitarian aid. Nonetheless, increasing accountability in all sectors of international aid and increasing expectations for the wellbeing of the world's downtrodden will eventually demand consistent and widespread documentation of humanitarian benefits. Existing epidemiological techniques can adequately do so, if only they were employed. The challenge will be to make this documentation occur through positive self-improvement motives rather than as a reactive response to criticism.
Authors contributions
LR, a field epidemiologist, wrote the theoretical parts of the paper and conducted the review of reports of 15 health-related programmes; CAH, a researcher and humanitarian worker, wrote the sections that relate to humanitarian assistance and the practice of impact assessment. This paper is based on a research project into the impact of humanitarian aid carried out by the Humanitarian Policy Group at the Overseas Development Institute.
Competing interests
The author(s) declare that they have no competing interests.
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| 15679909 | PMC544941 | CC BY | 2021-01-04 16:38:26 | no | Emerg Themes Epidemiol. 2004 Oct 7; 1:3 | utf-8 | Emerg Themes Epidemiol | 2,004 | 10.1186/1742-7622-1-3 | oa_comm |
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Emerg Themes EpidemiolEmerging Themes in Epidemiology1742-7622BioMed Central London 1742-7622-1-41567990510.1186/1742-7622-1-4Analytic PerspectiveThe role of the applied epidemiologist in armed conflict McDonnell Sharon M [email protected] Paul [email protected] Nadine [email protected] Ben [email protected] Mark 1MHW`@cdc.govNoji Eric [email protected] Centers for Disease Control and Prevention, Atlanta GA, USA2 Department of International Health, Boston University School of Public Health, Boston MA, USA3 Department of Homeland Security, Washington DC, USA2004 7 10 2004 1 4 4 5 10 2004 7 10 2004 Copyright © 2004 McDonnell et al; licensee BioMed Central Ltd.2004McDonnell 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
Applied epidemiologists are increasingly working in areas of insecurity and active conflict to define the health risks, suggest feasible means to reduce these risks and, monitor the capacity and reconstruction of the public health system. In 2001, The Carter Center and the United States Institute for Peace sponsored a conference within which "Violence and Health" was discussed and a working group on applied epidemiology formed. The group was tasked to describe the skills that are essential to effective functioning in these settings and thereby provide guidance to the applied epidemiology training programs.
Methods
We conducted a literature review and consultation of a convenience sample of practitioners of applied epidemiology with experience in conflict areas.
Results and conclusions
The health programs designed to prevent and mitigate conflict are in their early stages of implementation and the evaluation measures for success are still being defined. The practice of epidemiology in conflict must occur within a larger humanitarian and political context to be effective. The skills required extend beyond the normal epidemiological training that focuses on the valid collection and interpretation of data and fall into two general categories: (1) Conducting a thorough assessment of the conflict setting in order to design more effective public health action in conflict settings, and (2) Communicating effectively to guide health program implementation, to advocate for needed policy changes and to facilitate interagency coordination. These are described and illustrated using examples from different countries.
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Introduction
In 2004 it is estimated that there are 95 violent conflicts worldwide [1,2]. The profound consequences to the well-being of communities from these conflicts are disproportionately distributed. Ninety percent of those who die in war are civilians, half are female, and more children will die or be disabled than soldiers [2-9]. Prior to the 1990s, humanitarian assistance in the context of active violence was the domain of emergency medical services; public health and epidemiology focused on refugees and displaced populations [3,6,10,11]. As war became endemic in certain areas, primarily civil and geographically less demarcated, the international public health community was pressured to provide prevention and primary health services to the indigenous population as well [12,13]. The pervasiveness of war and the magnitude of its effects have led public health experts to advocate for directed strategies to prevent and mitigate its effects on communities [2,4,5,12-14]. These strategies are described in the WHO program called Health as a Bridge for Peace (HBP) and have included developing reference and training materials to support health workers in war settings [15-17].
In February 2001 the Carter Center and the United States Institute for Peace (USIP), in collaboration with CARE, Emory University and the Centers for Disease Control and Prevention (CDC), sponsored a meeting on "Violence and Health". The goals of the meeting were to determine the impact of violent conflict on public health and to advise public health training programs on means to enhance the work of public health professionals in settings of violent conflict.
During the meeting a specialty workgroup for "training public health care professionals" was formed and asked to focus on applied epidemiologists by describing their role in conflict prevention, mitigation, and documentation. Applied (or field) epidemiology was initially created by the US Centers for Disease Control and Prevention in the 1950s as a post-doctoral training program [18]. Over time this program has been adopted by more than 35 governments, the World Health Organization (WHO), and numerous schools of public health and non-governmental organizations (NGOs) [19]. The applied epidemiologist is trained to conduct high quality scientific studies and to translate findings into practical, effective public health programs.
Increasingly, applied epidemiologists are recruited to areas of insecurity and conflict to define the health risks, suggest feasible means to reduce these risks, and monitor the capacity and reconstruction of the public health system [20-22]. This trend reflects a growing demand from donors, governments, military, and humanitarian groups for credible information to support the planning and evaluation of health inputs in war. These realities have led to an emerging professional interest within public health on the causes and effects of conflicts [2,13,14,23-25].
Methods
Because of the increasing demand for applied epidemiologists in conflict, the workgroup leaders reviewed the relevant literature and sought further input from applied epidemiologists and public health practitioners experienced with conflict settings to better understand training needs in this setting. The literature review and discussions focused on the role of applied epidemiologists in violent conflict and what skills are needed to function effectively. The literature review included MEDLINE and Google keyword searches for "war", "conflict", "complex emergencies" and "epidemiology" combined with either "conflict", "disaster" and "refugees". The examination included specific training and reference materials developed to support Health as a Bridge for Peace [16,17]. The leaders expanded their workgroup to include other experts representing the Training in Epidemiology and Public Heal th Network (TEPHINET- a consortium of applied epidemiology training programs in 40 countries), the US Centers for Disease Control and Prevention (CDC), the World Health Organization (WHO), foundations, schools of public health, and numerous private organizations [20,21]. This paper presents the results of the literature review, as well as the answers provided by members of the group at the meeting and subsequently by members of the expanded workgroup via phone interviews about the implications of these recommendations for training field epidemiologists. Examples from the personal experience of the authors are presented and are referenced by giving the individual's initials.
Results
Applied epidemiologists have an important role in conflict prevention, mitigation, and reconstruction, but to be effective in these circumstances their training must emphasize knowledge about international law, human rights, and complex emergencies [16,17]. In addition, the skills needed must emphasize not only valid collection and interpretation of data but also highlight skills that fall into two general categories: (1) Conducting a thorough assessment of the conflict setting and players, which requires the collection, analysis and interpretation of qualitative and quantitative data to design effective public health action; and (2) Communicating effectively to guide health program implementation, to advocate for needed policy changes and to facilitate interagency coordination. We discuss each of these categories and illustrate each using examples from the experiences of workgroup members in actual conflict settings.
Conducting a thorough assessment of the conflict setting in order to design more effective public health action in conflict settings
The first contribution of the applied epidemiologist in conflict is to insist that a thorough assessment of the situation be done as soon as possible. Without such insistence the rush to begin programs without adapting interventions to local circumstances will prevail with predictably poor results [2,4,10]. The literature is replete with examples of quick humanitarian action with dire consequences due to poor assessment [10,24,25]. The need to precede the bulk of interventions with valid assessment data might best be summarized in the phrase "Don't just do something, stand there" (and first assess the situation) [20,26,27].
The use of validated qualitative research methods is essential to the processes of building trust and performing high quality assessments. Many workgroup experts suggested including social and behavioural scientists with practical field skills from the beginning; if this is not possible then their techniques for collecting data in the field should be adopted. Using ethnographic skills increases the accuracy and validity of assessments and allows the development of a common vocabulary of key terms for communicating with local people, even when using a translator [28,29]. Despite the inevitable concerns about time, qualitative assessment methods create only a minimal delay in quantitative investigations or services as they require relatively few respondents and can be done in the early stages of response and on a part-time basis. Information gathered in this way makes it less likely that data collected will be misused for political reasons and more likely that samples will be representative and services well targeted [10,26,27].
Once there is agreement that an assessment will be done- either prior to, or (more usually) simultaneously with emergency interventions- the role of the applied epidemiologist is to plan and coordinate the collection of valid and useful data. [19,20,26] The applied epidemiologist brings knowledge of how to achieve this in difficult and resource-poor circumstances by knowing whether and how to make compromises in study design, time, cost, logistics, and security while maintaining acceptable validity. Adequate preparation for the assessment, such as learning the priorities of communities and the language they use to describe health and illness, is invaluable. A good assessment defines the specific context for the health work and assures that the information obtained has a utility that justifies the cost and the risk to health workers and communities. Professionalism and impartiality during the assessment process can set the tone for and facilitate future work. If the role of the epidemiologist in collecting valid and useful data is understood by the community and other relief workers, there is a greater likelihood that the data will be appreciated as being impartial (even if certain groups have an interest in denying this).
The description of previous health programs is one source of useful information for assessments that can be obtained before fieldwork commences. Did previous programs engender trust or suspicion? Did they work, and if so, why? This information can indicate a community's ability to work together or with outside agencies. In addition, if a new program resembles a past program, it will inherit the community's view of the past program, for good or ill. For example, in planning a maternal and child health program in Afghanistan with WHO, we determined that communities that previously had good experiences with girls' schools were much more willing to consider other projects that educated or benefited girls (SMM). Thus, maps from the previous ministries of education and health showing the location of these schools provided initial leads that were followed up by interviews with community members.
Rapid survey methods are most often used to describe the current needs of a population in conflict [3,20]. They aim to describe:
• baseline health status, risks and determinants (e.g. mortality, morbidity, water purity, sanitation, vectors, food, and shelter)
• availability, quality, and use of health services [22]
• access to health care
• the security of a situation [21], and
• changes in the population (e.g. migration) and the conflict (i.e. location of the front lines) [3,21].
Field experience in previous conflicts enables the epidemiologist to know what data are most important to assist in determining risks and designing and assessing interventions in a specific situation. For an assessment to be useful, it must have clear objectives and resist the constant pressure to add more questions [21]. Negotiating questionnaire content among partners can be greatly assisted by agreeing on the final report format, its length and especially, the content of tables and graphs.
Despite the potential shortcomings of rapid surveys, systematically collected data that are appropriately interpreted are superior to anecdote. Additionally, by emphasizing and clearly documenting what sources of data are used to build a conclusion it can be re-evaluated over time. In 1990, patient logs compiled from multiple NGO-supported clinics within Afghanistan revealed that the majority of patient visits were for routine primary care services and not war-related injuries as was assumed. The result was a significant revision of the training curriculum for Afghan medics and physicians in more than 15 non-governmental health service programs (SMM).
Many of the challenges in generating useful, high-quality data can be addressed with innovative data collection methods and judicious interpretation [21]. In conflict settings, a rapid survey of a defined area should take about 1–2 days, and a report of its findings should be provided immediately to decision makers and service providers. For example, an immunization survey in Uganda determined that people in the safest and most easily served areas were un-immunized; it was concluded therefore, that people in hard-to-reach areas would also need immunization. This is an example of the application of the "best case" survey strategy [30].
There are many examples of innovative ways to select a sampling frame in the conflict setting [21]. Global Positioning Systems are increasingly used to identify areas for sampling. Alternatively, satellite imagery can be used to estimate population density and select a geographic area for sampling. The most common approaches for selecting participants in rapid population surveys during conflict are simple random sampling and cluster sampling. The former is easier to analyze and communicate but takes more time in the field. Cluster surveys are more complex to analyze and communicate but involve less field time. More important than which method for sampling is used is to recognize the methods potential biases and how these might limit its applicability [16,29].
In war, obtaining a denominator is very challenging [22,23,26,31,32]. Available population data vary widely in quality, and the movement of persons during conflict can result in inaccurate estimates. The population may be ideologically divided by the conflict, as well as widely dispersed, highly mobile, or in refugee camps [5]. The socio-political circumstances of a population can result in over- or underestimation of its size [11,24,29,31]. For example, women and children may be hidden for protection, or their mobility and access to health and social services severely restricted, resulting in a great underestimation of their numbers. During the civil conflict in Ethiopia in the 1990s, families sequestered young boys to prevent their recruitment as soldiers (SMM). Alternatively, population overestimates may occur when food, drugs, or other resources are rationed [21,29]. Multiple registrations of the same person and not reporting losses through death or departure are ways that families can increase or at least not reduce their resources for use or sale [31]. On the Thai-Khmer border in the 1980s the Khmer Rouge recruited members of other communities to increase their numbers for a census. Addressing these challenges requires an awareness of the forces on the population and the use of measures to accommodate to these forces. In Thailand the UN Border Relief Organization resorted to arriving without notice, calling the community in for registration, and marking those already registered with an indelible stamp to reduce over-counting (PB). In Afghanistan, discussions with community religious leaders revealed that the local mosque recorded households' numbers so as to equitably share food at Islamic holidays. Thus, religious leaders were able to provide an excellent estimate of the number of households and their size (SMM).
Accurate counts of births, deaths, and disease are also affected by social and political instability [31]. Conflict amplifies the risk of chronic and acute health problems while reducing the chance that the affected population will have access to health care [2,13,28]. These increases may go unnoticed, however, because of underreporting from disrupted health systems; demand might even appear to decline [26]. On the other hand, if local authorities believe that reporting disease will increase the flow of resources, such as drugs and equipment, they may inflate these numbers [26]. Rural physicians in the Philippines in 2001 described exaggerating reports of malaria cases in the dry season with the aim to stockpile anti-malarial medications to cover the needs during the malaria season (SMM). Similarly, individuals might feign illness to stock up on medicine if they believe that existing services will be unreliable. For these reasons, data from health services is frequently inaccurate during conflict and should be evaluated in the context of the pressures on the community to survive [10,11,13,21,23,24,29].
The epidemiologist will have to assess the level of access to health-care services for military and non-military personnel, particularly women, children, the elderly, and those with chronic medical conditions [2,11,26]. Defining age-, sex- and cause-specific mortality rates will help assess specific vulnerable groups and determine whether they need special help in accessing services [26,29]. Populations in conflict might suffer overcrowding, poor water and sanitation, and inadequate rations as well as being targeted for violence [2,5,11,13]. All of these can be assessed epidemiologically and determine what interventions should be provided and to whom they should be targeted.
Assessing the data from disease reporting systems is a core function of epidemiologists. If these systems are intact and can describe trends of priority diseases or conditions over time, maintaining them might be worth the effort [21,22]. Typically, however, conflict will interrupt health surveillance activities, including data collection, analysis, interpretation and dissemination, resulting in disease underestimates [2]. As a result, to answer high-priority questions, epidemiologists may need to employ surveillance strategies with less emphasis on routine reporting and more on surveys, sentinel sites, or sentinel populations [21,23,26].
Applied epidemiologists believe that the value of epidemiologic science must also be measured by whether and how it is used to create effective public health action [19]. Applying a public health approach to violence, war, and the factors that initiate and promote them may help identify effective prevention and response programs. For example, knowing the types of weapons used in a conflict makes it possible to predict the types of injuries that will occur [33,34]. At a larger scale, developing systematic predictors of violent conflict may allow earlier intervention, similar to food security and famine early warning programs that monitor the risk of malnutrition. In these programs the emergence of selected behaviours serves as a warning of population risk [35]. The long-term "health" effects on the public from social disruption, violence, poverty, oppression, and torture, are poorly understood, and even less is known about the effectiveness of our programs to address them [36].
Practicing epidemiology in conflict involves working in rapidly changing circumstances and uncertain security. Formal epidemiologic assessments and public health actions in war can only proceed where the environment is "permissive". The security situation for health workers, civilians and programs, as well as community participation and ownership of health programs, must be continuously monitored. Selected parameters that reflect the level of security, participation, demographics and disease should be discussed between partners and security agencies and re -evaluated frequently [2]. Health agencies must demand a level of security to function; however, their conduct on the ground may influence the extent to which they are afforded protection by local communities.
Acute and chronic war settings, while superficially similar, represent different challenges to the epidemiologist and can significantly affect the type of intervention selected [23]. In wars of short duration – such as Kuwait after the first Gulf war – the coping mechanisms of the population (for example, how they maintain their health and where they go for health services) are still based on peacetime conditions [32,37]. The epidemiologist may be able to focus on the pre-existing public health and surveillance system, which is likely to be partially intact for at least some parts of the population [26]. The epidemiologist can work with those local health workers and officials who are still in place in order to understand the local situation, define and prioritize health problems, and reestablish basic services in a form acceptable to local people.
A chronic war setting (e.g. Afghanistan) is more challenging [13,23]. Knowledge of the pre-war situation is still useful, but often little is left of the previous public health system, and people will have developed new coping mechanisms to deal with prolonged war. The epidemiologist must spend more time investigating the current situation and assist appropriate agencies to plan and build new systems that meet current needs. Because of the lack of predictability, security and centralized authority, all plans and programs will need to be tested at a smaller scale before expansion to a larger population. Building systems will take longer due to the lack of pre-existing resources and trained personnel. In Iraq, although the current conflict is "acute", it also follows ten years of sanctions after a previous war and a major change in leadership. Although much of the pre-2003 health infrastructure remains, the goal is not to replicate the old system with its poor supplies and limited access.
Communicating effectively to guide health program implementation, advocate for needed policy changes and facilitate interagency coordination
All aspects of communication are more difficult during conflict [10,26]. Pre-existing infrastructure may have been destroyed and, if not, it will be part of the struggle for power and control. Practical solutions to communication needs require flexibility and a higher proportion of the budget than is needed in more stable and secure situations. Radio, satellite, and cellular phones have advantages that make them very useful, but because of their cost these technologies are often out of reach for many programs. Aid organizations should collaborate to solve communication problems and share resources; donors need to encourage and reward this by allowing local coordination of grant funds to reduce redundancy. Included in communication skills are the abilities to facilitate inter-agency coordination and collaboration and to work with multiple constituencies. Coordination among program and donors offers an opportunity to eliminate waste and to encourage equity. In Uganda in 2001, local police allowed public health workers in isolated locations to use their radios to communicate surveillance information on outbreaks to the Ministry of Health (Personal communication P. Nsubuga 2001).
Ongoing consultation with communities is necessary for the integrity of programs and to monitor security. Epidemiologists can improve the quality of the information they receive and disseminate by developing alliances with partners who have a communication infrastructure in place- and people travelling into areas that are restricted or insecure. For example, contacting agencies that work in agriculture, health care, education, land-mine removal, and food provision about sharing or coordinating field staff that monitor and supervise projects can be helpful. Jointly planning the efficient use of field staff from all types of programs using checklists, and other tools to help the non-technical visitor be able to bring back useful information and support local communities can help these communities feel less isolated, reduce waste and increase the data sources about health programs and problems.
Workgroup members suggested that journalists may be potential sources of useful information, insofar as they have access to restricted areas. They may be able to:
• report on health-related activities or risks (e.g., immunizations, outbreaks, and unexpected behaviour in selected populations),
• provide reliable communication with all sides and help establish ceasefires for the provision of health services [38], and
• help control rumours by defining means to check information early and responding to misinformation quickly [22,26].
The different goals of the media and public health and the rapid turnover of journalists, however, may limit useful cooperation.
Epidemiologists in Colombia's Centro de Referencia Nacional sobre Violencia (CRNV) used national media coverage to highlight political homicides. They published information regarding deaths along one particular river in Colombia to raise awareness of the level of violence occurring there. This data-based approach drew less political resistance while focusing public attention on the deaths [39,40].
Effective communication strategies should target policy-makers from local to international levels. Epidemiologists can be an effective voice for public health if they maintain their credibility with policy-makers. Sometimes the most important role for the epidemiologist is to act as a witness to describe the local situation to an international audience of policy-makers and to advocate for action [2,10,27,41,42]. Using a public health approach the epidemiologist can describe the realities of war, its effects on individuals and communities, and the consequences of certain types of weapons, warfare and humanitarian approaches. We must think of war not as a natural consequence of life but as a preventable tragedy with multiple long-lasting implications. The decision to use violence must be examined openly and not sanitized [43].
A less rapid but possibly more respected method for bringing awareness of local health and social issues to national and international levels is publishing research in credible journals. Peer-reviewed work can be a tool to facilitate negotiations between governments, NGOs and communities, and to influence international and national level policy-makers.
Effective advocacy for public health includes the ability to use communication skills such as advocacy, consensus development, and negotiation to promote population health to policy-makers [23,26,28,42,44]. As much as possible, messages should be communicated directly to the local population rather than through political leaders or another entity, as the messages may be tainted or obscured by differing agendas. For example, in the Thai border refugee camps epidemiological data were interpreted and then translated into health messages that were communicated by program staff via megaphones while travelling around the community (PB). In 2003 the US military personnel and reservists in Iraq were responsible for providing humanitarian aid and direct interactions with civil authorities; a role for which they had no training (EJN). In addition, because of the military association, the information gathered was considered inherently biased and dismissed.
During conflict, health and information services that were once unified under a single health ministry can become fragmented with parts falling under the aegis of government, those resisting the government, or nongovernmental organizations [23,31]. Access to health care and humanitarian resources can be important in political battles at the local level [26]. Each party in a conflict wants control of hospitals, health workers, food, and medications so as to be able to offer them to the community, thus enhancing their credibility, and to aid its military efforts (SMM). In Colombia, some communities have lived in conflict for > 40 years despite local, national, and international efforts to find solutions to the chronic violence. Epidemiologists there described attempts to ameliorate animosities by involving local constituencies in health activities: the Church, NGOs, elected officials, and other leading organizations or personalities in the community (Personal communication Jorge Jara 2002). Local rather than national health authorities chose priorities, from outbreak investigations and interventions to data analysis to surveillance of violence and influenza. Through this process trust was built between the Colombia National applied epidemiology program and the local health authorities, thereby assuring access to the population that transcended partisanship. Parties on all sides of a political/military issue collaborated on a community health project, an unprecedented step.
Many workgroup members felt that negotiation skills were essential when working with multiple aid groups or the main parties in the armed conflict. For example, consensus building is useful when the health problems are not well defined and diverse groups must collectively identify and solve them. At the simplest level this includes the skill of running meetings and enhancing a group's utility and the understanding that disagreement might not necessarily represent failure. The goal is to help all parties understand that improving health meets the interests of all groups and to develop complimentary strategies. Too often decisions are rushed and participants pushed aside, leading to unsuccessful outcomes and future difficulties working together [44]. When disagreements are so deep that one or both sides will not accept any solution that benefits the other side, negotiation will be fruitless. Currently, in Iraq, addressing the emergency and reconstruction needs of the Iraqi people through a collaborative process that involves the full range of stakeholders – Iraqi, Coalition, UN, NGOs, Civil Affairs, Donors – remains out of reach (EJN).
To facilitate the coordination of health services, the epidemiologist needs to be familiar with the many agencies working in the target area, including governmental, nongovernmental, and United Nations agencies. The diversity of groups may make it difficult to establish a shared view of the situation, much less a collaborative plan of action. Additional obstacles to coordination may stem from competition for resources, lack of information or true differences in their philosophy about humanitarian aid and their ability to deliver it. Donors in particular can create a powerful force for coordination if they agree on priorities and processes. International donors can influence policy-makers by providing resources and technical support conditional on their commitment to the resolution of violent conflict [4]. It is the job of the applied epidemiologist to supply compelling credible information for these decisions. By providing credible information and establishing it as the foundation for action the epidemiologist can greatly assist coordination. Numerous workgroup members mentioned that being able to provide published medical and public health literature to government officials, donors and NGOs facilitated their efforts to coordinate agencies and to build consensus by providing an impartial standard based on scientific data [32]. One implication is that the epidemiologist needs rapid access to international literature, possibly via the Internet, even in war zones.
Summary
Applied epidemiologists can use their skills and position to promote positive population health policies and programs to address inequities that exacerbate conflict and violence [1,2,4]. In addition, credible on-site information can reduce the waste and harm of poorly planned humanitarian assistance [9,10,23-25]. Epidemiologists' work among policy-makers and the public uniquely positions them to communicate the effects of war, advocate for the population and assist in the reconstruction of health systems [9,15,23]. As impartial agents, epidemiologists can promote dialogue between conflicting parties, influence public opinion, facilitate projects that require cooperation, and coordinate multilateral responses to health and humanitarian needs [13,22,23]. Banning landmines, creating days of tranquillity for immunization, and surveillance of homicide resulting in limiting firearms in Colombia are examples of these ideas at work [38-40,44,45].
Providing health resources, including epidemiological support, should be part of a much larger diplomatic and humanitarian strategy. In fact, when a larger framework and commitment are missing, the health activities are unlikely to result in lasting changes and only increase the risks to the entire population. [26,47]. Epidemiologists are not diplomats nor should they be gratuitously sent into conflict situations as humanitarian band-aids. In addition, there are real risks that scientific information, meant to improve health, might be usurped for political aims rather than humanitarian purposes [14,27]. For example, in Colombia the data from health surveys undertaken with the best of intentions were used to locate populations for military action (Personal communication G. Suarez, 2002).
Epidemiologists need to advocate for focused field research on conflict resolution and violence prevention, and to evaluate the success of health programs in conflict [2,25,36]. It is no longer practical to conceptualize complex humanitarian emergencies like war into distinct phases [2]. In reality the settings for disasters, refugees, active conflict, and anxious peace are in constant transition. Defining and evaluating social and individual risk markers for violence may facilitate setting up early warning systems [46]. The reluctance to evaluate health programs in war stems from the assumption that doing anything is better than doing nothing and perhaps that good evaluation is too difficult. These flawed assumptions preclude progress and condemns us to rigid approaches resulting in the continued use of interventions of unknown effectiveness [13]. To paraphrase a military saying, "we are always treating the health problems of the last war".
Well-documented epidemiologic methods to assess conflict settings combined with ongoing re-assessment of the assumptions for interventions will greatly facilitate health program evaluation. In addition, measures of success for health programs need to reach much further into cultural competence, economic development, and social well-being. Based on the results of these studies more effective and cost-effective methods for public health response could be implemented in the future. With the number and intensity of current armed conflicts there is even greater urgency to begin this work.
The knowledge and skills described here are not typically part of epidemiology training. Yet they do fit with the epidemiologist's mission of gathering data and using it to improve the lot of populations. The materials developed to date to train health personnel working in war zones need more specificity and case examples. However, whether epidemiologists are able to respond to war largely depends on the availability of appropriate additional training and modelling by teachers and practicing epidemiologists [2,23]. Education programs designed for these goals would need to expand knowledge of human rights and international law, qualitative research methods, innovative ways to gather reliable population information during conflict, and effective methods to communicate this information. The working group strongly emphasized that these skills cannot be imparted solely in didactic courses, and training must include simulations with supervised field practice.
However well-intentioned the science and programs of epidemiology, we must be vigilant that epidemiology benefits public health, that it is not used to contribute to the prolongation of conflict and that it does not become part of it [13,22-25,33]. Insofar as epidemiologists influence decision-makers, we must strive to do so by adapting our work to the goals of peace through every means at our disposal. [33,42].
We should all strive for a time when, through the efforts of public health workers and others, war too will be eliminated.
- Jimmy Carter [2]
Acknowledgements
The authors would like to acknowledge the following people interviewed for their input on specific countries and for their time in developing this paper: Judy Carlson (Afghanistan, Cambodia), Dr Guillermo Herrera (Guatemala and head of the Central America and Caribbean FETP's)), Jorge Jara (Former Director Colombia applied epidemiology training program, currently field epidemiologist in Honduras), Joel Kuritsky (CDC and Carter Center); James Mendlein PhD (editing), Eric Noji (Iraq, Kuwait, and others too numerous to mention), P. Nsubuga (Uganda), M.C. Roces (Philippines); Carmen Sanchez-Vargas (Colombia); Gloria Suarez (Colombia, El Salvador); Nancy Jamieson (Afghanistan, East Timor), Helen Murphy (Indonesia, Afghanistan).
The Authors and their institutions have no financial or other conflicts of interests. There were no grants or outside funding for this work. This work was presented at the American Public Health Association meeting in Philadelphia PA, November 2002.
Author 1 (SMM) Co-led the original workgroup with the Carter Center, conceived of the study, designed the interviews of applied epidemiologists, supervised the literature review, and wrote the paper. Author 2 (PB) co-wrote the paper and assisted in interviews with applied epidemiologists. Author 3 (NS) participated in the design of the surveys, drafting the paper and the literature review. Author 4 (BB) participated in the design of the study, conducted interviews, and assisted in drafting the manuscript. Author 5 (MHW) Co-led the workgroup with the Carter Center and assisted in drafting the paper. Author 6 (EN) participated in the literature review, interviews, and drafting the final paper. All authors read and approved the final manuscript.
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Emerg Themes EpidemiolEmerging Themes in Epidemiology1742-7622BioMed Central London 1742-7622-1-51567991110.1186/1742-7622-1-5EditorialEpidemiology in conflict – A call to arms Tam Clarence C [email protected] Ben A [email protected] Olga [email protected] Egbert [email protected] Infectious Disease Epidemiology Unit, Department of Infectious & Tropical Diseases, London School of Hygiene & Tropical Medicine, London, UK2 Environmental and Enteric Diseases Department, HPA Communicable Disease Surveillance Centre, London, UK3 Department of Infectious Disease Epidemiology, Imperial College London, London UK4 Health Policy Unit, Department of Public Health Policy, London School of Hygiene & Tropical Medicine, London, UK2004 15 10 2004 1 5 5 5 10 2004 15 10 2004 Copyright © 2004 Tam et al; licensee BioMed Central Ltd.2004Tam 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 this first special theme issue, Emerging Themes in Epidemiology publishes a collection of articles on the theme of Epidemiology in conflict. Violent conflict is an issue of great sensitivity within public health, but more structured research and reasoned discussion will allow us to better mitigate the public health impacts of war, and place the public health community in a more informed position in discussions about possible interventions in future conflicts.
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"And there went out another horse that was red: and power was given to him that sat thereon to take peace from the earth, and that they should kill one another: and there was given unto him a great sword." – Revelations 6:4
Perhaps more than any other previous conflict, the recent war in Iraq has stirred the public health community, with numerous pages in general medical and scientific journals devoted to contributions condemning the basis of the war, condemning those condemning the war and condemning editors for delving into politics by publishing these condemnations [1]. For all this self-castigation, however, the public health message was notably absent from mainstream media and political discussion.
The issue of war is particularly sensitive in the field of public health, some might even say taboo. It is not seen as appropriate to openly denounce war. Perhaps we feel that becoming involved in what is largely perceived to be a political issue constitutes a threat to that most precious of our ideals, that of objectivity. Or maybe we are uncomfortable with the thought of being associated with military activities. Specifically, the opportunistic use of military language in public health issues has not been particularly welcome. Political calls for war on the societal problems of drugs and cancer have been accused of victimizing individuals and setting implausible goals for their prevention and control [2-6]. Ironically, we have yet to publicly declare war on the greatest of societal ills, war itself. The dilemma facing a scientific editor attempting to circumnavigate the turbulent waters of politics is eloquently summarized in a recent British Medical Journal editorial: to do nothing is as much a political decision as to challenge an issue head-on [7].
Given such an environment, readers may find it surprising that we should devote our first special issue to the subject of Epidemiology in conflict. Our reason for doing so is simple: regardless of the political context, war is bad for your health. The politicization of war within public health is unfortunate; re-framing public health questions within a political context prevents us from conducting informed discussion and finding rational solutions to them. Here, we draw a parallel with the early efforts to communicate the link between smoking and lung cancer. In his reflections on the subject [8], Ernst Wynder describes the opposition he encountered when trying to relay the findings from the first studies establishing the epidemiologic link. Opposition came not just from the conflicted interests of governments, the media and tobacco companies, but also from within the public health profession, which was at the time dominated by physicians, many of whom were smokers, were unused to the interpretation of epidemiologic data or simply thought the association to be implausible. Smoking was (and continues to be) a highly political issue, yet few would argue in retrospect that Doll, Hill, Wynder and the early proponents of the smoking-lung cancer association should, in the face of incontrovertible evidence, have done anything else than to publicize this link. Here the parallel ends, however. Given adequate knowledge about the risks to their health, an individual may freely choose to smoke and accept responsibility for any ensuing personal health consequences. Equally, they may choose to avoid these risks by not smoking altogether (although we stipulate that the effect of passive smoking is contentious here). An individual cannot choose not to go to war or have war inflicted upon them. Such decisions are carried out by governments, insurgents or other groups, many of which are not accountable to individuals. In this scenario, the role of academics and professionals, as well as professional associations and non-governmental organizations in informing governments and the public and raising issues that affect society as a whole becomes even more important. The duty of health professionals as advocates for public health is emphasized by Wynder and is equally applicable today and perhaps even more so to the issue of war:
"...the consensus of opinion among experts is not sufficient to create action unless such consensus is translated into preventive or control measures.... Scientists and physicians cannot be content with discoveries until their beneficial or protective outcome for the population has been fully realized. This means that the members of the scientific and medical community must become more proactive in public health matters [8]."
The issue of consensus is important, but difficult. Some will argue that war is inherently bad for public health and should always be opposed. Others will consider some wars justifiable if they address gross injustice or human rights abuses. And yet others, in line with the Geneva Conventions, will see wars, just or not, as inevitable and will want to focus on mitigation of human suffering.
Regardless of one's viewpoint, each of these positions needs to be supported by an evidence base with answers to the questions of when, how and why war is bad for public health, as well as how the adverse health effects of war may be prevented. Therein lies the greatest challenge for epidemiologists. Conflict situations deny us access to data and dissolve the health infrastructures on which we rely for the collection of epidemiologic information. In the face of such challenges, the authors of the articles in this special issue deal with a broad range of issues of great relevance to epidemiologists.
Mock et al. [9] argue that the interface of HIV/AIDS and conflict is more complex than is usually assumed. It is often said that war exacerbates the HIV epidemic, but the ecologic evidence suggests that this is not always the case. The authors examine the complexities of this issue and analyze how conflict can both exacerbate and retard the spread of HIV.
McDonnell et al. [10] evaluate the role of epidemiologists in conflict settings. Present barriers to effective engagement stem from the fact that epidemiologists do not receive training on issues pertinent to their operating in conflict-affected areas. Perhaps most important are appropriate communication skills to enable epidemiologists to present their message clearly as health related rather than political.
Roberts and Hofmann [11] place the work of humanitarian agencies under the epidemiologist's gaze. All too often such agencies, with the best intentions, measure success in terms of process – how many meals were handed out, how many vaccinations were given? From a health perspective this is only part of the story. Did these actions really have a positive impact on health? The difficulties of collecting such information mean that humanitarian interventions rarely incorporate tangible, impact-driven outcomes as priorities. This article proposes a framework for assessing the impact of aid on health.
These papers provide a foundation on which we hope authors will continue to build so that a comprehensive range of relevant topics on this subject may be compiled. There remain many issues to be addressed by the epidemiology community (see figure). Some of these issues involve challenges so great that we have perhaps not even begun to think about how we might start tackling them. We encourage authors to continue submitting articles on the theme of Epidemiology in conflict, so that we may be kept informed of developments in the field and promote the public health perspective in discussions about future conflicts.
Figure Conceptual framework for Epidemiology in conflict
We hope readers intending to take up these challenges will be informed, inspired and provoked into action. This is a call to arms, not against the barriers of physical inactivity and excessive caloric intake, the adaptability of infectious agents or the subtleties of gene-environment interactions, but against the sheer brutality of human beings killing each other. We encourage readers to research and discuss the humanitarian and public health consequences of this social disease. The knowledge gained will allow us to better mitigate the public health impacts of war, and place the public health community in a more informed position in discussions about possible interventions in future conflicts. The pen may yet prove to be mightier than the sword, but only as long as it keeps writing.
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Mock NB Duale S Brown LF Mathys E O'Maonaigh HC Abul-Husn NKL Elliott S Conflict and HIV: A Framework for Risk Assessment to Prevent HIV in Conflict-Affected Settings in Africa Emerg Themes Epidemiol 2004 1 6 15679919
McDonnell SM Bolton P Sunderland N Bellows B White M Noji E The Role of the Applied Epidemiologist in Armed Conflict Emerg Themes Epidemiol 2004 1 4 15679905 10.1186/1742-7622-1-4
Roberts L Hofmann C Assessing the impact of humanitarian assistance in the health sector Emerg Themes Epidemiol 2004 1 3 15679909 10.1186/1742-7622-1-3
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Emerg Themes EpidemiolEmerging Themes in Epidemiology1742-7622BioMed Central London 1742-7622-1-61567991910.1186/1742-7622-1-6Analytic PerspectiveConflict and HIV: A framework for risk assessment to prevent HIV in conflict-affected settings in Africa Mock Nancy B [email protected] Sambe [email protected] Lisanne F [email protected] Ellen [email protected]'Maonaigh Heather C [email protected] Nina KL [email protected] Sterling [email protected] Tulane University Center for International Resource Development, New Orleans, United States2 Department of International Health and Development, Tulane University School of Public Health and Tropical Medicine, New Orleans, United States2004 29 10 2004 1 6 6 8 10 2004 29 10 2004 Copyright © 2004 Mock et al; licensee BioMed Central Ltd.2004Mock 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 sub-Saharan Africa, HIV/AIDS and violent conflict interact to shape population health and development in dramatic ways. HIV/AIDS can create conditions conducive to conflict. Conflict can affect the epidemiology of HIV/AIDS. Conflict is generally understood to accelerate HIV transmission, but this view is simplistic and disregards complex interrelationships between factors that can inhibit and accelerate the spread of HIV in conflict and post conflict settings, respectively. This paper provides a framework for understanding these factors and discusses their implications for policy formulation and program planning in conflict-affected settings.
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Introduction
Of the obstacles to development in the sub-Saharan African (SSA) region, perhaps none has had a more profound impact than the dual burdens of HIV/AIDS and conflict. During the past quarter century, no region of the world has been more acutely affected by large-scale violent conflict than SSA. Almost all SSA countries have directly experienced, or border a country that has directly experienced, violent conflict. The number of states engaged in significant violent conflicts doubled between 1989 and 2000, from 11 to 22 [1,2]. A "conflict belt" stretches from Angola to the Horn of Africa, while peace in western Africa remains elusive.
The same quarter century has seen the development of an even more pervasive crisis – the HIV/AIDS pandemic. SSA continues to account for the large majority (66%) of the world's HIV/AIDS cases [3], even though its population comprises only 10% of the world's total population. There is great diversity across Africa in the levels and trends of HIV infection. While stabilization of the epidemic appears to have begun in several countries, population prevalence rates across most of the continent continue to increase, including among rural populations and sociodemographic groups not previously considered at elevated risk.
Although much has been written about HIV and conflict individually, surprisingly little has been written about the dynamics of the relationship between the two crises. In fact, an extensive keyword search of the internet and peer-reviewed journal databases turned up fewer than 100 references to the intersection of HIV and conflict. Virtually all studies are descriptive and only one utilized hypothesis-testing analytical strategies [4]. Although constraints on conducting such research are considerable, research on the interface between the two crises is of critical importance. A clearer understanding of the dynamics of the interface between conflict and HIV is crucial for the development of effective and efficient strategies to reduce population risk.
Some literature on conflict-affected countries in SSA underscores mechanisms by which HIV/AIDS and violent conflict may exhibit bi-directional causal associations on the population level. HIV/AIDS has been recognized to play a potential role in creating conditions conducive to violent conflict on the continent, although there is as yet no good empirical evidence to that effect. A policy forum held at the United States Institute for Peace [2] concluded that the pandemic will emerge as a deeply destabilizing force across the social, political, and economic landscapes of Africa. For example, large numbers of AIDS orphans and vulnerable children strain the social support networks in many southern African countries [5,6]. The psychological trauma and the lack of parental affection and supervision experienced by AIDS orphans put them at risk of involvement with criminal or antisocial activities (e.g. as child soldiers), or activities that serve to further their risk of contracting and further propagating the disease (e.g. as child prostitutes). The extent of the risks faced by AIDS orphans – e.g. risks for anti-social and criminal behavior later in life – needs to be sufficiently researched. By causing elevated AIDS-related mortality among the 15–45 year-old age group, the illness degrades human capital, undermines household capacity and stability, reduces economic productivity, and robs social and political institutions of intellectual resources [7].
On the other hand, violent conflict clearly influences the epidemiology of HIV. To date, the literature emphasizes conflict as a risk factor for HIV transmission. Conflict destroys social and physical infrastructure, resulting in untreated sexually transmitted infections (STIs), poor health and malnutrition and, as a consequence, increased risk of transmission in the event of viral exposure. The migration and poverty created or exacerbated by conflict may result in increased exposure opportunity through: (1) increased prevalence of casual or commercial sexual activity; (2) increased interactions among civilians and combatants/military personnel, known for their high risk behaviors; (3) the development of cultures of violence that promote sexual violence and predation; (4) mass migration, which increases sexual mixing among populations; and (5) the destruction of public health education mechanisms (e.g. mass media, health facilities, and formal education), which negatively affects public health-related knowledge, attitudes, and practices [8,9].
However, the epidemiologic data despite their limitations2, suggest that the relationship between conflict and HIV levels may be more complex than this general picture of joint potentiation implies. With few exceptions, countries that have experienced widespread violent conflict have apparently lower levels of HIV infection compared to those that have experienced relative peace (compare figures 3c and 3d). While many factors operate during conflict to increase vulnerability of affected populations to HIV, exposure opportunities at an aggregate level may actually be lower, therefore decreasing HIV risk. Alternatively, when conflict subsides, exposure opportunity may increase, leading to the potentially explosive spread of HIV. At the end of the Angola conflict in 2002, the country HIV prevalence was relatively lower than the rates in other Southern African countries, which suggests that conflict may have slowed HIV spread in this case [10].
Figure 3 Maps of Africa
The present paper evolved out of the observation that available evidence for conflict-affected countries in SSA suggests a complex set of possible net effects of conflict on the HIV epidemic, with multiple and quite variable outcomes. The implications of the analysis and findings have great significance for developing policies and programs to confront HIV in the context of conflict. These are discussed along with recommendations for improved policies and programs to address this important constraint to African health development.
A Framework for Understanding the Interrelationships between Conflict and HIV
Our framework develops two important aspects of the problem of HIV and conflict in SSA. These are:
• the importance of ecological in addition to individual risk factor explanatory models, and
• the importance of comparing the influences of conflict both during and post conflict.
We also develop the notion that conflict is a complex social phenomenon and that its effects are highly contextualized.
To articulate a framework for understanding the interface between the two crises, the concepts of vulnerability, hazard exposure opportunity and risk are useful. These are borrowed from the disaster literature [11] but are germane for understanding the evolution of HIV/AIDS as a crisis. Vulnerability is the ability of a population to withstand hazards or shocks to the system when they are present. Classical vulnerability factors include poverty, low education/knowledge, poor social infrastructure and attitudinal factors. Additional factors specific to conflict and HIV include levels of economically-motivated sex, levels and types of civil-military interactions, migration patterns, sex-related knowledge and attitudes, and population health status (particularly STIs and nutrition). Vulnerable groups in conflict settings may include child-headed households, child soldiers, unaccompanied children, women, demobilized soldiers, and repatriating refugees. Vulnerability means that, when exposed to a hazard, an individual or group is more likely to experience adverse effects (risk).
Hazards or shocks to human communities such as conflict affect the HIV exposure opportunity of members of these affected communities. HIV exposure opportunity can be mediated by war or other ways in which these communities interact with other communities (regional human ecology), which ultimately affects disassortive mixing (i.e. mixing of population groups of differing levels of HIV prevalence). In Southern Africa, the regional ecology is one that favors extraordinarily high HIV risk through, for example, high population density, good physical infrastructure, economically-motivated mobility, high poverty levels, and large wealth disparities within and between countries. By lowering exposure opportunity, war may lead to isolation from the general regional ecology and, therefore, to lower HIV risk at the population level. Alternatively, war leads to a changed and often more intense mixing of mobile military populations and civilians, which can increase HIV risk through disassortive mixing.
Figure 1 illustrates how conflict shocks may affect vulnerability and exposure opportunity to affect population HIV risk. Conflict shocks can weaken a community's ability to avoid HIV exposure/infection (vulnerability), or it can influence exposure opportunity itself. Vulnerability and exposure are the basic determinants of population-level HIV risk. Underlying determinants here include violent conflict and the regional ecology of HIV, which exhibit measurable effects on both vulnerability and exposure opportunity (and interact with each other). Finally, HIV infection goes on to influence the progression of conflict and the social ecology of HIV in a feedback loop. The model has utility for structured inquiry into the effects of conflict on HIV. It draws attention to mechanisms by which vulnerability and risk of exposure, and therefore risk of transmission, may be increased or decreased during conflict. The relative importance of each of the components, and therefore the ultimate effect of conflict (i.e. augmenting or reducing risk), will be highly context specific.
Figure 1 Conceptual Framework of Principal Causes of HIV Risk in Conflict-Affected Populations
Figure 2 illustrates that there is an important time dimension to consider as well. The most basic distinction of interest is that of conflict and post conflict, although these are rarely clear-cut distinctions. What is important to note here is that vulnerability increases during conflict and rarely decreases rapidly after conflict subsides because of the profound societal changes that generally occur during conflict. On the other hand, opportunity for exposure to HIV may change dramatically post conflict due to decreased population isolation and rapid improvement in freedom of movement. These changes will likely be most acute in countries with high economic potential, such as Angola, Côte d'Ivoire and the Democratic Republic of Congo. The juxtaposition of high vulnerability and increased exposure opportunities post-conflict can lead to explosive growth in the epidemic. This already has occurred to some extent in Mozambique [12].
Figure 2 HIV Vulnerability and Exposure Opportunity in Relation to Conflict Phase
This finding is critical in that it explains in part why, in some chronic conflict settings, apathy has developed towards HIV because seroprevalence levels may be low. However, the potential for rapid progression of the infection post conflict due to high vulnerability argues for a much more aggressive and deliberate approach to HIV in post-conflict settings.
Conflict as an Analytical Factor
Considerable research has been conducted to date to construct comprehensive typologies of conflict. Several well-established research initiatives, such as the Correlates of War Project [13], have developed typologies of conflict for application to conflict early warning and prevention efforts. These typologies categorize conflicts based upon variables associated with either the determinants of conflict or the manifestations of conflict. Typologies that focus on the determinants of conflict categorize conflicts primarily based upon characteristics of the political, legal, economic, social, or cultural environments that are associated with greater risk of conflict. Examples of such factors include [14]:
• political/legal status and structure of warring parties (e.g. state, non-state, internal, or external; military capabilities and levels of discipline of combatants);
• economic context (e.g. economic disparities, distribution of poverty, and access to critical resources);
• nature of political system (e.g. democratic or authoritarian);
• self-identified characteristics distinguishing warring parties (e.g. ethnic, religious, class, regional, or social identity);
• characteristics of physical environment as it affects the conflict (e.g. mountainous or flat); and
• purpose of conflict (e.g. motives of combatants, points of contention, and ideological differences).
In contrast, some approaches focus on the manifestations of conflict, defining categories in terms of measurable aspects of the conflict and its effects on populations. Examples include:
• geographic scale of conflict (e.g. sub national, national, or international; inter-state, extra-state, or intra-state);
• time-scale of conflict (e.g. duration and onset);
• types of military technology, tactics, and funding methods employed;
• involvement of civilians;
• levels of mortality (e.g. battle-related and indirect);
• levels of other negative health outcomes (e.g. morbidity and malnutrition); and
• levels of displacement (e.g. internal or cross-border).
A determinant-based typology has more direct application to the critical areas of conflict early warning and prevention, despite the analytical complexities derived from the high degree of interdependence (i.e. interaction) among these determinants [15]. However, most existing classification schemes fail to incorporate an emphasis on the public health impacts of conflicts other than battle-related fatalities. For example, other categories of war-related mortality, such as civilian deaths, deaths following famine or disease and deaths that are allowed to occur for political reasons, are often omitted. Similarly, morbidity, malnutrition and displacement among civilians, highly characteristic of post-Cold War conflicts in SSA, have been largely overlooked [14]. These population-level results of conflict appear, in many cases, to help explain the links between conflict and HIV/AIDS progression. Therefore, the present framework draws from both types of typologies for maximum utility regarding the problem of HIV and conflict, recognizing the complex nature of conflict as an epidemiologic study factor.
Aspects of Violent Conflict that Directly Affect the HIV Epidemic
Despite large cross-cultural and historical variation in the determinants and manifestations of conflict, and variation in the systems that have been developed to analyze conflict, a model can be developed specific to inquiry about the public health impacts of conflict on HIV epidemiology. Conflict can be analyzed in terms of the following dimensions, all of which may help to shape its influence on HIV risk.
Time-scale of conflict
Both the duration of the conflict and the point of conflict onset relative to the progression of the epidemic should be considered. Chronic conflict, for example, may engender profound societal changes that are manifested in immediate increases in population risk of HIV. For example, widespread impoverishment resulting from war may fuel high levels of commercial sex as a survival strategy. Similarly, displaced populations are believed to be at greater risk of sexual violence or sexual activity as a way to negotiate access to key resources for survival. A recent finding regarding the behavior of humanitarian aid workers in western Africa tragically supports this assertion [16].
Longer-duration conflicts generally lead to greater cumulative effects of conflict on social infrastructure. Populations may be isolated from modern communications for a long period of time, resulting in much greater naïveté regarding the epidemiology and prevention of the disease. In low-prevalence conflict settings this may not impact transmission significantly; post conflict, however, this may exacerbate vulnerability.
Alternatively, chronic conflict may result in lower exposure opportunities following reduced social mixing due to isolation and limited population mobility. During the war in Angola, for example, mobility was limited and most of the population was concentrated in small "islands of security" around provincial capitals. HIV seroprevalence remained low as a result.
Equally, the point of conflict onset relative to the progression of the epidemic may determine the degree to which conflict may result in sustained low seroprevalence levels for the duration of the war. In Rwanda, for example, sentinel surveys among pregnant women and STI patients demonstrated prevalence rates exceeding 30% before the 1994 genocide [17]. Because HIV was already so prevalent, community isolation did not likely curtail the local spread of the disease. In contrast, prevalence rates in Mozambique in sentinel surveys of high-risk populations appear to have remained relatively low until the war ended, at which point the resumption of normal patterns of social mixing occurred alongside a marked rise in prevalence rates [17]. Thus, when initial low prevalence rates are combined with isolation for long periods of time, HIV progression at the population level may be considerably attenuated.
Characteristics and involvement of parties involved in conflict
The political/legal status, differential HIV prevalence rates, relative size, motives, and tactics characterizing combatants and affected civilian populations may all shape the progression of the epidemic. In terms of political/legal status, combatant groups may comprise government military forces, paramilitary forces, organized rebel groups, or highly fragmented rebel groups. Available data suggest but do not conclusively confirm the oft cited axiom that significant differentials in seroprevalence can exist between government military and civilian populations in Africa [18]. More recent findings suggest that this too is contextualized [19]. Almost no data is available regarding other types of armed groups.
Modern conflict in SSA often inflicts a high degree of sexual violence upon civilian populations that, if civil-military seroprevalence differentials exist, will result in increased mixing and HIV spread. Additionally, commercial sexual activity and other commercial activities may follow soldiers, again facilitating HIV spread. The size and motives/tactics of armed groups may determine in part the types of interactions (and violence of those interactions) with the civilian population.
Geographic scale and dynamics of conflict
The scale and geographic focus of the conflict highlight geographic areas that may be differentially affected by the conflict or the epidemic. In terms of scale, conflicts may take place on a regional, national, or sub-national level. Most post-Cold War conflicts in SSA involve civil conflict; many conflicts have international and regional dimensions. The war that started in the Democratic Republic of Congo (DRC) in 1998 drew armies from at least seven African countries experiencing wide variations in their levels of HIV prevalence. The concept of the geographic focus of the conflict highlights the fact that the effects of war are unlikely to be homogeneous in any situation. This gives rise to distinct local ecologies that may experience lower HIV vulnerability and exposure to HIV hazards through the factors listed above, particularly isolation and population mixing associated with military and forced migration. For example, the Ethiopian conflict resulted in a large concentration of military along the Ethiopian-Eritrean border and accompanying civilian commercial activity. In some cases, such as Mozambique, conflicts have resulted in intensified and highly localized trading corridors between outside countries of higher seroprevalence and lower prevalence areas.
Specific Mechanisms through which Conflict Influences HIV Risk
Factors that May Decrease Risk
Factors may decrease risk by decreasing vulnerability, or more commonly, by decreasing exposure opportunity. The following factors are postulated to accompany conflict in post-Cold War SSA and may explain how conflict may actually limit the spread of the epidemic during the period of conflict.
Factor 1: Increased isolation of communities
Conflict isolates communities by destroying transport systems, making travel unsafe, and disrupting the market-based activities that encourage economic migration. Conflict may freeze normal cross-border migration, as in the case of Ethiopia and Eritrea. On the sub-national level, civil conflict may isolate rural communities controlled by armed factions, as in Angola, or limit population movement because of the loss of transport infrastructure, as in the Democratic Republic of Congo. Where conflict is chronic, the effect of limiting population movement and therefore population mixing could be a very significant contributor to the relatively lower levels of HIV found among affected populations.
Factor 2: Increased death rates among high risk groups
Another aspect of conflict that may decrease population HIV risk is differential mortality among high-risk groups. It is well established that mortality among adult males is elevated in conflict-affected settings. Post-genocide Rwanda has shown a demographic shift such that among adults between the ages of 15 and 54 years, women far outnumber men [20]. In addition, poor nutrition and the lack of access to health services probably decreases the survival time of HIV-infected individuals. A recent study in Guinea Bissau demonstrated a strong differential in mortality between war-affected cohorts of a tuberculosis treatment program according to HIV status [21].
Factor 3: Decreased casual sex associated with trauma and depression
Available evidence points to the unexplored possibility that conflict may result in decreased sexual activity, most commonly as a result of psychological sequelae of trauma such as post-traumatic stress disorder (PTSD). It has been shown that absent or low libido in those with post-traumatic stress disorder can be as high as 69% [22]. In those patients with general depression, reduced libido is present in up to three quarters of patients [23]. An elevated level of depression in conflict-affected settings has been documented [24]. Research in Rwanda in the late 1990s investigated the prevalence of clinical depression (as per the Diagnostic and Statistical Manual for Mental Disorders, DSM-IV criteria, measured using an adapted version of the Hopkins Symptom Checklist); the prevalence of a local severe depression-like syndrome of "mental trauma" (guhahamuka); and the prevalence of a local, less acute syndrome of "severe grief" (agahinda gakabije). In the commune that experienced significantly lower levels of violence during the genocide (Butamwa), only 5.6% of adults had depression, and 31.9% of adults had agahinda gakabije. In the commune that experienced widespread violent conflict during the genocide, almost one-fifth (17.9%) of adults had depression and almost half (41.8%) of adults had agahinda gakabije [24]. The results, though inconclusive, suggest that depression-like syndromes may be highly prevalent in some settings.
Factor 4: Disruption of sexual networks following conscription or displacement
Conflict may lead to the disruption of sexual networks associated with forced migration and/or the conscription of husbands and sexual partners. Females may have fewer opportunities for casual sex, though this factor remains unexplored in the research literature. This may hold most true in displaced communities, which are often comprised disproportionately of women and children.
Factors that May Increase Risk
Accelerating factors in this model constitute factors that tend to enhance the spread of HIV by worsening dimensions of population vulnerability or increasing HIV exposure opportunity.
Factor 1: Increased interaction among military and civilians
Conflict has been demonstrated to result in the increased sexual mixing of military with civilian groups, especially in areas of high military concentration for extended periods of time [25]. In post-conflict settings, demobilization of combatants may also result in disassortive mixing. It has been documented that in the African context, some military groups have higher HIV risk than the general population [26], although this has not been demonstrated conclusively in all contexts. Although military seroprevalence data are not typically available in the public domain, a recent analysis suggests that seroprevalence levels are commonly at least 5% higher among military than their civilian counterparts in Africa [18]. It is important to note, however, that military infection levels vary greatly between and within military organizations. In Cambodia, for example, household surveys estimate prevalence rates of police and military personnel at 8%, while the rate among the civilian population is 2.7% [27,12]. Military recruits in Myanmar, however, appear to have prevalence rates similar to that of the general public [27]. In a large HIV prevalence study in Ethiopia, prevalence among urban military was 7.2%, while among urban civilians the level was 6.4%. Rural military registered a lower rate than the general rural population [28].
Factor 2: Increased levels of commercial or casual sex
Impoverishment coupled with discrimination against women and the erosion of traditional behavioral norms may give rise to high levels of sex driven by economic motives in conflict and transitional societies. This again is frequently cited as an important consequence of war, but is difficult to quantify. What is clear is that African countries that have experienced war have lasting socioeconomic effects. For example, Mozambique and Angola are among the three lowest ranking countries on all components of the Human Development Index among Southern African countries [29]. Infant mortality in these two countries rank among the highest in the world. Among refugee and displaced populations poverty results from the severing of livelihood strategies and catastrophic asset loss. Semi-permanent refugee populations that relocate near population centers may be forced to participate in commercial sex in order to ensure household livelihoods or, in extreme cases (e.g. poor female-headed households), to exchange sex for food and other assets [30-32]. In addition, high levels of female illiteracy affect the ability of women to seek alternative forms of livelihood. In northern Uganda, many separated and widowed female Sudanese refugees began brewing and selling beer to sustain themselves and their children. This unfortunately led to an "increase in unprotected sex with multiple partners while under the influence of alcohol" [33]. These vulnerabilities characterize populations in both conflict and post-conflict phases.
Factor 3: Decreased availability of reproductive health and other health services
Destruction of the health sector is a common feature of conflicts and its reconstruction has not been rapid in most SSA countries. Massive loss of health infrastructure – both personnel and physical infrastructure – is common in conflict-affected countries of Africa. Mozambique lost the majority of its clinics during the civil war [34]. Rwanda was estimated to have lost more than 80% of its health personnel through death or flight during the genocide [35]. Access and utilization failures result in lack of treatment for STIs and, subsequently, poor health, which may result in greater population-level HIV risk. In Guinea-Bissau, for example, interruption of TB treatment for HIV patients due to the civil conflict was directly responsible for the significantly higher mortality rate experienced by HIV patients. Those who had completed TB treatment before the outbreak of the war, showed no differential increase in mortality [21]. Lack of infrastructure and changing utilization patterns also handicap surveillance efforts [9]. This may delay recognition of HIV, as well as other diseases, as a public health problem.
Factor 4: Decreased utilization of reproductive health and other health services
Not only is infrastructure lost, but utilization patterns also are affected by violent conflict. People may not be able to get to health care services because of ambient danger. They may also be reluctant to use services because they distrust providers. This may have been a factor in Rwanda, for example, where providers were particularly implicated in genocide activities during the war [36]. Another deterrent to use is the tendency for conflict-affected populations to become more reliant on self-care, traditional health systems, or emergent predatory health providers, as has been documented in several instances [37].
Factor 5: Increased levels of malnutrition
While quality evidence-based research demonstrating direct links between malnutrition and increased susceptibility to HIV seroconversion is scarce, the importance nutrition plays in limiting infection and regulating the immune system is well documented. Malnutrition and micronutrient deficiencies impair immune function, thereby increasing vulnerability to infections in general. Vitamin A, for example, has been shown to play an important role in immune function, including the maintenance of mucosal epithelia, the growth of immunogenic cells and antibody response [38]. Supplementation with vitamin A increases levels of natural killer cells in HIV infected children [39] and decreases morbidity due to other diseases such as measles and malaria [40,41]. Similarly, vitamin E is associated with neutrophil phagocytosis and lymphocyte proliferation [42]. Such deficiencies are associated with accelerated disease progression in HIV patients [43], greater risk of vertical transmission of HIV [44,38] and increased HIV loads [45,46]. It should be noted, however, that other studies have concluded that vitamin A deficiency is not associated with increased vertical transmission [48-52].
In addition, malnutrition levels are generally a good indicator of health status and social equity. High levels of micronutrient deficiency and general malnutrition are widely documented in conflict-affected populations, resulting in decreased resilience to infections. It is known that poor nutritional status is associated with vulnerability to progression from tuberculosis infection to disease. The immunosuppression associated with HIV infection is a major risk factor for the progression of latent tuberculosis infection to active disease and death [53]. The increased level of HIV in a community will certainly contribute to a high dual HIV-TB burden.
Factor 6: Decreased use of means to prevent HIV transmission
The isolation and poverty caused by war often results in decreased knowledge of, and access to, means to prevent HIV transmission. Population probability surveys available through the Demographic and Health Surveys, UNICEF's Multiple Indicator Surveys, and specialized HIV behavioral surveys show that knowledge levels and condom use are quite low in conflict-affected countries [9] and that they contrast with regional and sub-regional norms, especially in cases of protracted and widespread conflict. Depressed knowledge levels reflect the failure of mass media campaigns, formal education, and literacy and clinic-based education activities during and following conflict. As illustrated in Table 2, general awareness of HIV as a health threat often significantly surpasses in prevalence the awareness of specific measures that may be taken to prevent transmission, such as the utilization of condoms and the avoidance of multiple sexual partners.
Table 2 Knowledge, Attitudes and Practices Related to HIV/AIDS, Selected Countries in SSA
Country % heard of HIV % know no ways to prevent % know condom use
Mozambique (DHS, 1997) 82.2 65.8 15.4
Eritrea (DHS, 1995) 80.6 24.2 34.6
Ethiopia (DHS, 2000) 84.7 31.5 33.5
Sierra Leone (MICS, 2000) 54.0 -- 27.0
Somalia (MICS, 2000) 36.6 88.3 2.8
Sources: Marco-International, Demographic and Health Surveys (DHS) . UNICEF, Multiple Indicator Cluster Survey (MICS) .
Additionally, knowledge levels tend to exhibit marked intra-national variability, particularly among socially and economically marginalized groups. This variability may be enhanced in conflict-affected countries through regional isolation [54].
The case of Rwanda suggests that conflict may affect contraceptive use and desired family size. Popular wisdom suggests that post-conflict populations may want to repopulate and, to an extent, that tendency is supported by examination of Demographic and Health Survey (DHS) data collected before and after the genocide of 1994. In 1992 and 2000, the DHS documented rates of awareness of HIV exceeding 80% and awareness of modern methods of contraception exceeding 90% [20,55]. Despite the relatively high levels of exposure to information, condom use remained low in 2000, though use is significant in higher risk groups. While this may be due to the disruption of social marketing campaigns and limited access to condoms, desired fertility may have increased since the war, thereby reducing the utilization of modern methods of contraception. From 1992 to 2000, women's rates of utilization of modern methods of contraception decreased from 8.6% to 2.7% (12.9% to 4.3% for women "in union"). During the same period, the percentage of women reporting a desire to have six or more children increased from 14.9% to 28.6% [20,55]. These data highlight the fact that the constraints to adoption of condom use remain significant on the population level and may in part represent a desire to repopulate. In these cases, HIV prevention programs will need to be carefully targeted to take into account this underlying dynamic.
Factor 7: Increased population mixing following large internal or regional population movements
On the whole, forced migration may increase HIV risk, as forced migration movement tends to be in a rural-to-urban direction, which greatly enhances the possibility of disassortive mixing. In addition, the migration process is often associated with high levels of physical danger and exposure to sexual violence. Where forced migrants remain in highly insecure areas, migration may be associated with frequent assaults by combatants. In addition, migration patterns may be quite fluid, enabling dislocated populations to move back and forth between higher and lower risk areas, thus increasing disassortive mixing. Research suggests that urban rates of casual/commercial sexual activity tend to exceed rural rates and that exchange of rural/urban populations tends to undermine traditional norms governing sexual activity in rural areas [56]. It has been shown that war can increase partner exchange, as relationships generally tend to be shorter-term, thereby increasing the reproductive rate of the disease [57,58]. Due to the positive relationship between frequency of STIs and HIV, frequent partner exchange and its associated increased risk of STI infection, HIV transmission is further exacerbated [59].
Fluid population movements are particularly common during prolonged conflicts in border areas that might give rise to population mixing and then repatriation. In the case of Rwanda, research illustrates that the post-war period has seen a sharp decrease in the HIV prevalence differential between rural and urban women attending antenatal clinic services in Kigali [60]. The trend may be due in large part to migration between rural and urban communities, combined with the high level of sexual violence inflicted on rural women during the genocide.
Factor 8: Emergence of norms of sexual predation and violence
Coupled with increased population movement is the frequent emergence of norms of sexual predation and sexual violence within conflict-affected areas. The phenomenon of rape as a war tactic is increasingly being recognized and documented. The widespread infliction of sexual violence upon women during the Rwandan genocide and its aftermath illustrates the extent to which sexual violence may be utilized as an instrument of war. While exact figures are unavailable, it is estimated that between 250,000 and 500,000 cases of rape occurred during the conflict [61,62]. In Liberia, 49% of women surveyed reported at least one act of sexual or physical abuse by either a soldier or a fighter during the civil war [63]. In Sierra-Leone, 9% of those surveyed reported a war-related incident of sexual violence [64]. Similar accounts have been reported across the world including Kosovo, Azerbaijan, Iraq and others.
In cases of HIV resulting from sexual violence, the shame and social stigma attached to rape prevent women from seeking testing or care. It should be noted that in some conflict-affected populations, research suggests discriminatory cultural attitudes and practices. In Rwanda, research in 1990 found that men control sexual decision-making, and that HIV positive women were more likely to report coercive sex and violence with their sexual partner than HIV negative women [65]. In Angola, of the 38% of women who had been physically abused, 69% had been abused by their husband or boyfriend and 23% by their mother or father [66]. Therefore, HIV treatment and prevention programs need to address the sociocultural determinants of unequal power sharing in sexual partnerships. It should also be noted that in some African cultures including Rwanda, mourning rituals involve intercourse between the widow or widower and another individual. Widows may be forced to have intercourse with a close male relative (i.e. brother or cousin) of her deceased husband to achieve the purification intended from the ceremony [67]. This ceremony, which may occur more frequently during periods of elevated mortality in conflict situations, places the economically vulnerable widow at an elevated risk of HIV transmission.
Factor 9: Fragmentation of families and resultant vulnerable household structures
Like HIV, conflict decimates family structures through mortality and dislocation, and results in lasting effects on society [1]. Conflict-affected populations typically have higher rates of child-headed households (e.g. orphans). They may also have higher dependency ratios because of greater numbers of female-headed households and, in some cases, higher levels of handicap and fewer able-bodied men. When juxtaposed with proximity to economically remunerative sex, this change in household ecology would favor greater vulnerability.
Putting it Together at a Regional Level: Social Ecology of HIV and Conflict
The models and mechanisms identified above help to develop an explanatory model built on a foundation of social ecology. A social ecology approach argues for models of explanation that explore the interactions of multiple causes (environmental, social, and biological) working through different mechanisms and at differing levels of scale (individual, household, community etc.) with emphasis on dynamic change over time. The social ecology of the epidemic is defined in terms of sociodemographic, socioeconomic, structural, contextual, biologic and behavioral variables that facilitate or inhibit progression of the epidemic [68-70]. The ecological perspective explains how conflict might impede the progress of the epidemic and it provides clues as to regional strategic factors that may exacerbate risk in the post-conflict setting. While the epidemiologic literature postulates a number of factors that we do not elaborate here (such as comorbidity and religious practice) these factors also are likely to be important. For the sake of illustrative simplicity, we examine four key variables not commonly cited that are more closely linked to the conflict-HIV axis: (1) population density, (2) level and geographic distribution of economic growth, (3) the prevalence of poverty, and (4) the existence of physical infrastructure, particularly transport infrastructure.
One key feature of mapped data is the relative correspondence of population density, road infrastructure, economic productivity, and HIV risk in Southern Africa (see Figures 3a,3b,3c). Central Africa is marked by more clustered populations and sparse road infrastructure, especially in Congo and Angola – those areas that have experienced chronic conflict. Coastal West Africa has high population density and road density but comparatively low HIV seroprevalence. This may be due in part to poor road quality and lack of road connectedness to areas of high HIV risk. Also the economic productivity of western Africa is much lower than that of southern Africa. Therefore, economic migration patterns may not be as intense. As mentioned earlier, other factors, such as the moderating influence of religion and biological co-factors (e.g. circumcision, and STI rates), may also be important. It is also possible that conflict in central Africa may have moderated HIV risk by buffering the west coast of Africa from movement of HIV up the coast from southern Africa.
Population density may directly influence the occurrence, severity, and spread of violent conflict and HIV. Where population density is lower, population mixing associated with conflict may be more sporadic (as opposed to consistent and sustained). Population density paired with excellent road infrastructure may have a synergistic effect on HIV risk. This may, in part, explain the pattern of risk seen in southern and eastern Africa.
Economics and poverty are important drivers. Although higher economic output is associated with higher levels of HIV in general, the poor are increasingly affected for a variety of well-accepted reasons. From this perspective, Southern Africa remains the most problematic with respect to the potential for rapid northward spread of HIV. Angola and Mozambique are characterized by high poverty, economic potential, and in the case of Angola, the potential to be a gateway to West Africa. One factor that may be driving the epidemic is the interaction between poor women and rich men (or men with some income, such as the military and truck drivers) as exemplified by Drain et al. [68] who found that income inequality was independently associated with HIV seroprevalence.
The ecology of HIV and conflict also results in greater micro-level variation in the determinants of HIV risk. Mock and Drapcho [37] showed that regional variability and the design effects (sample variation across clusters/villages) associated with measures of nutrition and mortality were far greater among post-conflict countries surveyed by DHS than are typically found in more stable settings. This is plausible given that conflict does not have a consistent effect in all areas of countries. Also, it would be expected that functioning social systems found in stable settings might have an equalizing effect on health status.
How has the International Community Responded to the Joint Effects of HIV and Conflict?
Sadly, the international community has compartmentalized its responses to HIV and conflict – as well as its relief, recovery, and development programming instruments – which precludes an integrated and aggressive attack on the epidemic. As a result, very little has been learned in terms of designing and implementing programs to address HIV among conflicted-affected populations, with some exceptions [71].
The current structure of development assistance results in poor availability of resources to address HIV in conflict settings, as these are traditionally the domain of humanitarian assistance. To date, though much lip service is paid to the concept of developmental relief, the bulk of all humanitarian assistance is supply-side delivery of immediate survival commodities and services. This approach continues to dominate even though humanitarian assistance may be provided to conflict-affected population for years and decades, and even when displacement may provide unique opportunities to reach populations that might otherwise be inaccessible.
Similarly, the immediate post-conflict period is typically serviced by transition programs that emphasize demobilization, reinsertion, reintegration and development of the foundations of governance. While these are clearly important considerations, a problem as severe as HIV cannot be compartmentalized as a development problem (i.e. not a humanitarian or transition-period concern). And indeed, problem-focused activities such as HIV prevention might be a motivating cause for the stimulation of civil society groups.
Even internal to the development community, the problem of HIV is not linked to the problem of conflict, but rather these two issues are seen and treated as unrelated concerns. Different strategies and partners are managed by separate bureaucratic units that have rare interactions. Such compartmentalization of both conflict and HIV along humanitarian/development lines and internal to development management has resulted in fragmented approaches to addresses these important and interactive influences on the health development of SSA.
Similarly, the transitional period should be assessed for opportunities to creatively integrate HIV-related interventions into priority program strategies to promote economic recovery and social rehabilitation. Examples of such integrated programs include integrating HIV education into micro-credit or economic development programs and incorporating HIV risk reduction strategies into demobilization and social integration programs. Demobilized ex-combatants might also be incorporated in educational and health extension programs such that they become part of the solution instead of the problem.
Humanitarian assistance provides numerous opportunities for the cross-sectoral integration of effective HIV-directed initiatives, including the opportunity for synergistic amelioration of adverse health outcomes.
Even though conflicts typically last for years or decades, countries affected by conflict continue to receive assistance, primarily for immediate survival needs, through humanitarian assistance efforts. In post-accord transition programs, the emphasis is on establishing democratic rule, governance, and re-integration. Sequential application of traditional humanitarian and transition interventions ignores the regional context of high HIV risk. Since HIV risk may be lower in conflict settings while vulnerability to HIV is increasing, this programming approach results in a major constraint to timely, prevention-oriented intervention.
Implications for Policies and Programs
Our analysis suggests that the relationship between conflict and HIV is complex and contextualized; however, important general conclusions can serve as a basis for action. The analysis demonstrates that while vulnerability to HIV is heightened as a result of conflict, exposure opportunities may be significantly reduced. This may lead to important opportunities for interventions to keep HIV prevalence rates low in the affected settings (i.e. to prevent HIV in relatively lower-prevalence countries). More urgently, post-conflict changes in exposure opportunities could result in explosive epidemic waves. The case of Angola is particularly troublesome given its strategic location and economic potential.
Conflict and HIV are inter-related problems that demand a clear strategy and coordinated use of humanitarian and development assets. This means that objectives for addressing these problems should drive the response, and conflict should be viewed as a key determinant of HIV risk. The HIV/AIDS epidemic is a major constraint to development in Africa. Programs and bureaucracies should more clearly align along a consistent vision of HIV prevention and mitigation in both conflict and post-conflict settings.
A portion of the Declaration of Commitment on HIV/AIDS, adopted at the United Nations General Assembly Special Session on HIV/AIDS (UNGASS) on June 27, 2001, articulates strategies and goals to address HIV/AIDS in conflict and disaster-affected regions. It calls for the development and implementation of national strategies that incorporate HIV/AIDS awareness, prevention, care, and treatment elements into programs or actions that respond to emergency situations. The declaration recognizes that populations destabilized by armed conflict, humanitarian emergencies, and natural disasters – including refugees, internally displaced persons and, in particular, women and children – are at increased risk of exposure to HIV infection, and calls for HIV/AIDS components to be factored into international assistance programs where appropriate [72].
As international, regional and national agencies strive to abide by the Declaration of Commitment on HIV/AIDS, the framework provided here will be useful in the identification of determinants of HIV risk. We recommend that factors affecting HIV risk in conflict settings be systematically assessed as a basis for strategic planning to address HIV in conflict. The assessment should include vulnerability profiling, exposure risk assessment, and characterization of mechanisms through which conflict affects HIV risk. It is particularly important to pre-empt disassortive mixing associated with post-conflict improvements in mobility and resettlement. The assessment should be conducted at the different planning levels (i.e. regional, national and sub-national).
Conflict risk assessment should also be a key component so that synergistic programming between conflict and HIV initiatives can be achieved. Examples of these include:
• potentiating civil society organizations around the HIV problem, especially in areas of high prevalence;
• incorporating HIV prevention as a key element in demobilization and reinsertion initiatives, including the possible use of ex-combatants as HIV educators/mobilizers; and
• aggressive and progressive approaches to poverty alleviation and reduction. Poverty is particularly problematic in chronic conflict contexts, even when societies have high development potential (e.g. the DRC and Angola). Aggressive development programs are probably the most central strategy to confronting HIV and conflict. HIV prevention and conflict resolution can be inserted into a number of the specific components of these programs. Targeting women- and child-headed households, and making it economically feasible for families to send youth to school are also of particular importance.
Our analysis stresses the importance of maintaining a global perspective, while at the same time recognizing that micro-planning may be even more important in conflicted-affected settings than in stable settings. This is because local ecologies may be more diverse due to the varying spatial and temporal effects of conflicts. Indeed, most countries that have experienced large-scale conflict have had quiet zones where life was relatively stable, even during the course of large-scale war. These stable areas could absorb major non-emergency initiatives. In post-conflict settings, these micro-level differences persist, while others, such as differing levels of infrastructure destruction, ethnic tensions and pockets of high HIV prevalence, may require highly specific approaches.
These findings also suggest somewhat different approaches to HIV surveillance in conflict-affected settings. First, a more deliberate attempt should be made to support surveillance in countries and areas affected by conflict so that a better evidence base is developed. Unfortunately, a review of the UNAIDS surveillance database revealed that conflict-affected countries have little or no systematic surveillance, despite the existence of relatively stable areas [73]. This may be due to the fact that lab testing and quality control for HIV surveillance is generally centralized. Perhaps a more de-centralized approach would better ensure continued data collected in conflict-setting such that at least some regions of a country would continue to collect data during conflict. Higher micro-level variation and greater social change in the post-conflict setting argues for more finely graded surveillance. Also, surveillance should take into account the differing risk groups resulting from conflict, and the dynamics of exposure opportunity that occur as a result of opening international borders, rapidly evolving trade corridors and improved internal mobility.
Finally, we argue that information strategies, including HIV/conflict risk assessments and surveillance, should be rationally planned and implemented as a basis for intervention planning and program evaluation during the early phases of conflict response. Seroprevalence or other proxy measures of HIV infection must be monitored more deliberately as a part of surveillance. Conflict-HIV vulnerability/risk assessment tools can be developed based on the factors enumerated above. They should be applied periodically to more effectively respond to a dynamic setting. Improved HIV status information together with these planning data should foster a more evidence-based approach to preventing and mitigating HIV in conflict settings. We call for mainstreaming HIV/AIDS prevention and care policies into conflict prevention, peacekeeping operations, humanitarian responses to crises, post conflict reconstruction planning, implementation and evaluation.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
Dr. Mock responsible for overall framework and approach for this paper. Drs. Duale and Brown contributed HIV literature synthesis. Ms. Mathys, O'Maonaigh, Abul-Husn and Elliot extracted literature. All authors read and approved the manuscript.
Table 1 HIV/AIDS Risk in Selected Sub-Saharan Africa Countries
People living with HIV/AIDS
% Adults 1999,1 20012 Women2 15–49, 2001 Children2 0–14, 2001 Cumulative AIDS Rate, per 1,000 (year)1 Estimated Num. of Death due to AIDS, 20013 Estimated Num. of AIDS Orphans 20014
ANGOLA 2.8, 5.5 190,000 37,000 0.2 (1997) 24,000 104,000
BOTSWANA 35.8, 38.8 170,000 28,000 4.7 (1998) 26,000 69,0003
BURKINA FASO 6.4, 6.5 220,000 61,000 1.0 (1997) 44,000 268,000
BURUNDI 11.3, 8.3 190,000 55,000 1.7 (1996) 40,000 237,000
CONGO 6.4, 7.2 59,000 15,000 3.9 (1998) 11,000 78,000
CONGO DR 5.1, 4.9 670,000 170,000 0.8 (1998) 120,000 927,000
COTE D' IVORIE 10.8, 9.7 400,000 84,000 2.6 (1996) 75,000 420,000
ERITREA 2.9, 2.8 30,000 4,000 1.3 (1998) 350 24,000
ETHIOPIA 10.6, 6.4 1,100,000 230,000 1.3 (2000) 160,000 989,000
GUINEA 1.5 (1999) 29,0005 2,7005 0.6 (1998) 5,600 29,000
KENYA 14, 15.0 1,400,000 220,000 2.7 (1998) 190,000 892,000
LIBERIA 2.8 (1999) NA 2,000 0.1 (1998) 4,500 39,000
MOZAMBIQUE 13.2, 13.0 630,000 80,000 0.6 (1998) 60,000 418,000
NAMIBIA 19.5, 22.5 110,000 30,000 4.1 (1997) 13,000 47,0003
NIGERIA 5.1, 5.8 1,700,000 270,000 0.2 (1999) 170,000 995,000
RWANDA 11.2, 8.9 250,000 65,0003 2.2 (1997) 49,000 264,000
SIERRA LEONE 3.0, 7.0 90,000 16,000 0.0 (1996) 11,000 42,000
SOUTH AFRICA 19.9, 20.1 2,700,000 250,000 0.3 (1996) 360,000 660,0003
TANZANIA 8.1, 7.8 750,000 170,000 3.2 (1998) 140,000 815,000
UGANDA 8.3, 5.0 280,000 110,000 2.5 (1997) 84,000 884,000
ZAMBIA 20, 21.5 590,000 150,000 5.0 (1997) 120,000 572,000
1 International Program Center, Population Division, U.S. Census Bureau. Updated June 2000. HIV/AIDS Surveillance Data Base. "HIV /AIDS Country Profiles." Online [accessed April 5, 2002].
2 United Nations Development Program. 2002. "Human Development Indicators 2002." Aggregates calculated for the Human Development Report Office by UNAIDS. Online [accessed January 14, 2003].
3 UNAIDS, WHO. Updated 2002. "Epidemiological Fact Sheets on HIV and Sexually Transmitted Infections." [accessed January 22, 2003].
4 Joint USAID/UNICEF/UNAIDS Report. 2002. "Children on the Brink." Appendix I. Online [accessed January 14, 2003].
5 United Nations Development Program. 2001. "Human Development Indicators 2001." Aggregates calculated for the Human Development Report Office by UNAIDS. Online [accessed January 14, 2003].
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J Autoimmune DisJournal of Autoimmune Diseases1740-2557BioMed Central London 1740-2557-1-11567990610.1186/1740-2557-1-1EditorialWhy do we need a new journal in autoimmunity? D'Cruz David 1david.d'[email protected] Vitaly [email protected] The Rayne Institute, St Thomas' Hospital, London, UK2 St. Luke's-Roosevelt Hospital Center and Columbia University, New York, NY2004 13 10 2004 1 1 1 12 9 2003 13 10 2004 Copyright © 2004 D'Cruz and Ablamunits; licensee BioMed Central Ltd.2004D'Cruz and Ablamunits; 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 new online Journal of Autoimmune Diseases is created as an independent open access journal. In addition to the obvious advantages of the open access, the Journal will practice a double-blind reviewing of the manuscripts, which means that both the reviewers and the authors remain anonymous to each other. We believe that such a policy will reduce the influence of personal and other non-scientific factors on the reviewer's decision making.
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Autoimmune diseases are common and can affect virtually every organ in the body. They range from organ specific diseases such as thyroiditis or type 1 diabetes to life-threatening multi-system diseases such as systemic lupus erythematosus and the systemic vasculitides. Clinicians from every field of medicine may encounter these patients and both generalists and specialists need to keep up to date with clinical and experimental developments in autoimmunity. The advent of targeted therapies for example the anti-TNF-α agents for the treatment of rheumatoid arthritis exemplify the application of basic science research that can lead to effective therapies.
Inspired by the publisher, BioMed Central, a group of enthusiastic professionals gathered to launch the Journal of Autoimmune Diseases. There are several journals devoted to autoimmune diseases already, with at least four having the word "autoimmunity" in the title. In addition, a number of immunological journals and journals that specialise in particular diseases publish papers on autoimmunity. So is there a need for yet another journal?
The Editorial Board of Journal of Autoimmune Diseases certainly thinks so. This Journal is one of the new breed of online-only journals which are proving extremely successful. But it is not the magic word "internet' that makes the difference. Two distinct features provided by, BioMed Central and the Editorial Board offer special advantages to readers and authors.
Open Access
The aim of publishing is to share information with the community. Authors are also keen to know that their work is being read. One of the syndromes of authorship has been the little card politely requesting a reprint. This card is sometimes forgotten or filed in the circular filing cabinet thus inducing considerable guilt both in the paper's author and the recipient. The era of reprint requests may be drawing to a close since most of the journals can now be found on-line. However, the access to a paper on-line often requires a subscription or a purchase of an individual article ("pay-per-view"). The high publication costs of printed journals deprive their publishers of the generosity of complete free access, but this tends to limit the dissemination of research. An alternative approach provided by BioMed Central is to make access totally free for everybody, as part of their Open Access policy [1]. It means that Journal of Autoimmune Diseases is universally and freely available online to everyone, its authors retain copyright, and it is archived in internationally recognised free repositories such as PubMed Central [2]; e-Depot [3]; Potsdam [4] and INIST [5].
Journal of Autoimmune Diseases enables scientists from countries and institutions with limited funds to read the same material as wealthier ones [6]. This has become possible because lower publication costs can be covered by the authors. The advantage for the authors is also obvious: access to their work is much more freely available throughout the world [7].
Double-blinded peer review
The Journal of Autoimmune Diseases aims to provide a high standard of double-blinded peer review, in which the reviewer's name is not disclosed to the authors, and the authors remain anonymous to the reviewers. A few journals already use this system, so why do we believe that this will benefit the scientific community?
We have all come across situations where an honest, laborious piece of work was hard to publish. Some journals reject up to 95% of manuscripts [8], and this includes good papers. The problem here is that the decision made by a reviewer is not always based solely on the scientific merit of the paper. This may cause either of two antiscientific consequences, unfair rejection or unfair acceptance.
It is the nature of human beings that if a reviewer does not like you personally, a good paper is rejected or additional possibly unnecessary experiments are required which will take a year or two. Unfair rejection can occur due to a conflict of interest, be it personal or financial. For instance, an individual may be rejected on the basis of ethnicity [9], so may his paper be.
An unfair acceptance takes place when the reviewer is benevolent to a weak paper that has an outstanding name as the last author. Some authors may successfully exploit this weakness of a reviewer's human being by placing the famous name intentionally. This results in a ghost authorship of which the celebrity may be unaware! One of us remembers an anecdotal conversation he heard a few years ago in one of the universities:
Dorit (a secretary to the Professor): Robert? There is a new paper of yours I see on Medline. Do you wish me to update your publication list?
Professor: Who are the other authors?
(Dorit reads 20 last and first names)
Professor: Hm-m-m...I don't know any of these people. What is the title there?
(Dorit reads the title)
Professor: I don't remember even discussing anything like that or being consulted...
Dorit: Should I ignore it then?
Professor: Could you print it out for me? I will read it first. If the paper is good enough...Well, then I'll have nothing to do but to add it to my list of publications.
What are the remedies then? Two policies that could help to overcome these difficulties are completely open peer review and completely anonymous, or double-blind peer review. In open peer review [10], the reviewers' names are disclosed to the authors and vice-versa, ensuring accountability. Obviously, this might help against unfair rejections, but not against unfair acceptance. In addition, there is a danger that younger reviewers will be intimidated and the political power of the established will be increased [8]. We believe that a double-blinded review process will be much more effective in helping to avoid rejection of good papers and acceptance of unsound manuscripts for subjective, political or other non-scientific reasons.
Journal of Autoimmune Diseases
We hope to attract clinical and basic science reports from leading and innovative authors. We call for established authors to publish with us in order to be easily accessible by all other scientists, to stop fighting with the windmills of the elite journals, to write detailed and clear papers in the unlimited space provided by internet. We would also strongly encourage more junior authors who are making their way in this field to consider publishing high quality science in the Journal of Autoimmune Diseases – we all need that important first paper to get going. The Editorial Board has been carefully selected from leading authorities in their own fields to help achieve our ambitious aim of developing a high class international journal that is accessible by all who have internet access and we look forward to receiving your submissions.
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PubMed Central
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INIST
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Lawrence S Free online availability substantially increases a paper's impact Nature 2001 411 521 11385534 10.1038/35079151
Lawrence PA The politics of publication Nature 2003 422 259 261 12646895 10.1038/422259a
Watzman H Oxford professor accused of discrimination over e-mail Nature 2003 424 7 12840724
Gura T Scientific publishing Peer review, unmasked Nature 2002 416 258 260 11907547 10.1038/416258a
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J Autoimmune DisJournal of Autoimmune Diseases1740-2557BioMed Central London 1740-2557-1-21567990710.1186/1740-2557-1-2ReviewAutoantibodies and autoantigens in autoimmune hepatitis: important tools in clinical practice and to study pathogenesis of the disease Zachou Kalliopi [email protected] Eirini [email protected] George N [email protected] Research Laboratory of Internal Medicine, Department of Medicine, Larissa Medical School, University of Thessaly, Larissa 41222, Greece2 Academic Liver Unit, Department of Medicine, Larissa Medical School, University of Thessaly, Larissa 41222, Greece2004 15 10 2004 1 2 2 14 12 2003 15 10 2004 Copyright © 2004 Zachou et al; licensee BioMed Central Ltd.2004Zachou 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.
Autoimmune hepatitis (AIH) is a chronic necroinflammatory disease of the liver characterized by hypergammaglobulinemia, characteristic autoantibodies, association with HLA DR3 or DR4 and a favorable response to immunosuppressive treatment. The etiology is unknown. The detection of non-organ and liver-related autoantibodies remains the hallmark for the diagnosis of the disease in the absence of viral, metabolic, genetic, and toxic etiology of chronic hepatitis or hepatic injury. The current classification of AIH and the several autoantibodies/target-autoantigens found in this disease are reported. Current aspects on the significance of these markers in the differential diagnosis and the study of pathogenesis of AIH are also stated. AIH is subdivided into two major types; AIH type 1 (AIH-1) and type 2 (AIH-2). AIH-1 is characterized by the detection of smooth muscle autoantibodies (SMA) and/or antinuclear antibodies (ANA). Determination of antineutrophil cytoplasmic autoantibodies (ANCA), antibodies against the asialoglycoprotein receptor (anti-ASGP-R) and antibodies against to soluble liver antigens or liver-pancreas (anti-SLA/LP) may be useful for the identification of patients who are seronegative for ANA/SMA. AIH-2 is characterized by the presence of specific autoantibodies against liver and kidney microsomal antigens (anti-LKM type 1 or infrequently anti-LKM type 3) and/or autoantibodies against liver cytosol 1 antigen (anti-LC1). Anti-LKM-1 and anti-LKM-3 autoantibodies are also detected in some patients with chronic hepatitis C (HCV) and chronic hepatitis D (HDV). Cytochrome P450 2D6 (CYP2D6) has been documented as the major target-autoantigen of anti-LKM-1 autoantibodies in both AIH-2 and HCV infection. Recent convincing data demonstrated the expression of CYP2D6 on the surface of hepatocytes suggesting a pathogenetic role of anti-LKM-1 autoantibodies for the liver damage. Family 1 of UDP-glycuronosyltransferases has been identified as the target-autoantigen of anti-LKM-3. For these reasons the distinction between AIH and chronic viral hepatitis (especially of HCV) is of particular importance. Recently, the molecular target of anti-SLA/LP and anti-LC1 autoantibodies were identified as a 50 kDa UGA-suppressor tRNA-associated protein and a liver specific enzyme, the formiminotransferase cyclodeaminase, respectively. Anti-ASGP-R and anti-LC1 autoantibodies appear to correlate closely with disease severity and response to treatment suggesting a pathogenetic role of these autoantibodies for the hepatocellular injury. In general however, autoantibodies should not be used to monitor treatment, predict AIH activity or outcome. Finally, the current aspects on a specific form of AIH that may develop in some patients with a rare genetic syndrome, the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED) are also given. Autoantibodies against liver microsomes (anti-LM) are the specific autoantibodies detected in AIH as a disease component of APECED but also in cases of dihydralazine-induced hepatitis. Cytochrome P450 1A2 has been identified as the target-autoantigen of anti-LM autoantibodies in both APECED-related AIH and dihydralazine-induced hepatitis. The latter may indicate that similar autoimmune pathogenetic mechanisms can lead to liver injury in susceptible individuals irrespective of the primary defect. Characterization of the autoantigen-autoantibody repertoire continues to be an attractive and important tool to get access to the correct diagnosis and to gain insight into the as yet unresolved mystery of how hepatic tolerance is given up and AIH ensues.
Antibodies against Liver Cytosol 1 Antigen (anti-LC1)Antibodies against Soluble Liver Antigens or Liver Pancreas (anti-SLA/LP)Antinuclear Antibodies (ANA)Antineutrophil Cytoplasmic Autoantibodies (ANCA)Autoimmune HepatitisCytochrome P450 2D6Cytochrome P450 2A6Cytochrome P450 1A2Hepatitis CHepatitis DLiver-Kidney Microsomal Autoantibodies (anti-LKM)Liver Microsomal Autoantibodies (anti-LM)Smooth Muscle Autoantibodies (SMA)
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1. Introduction
Autoimmune hepatitis (AIH) is a rare chronic liver disease of unknown etiology. The estimated prevalence of AIH in Northern European countries is approximately 160–170 patients/106 inhabitants [1,2]. The disease predominates among women and is characterized by hypergammaglobulinemia even in the absence of cirrhosis, characteristic autoantibodies, association with human leukocyte antigens (HLA) DR3 or DR4 and a favorable response to immunosuppressive treatment [3-5]. The onset of AIH disease is usually insidious, with unspecific symptoms, such as, fatigue, malaise, arthralgias, and fluctuating jaundice, right upper quadrant pain or lethargy [5-8]. However, a substantial proportion of patients may have no obvious signs or symptoms of liver disease, while occasionally the presentation may be severe and almost identical to an acute or fulminant episode of viral hepatitis [5-8]. Although AIH brings in mind the archetypal patient being a young female with endocrine abnormalities, there is nowadays increasing evidence that the disease can also affect males and can present at almost any age (the large majority of patients being between 50 and 70 years of age) [6-12].
Liver histology is not pathognomonic for AIH and there is no single serologic test of sufficient specificity for the diagnosis of AIH as for the diagnosis of viral hepatitis A to E. Although the presence of autoantibodies is one of the distinguishing features of AIH, there is no single autoantibody with the diagnostic significance and specificity that antimitochondrial autoantibodies (AMA) demonstrate for the diagnosis of primary biliary cirrhosis (PBC). For this reason, autoantibodies can not be employed as a single marker for the diagnosis of AIH. It is rather a diagnosis reached by the exclusion of other factors leading to chronic hepatitis that include viral, toxic, genetic and metabolic causes [6]. Under this context, it is clear that sometimes AIH may be difficult to diagnose [7,8]. In 1992, the International Autoimmune Hepatitis Group reported a descriptive set of criteria that could be applied in the routine clinical practice for the diagnosis and classification of patients as having either 'definite' or 'probable' AIH [13]. In addition, a diagnostic scoring system was devised to provide an objective method for selection of relatively homogeneous groups of patients for research purposes [13]. The same group has remarkably simplified the descriptive set of criteria and the diagnostic scoring system in late 1998 (Tables 1 and 2) [6]. The diagnostic score demonstrates that the presence of defined autoantibodies is an integral part of the diagnosis of AIH but not its single diagnostic tool [6,14].
Table 1 Revised Scoring System for the Diagnosis of Autoimmune Hepatitis6.
Parameter/Features Score
Gender
- Female/Male +2/0
Degree of elevation above upper normal limit of ALP vs. aminotransferases
- <1.5 +2
- 1.5 – 3.0 0
- >3.0 -2
Total serum globulins, γ-globulins, or IgG above normal
- >2.0 +3
- 1.5 – 2.0 +2
- 1.0 – 1.5 +1
- <1.0 0
ANA, SMA or LKM-1 (titers by immunofluorescence on rodent tissues or HEp2-cells)
- >1 : 80 +3
- 1 : 80 +2
- 1 : 40 +1
- <1 : 40 0
- AMA positive -4
Hepatitis viral markers (IgM anti-HAV, HBsAg, IgM anti-HBc, anti-HCV and HCV-RNA)
- Positive/Negative -3/+3
Recent or current use of known or suspected hepatotoxic drugs
- Yes/No -4/+1
Average alcohol intake
- <25 g/day +2
- >60 g/day -2
Other autoimmune disease(s) in patient or first degree relatives
- Yes/No +2/0
Optional additional parameters (should be allocated only if ANA, SMA or LKM-1 are negative)
- HLA DR3, DR4, or other HLA with published association with AIH) +1
- Seropositivity for any of ANCA, anti-LC1, anti-SLA/LP, anti-ASGPR and anti-sulfatide +2
Liver histology
- Interface hepatitis +3
- Predominant lymphoplasmacytic infiltrate +1
- Rosetting of liver cells +1
- None of the above -5
- Biliary changes -3
- Other changes -3
Response to therapy (as defined in Table 2)
- Complete/Relapse +2/+3
Definite AIH if greater than 15 before treatment or greater than 17 post-treatment; probable AIH if varies between 10–15 before treatment or 12–17 post-treatment. ALP: alkaline phospatase, IgM anti-HAV: hepatitis A virus IgM antibody, anti-HCV: hepatitis C virus antibody, HBsAg: surface antigen of hepatitis B virus, IgM anti-HBc: IgM antibody against the nuclear antigen of hepatitis B virus. Other abbreviations are the same as shown in the text.
Table 2 Definitions of Response to Therapy.
Response Definition
Complete Either or both of the following: marked improvement of symptoms and return of serum AST or ALT, bilirubin and immunoglobulin values completely to normal within 1 year and sustained for at least a further 6 months on maintenance therapy, or liver biopsy specimen at some time during this period showing at most minimal activity. or Either or both of the following: marked improvement of symptoms together with at least 50% improvement of all liver tests during the first month of treatment, with AST or ALT levels continuing to fall to less than twice the upper normal limit within 6 months during any reductions toward maintenance therapy, or a liver biopsy within 1 year showing only minimal activity.
Relapse Either or both of the following: an increased in serum AST or ALT levels of greater than twice the upper normal limit or a liver biopsy showing active disease, with or without reappearance of symptoms, after a "complete" response as defined above. or Reappearance of symptoms of sufficient severity to require increased (or reintroduction of) immunosuppression, accompanied by any increase in serum AST or ALT levels, after a "complete" response as defined above.
AST: aspartate aminotransferase, ALT: alanine aminotransferase.
In recent years however, significant progress has been made in the characterization of liver-related target-autoantigens. This has led to the notion that some of the major target-autoantigens in AIH are active enzymes of the human hepatic and non-hepatic microsomal xenobiotic metabolism [14-16]. The latter serve as a means to investigate this still enigmatic liver disease. This article will focus on the data that have evolved in the course of the characterization of autoantibody-autoantigen "system" in AIH by giving the current aspects on the role and significance of this "system" in the differential diagnosis and study of pathogenesis of AIH.
2. Classification of AIH
According to the pattern of autoantibodies detected in AIH patients, a subclassification of the disease into three types was proposed in 1994 [17]. AIH type 1 (AIH-1) is characterized by the presence of antinuclear antibodies (ANA) and/or smooth muscle autoantibodies (SMA) which may associate with perinuclear anti-neutrophil cytoplasmic antibodies (p-ANCA) [3,5,6,14,15]. AIH type 2 (AIH-2) is characterized by the detection of specific autoantibodies against liver and kidney microsomal antigens (anti-LKM type 1 or infrequently type 3) [14-16,18] and/or antibodies against liver cytosol type 1 antigen (anti-LC1) [14,15,19]. AIH type 3 (AIH-3) is characterized by autoantibodies against soluble liver antigens (anti-SLA) [20] or to liver-pancreas antigen (anti-LP) [21,22].
The serological diversity of autoantibodies found in AIH supports the aforementioned subclassification and provides a framework for the scientific analysis of this heterogeneous disease group [5,15]. It also demonstrates that AIH may not be a single disease with a single underlying mechanism but most likely is a group of diseases with a similar clinical presentation [14,15]. This is further substantiated by the finding of an unusual form of AIH in 10–18% of patients with a rare autosomal recessive disorder, the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED) [23-25]. This syndrome is characterized by chronic mucocuteneous candidiasis, ectodermal dystrophy and autoimmune tissue destruction particularly of the endocrine glands (hypoparathyroidism, adrenocortical failure and gonadal failure in females) [26-29].
However, due to recent clinical, serologic and genetic findings, it has been suggested that anti-SLA seropositive patients do not define a subgroup of AIH, but rather belong to the AIH-1 group [30-32]. For this reason, subdivision into AIH-1 (ANA, SMA, p-ANCA and/or anti-SLA/LP positive) and AIH-2 (anti-LKM-1, anti-LKM-3 and/or anti-LC1 positive) is in common usage (Table 3). Apart from serological differences, AIH-2 seems to be clinically and genetically distinguishable from AIH-1 [5,8,14]. Indeed, patients with AIH-2 are younger at presentation, usually have higher levels of bilirubin and transaminases, and are characterized by more severe disease than patients with AIH-1 [5,6,14,18,33]. In addition, contrary to what has been recorded in patients with AIH-1, no sustained remission has been observed after the discontinuation of immunosuppressive therapy in patients with AIH-2 [4,33]. Taking into consideration the genetic markers, it has been found that the association between HLA DR3 and AIH-2 is rather weaker than that reported in AIH-1, while an association between HLA DQ2 and AIH-2 has been reported [14,18,33,34]. However, AIH-2 patients represent only a small proportion of the total cases of AIH [3-6]. In addition, the long-term outcome of the affected patients appears to be similar both in AIH-1 and AIH-2 [6,33]. Therefore, the classification of AIH in these two major subgroups is still uncertain and controversial [6,8,13].
Table 3 Classification of Autoimmune Hepatitis (AIH) According to Autoantibodies Detection
Type of AIH Characteristic autoantibodies
AIH-1 ANA, SMA, p-ANCA anti-ASGP-R, anti-SLA/LP
AIH-2 anti-LKM-1, anti-LKM-3, anti-LC1, anti-ASGP-R
Abbreviations are the same as shown in the text.
3. Detectable Autoantibodies in AIH-1
3.1. Anti-nuclear antibodies (ANA) and smooth muscle autontibodies (SMA)
ANA and/or SMA are almost exclusively requisites for the diagnosis of AIH-1 [3,5,6,14,15,30]. In typical cases of AIH-1, these autoantibodies are detected in significant titers (≥1;80 in adults and ≥1:40 in children) in almost half of Caucasians patients with AIH-1, while ANA alone are detected in 15% and SMA alone in 35% [5,30,35].
The most frequent and conventional method for ANA detection is the indirect immunofluorescence (IIF) assay on cryostat sections of rodent tissues and HEp-2 cells slides (Figures 1 and 2) [14,15,35,36]. Different patterns of fluorescence are found by this assay due to the large variability of target-autoantigens in the nuclei of HEp-2 cells that have been recognized [35-40]. Actually, ANA are found to be directed against single or double stranded DNA, tRNA, SSA-Ro, snRNPs, laminins A and C, cyclin A or histones [35-40]. Most commonly, a homogenous (34–58%) or speckled (21–34%) immunofluoerescence pattern is demonstrable [14,15,35-37]. So far however, neither a liver-specific nuclear antigen nor a liver-disease-specific ANA has been identified. For this reason, subtyping of ANA in cases of AIH-1 seems to have limited clinical implication and diagnostic relevance in routine clinical practice [6,14,15,35-40].
Figure 1 High titer antinuclear antibodies (ANA) of the homogeneous pattern by indirect immunofluoerescence on immobilized HEp-2 cells in a female patient with autoimmune hepatitis type 1 (AIH-1). Homogeneous ANA are frequently found in AIH-1 (original magnification 40×).
Figure 2 Typical staining of antinuclear antibodies in the serum of a patient with autoimmune hepatitis type 1 visualized by indirect immunofluoerescence on cryostat sections of rat liver (original magnification 40×).
SMA are detected by IIF on rodent liver and kidney, due to staining of vessel walls (Fig. 3), and stomach due to staining of the muscle layer (Fig. 4). SMA are directed against structures of the cytoskeleton such as actin, troponin, vimentin and tropomyosin. In AIH-1, SMA are predominantly directed against F-actin [41]. The latter seems to be diagnostically more relevant in pediatric patients where SMA may be the only marker of AIH-1 even in titers as low as of 1:40 [14,15]. Czaja et al [41] have suggested that antibodies to actin are associated with a younger age of disease onset, the presence of HLA-A1-B8-DR3 haplotype and a greater frequency of treatment failure, death from liver disease and earlier requirement for transplantation than actin-antibody negative AIH-1 patients.
Figure 3 Smooth muscle antibodies by indirect immunofluoerescence on rat kidney (from a female patient with autoimmune hepatitis type 1). The immunofluorescence involves smooth muscle fibers within blood vessels (original magnification 40×).
Figure 4 Smooth muscle autoantibodies by indirect immunofluoerescence on rat stomach (serum from a female patient with autoimmune hepatitis type 1). The immunofluorescence involves smooth muscle fibers within muscularis mucosa (original magnification 40×).
ANA and/or SMA – usually in low titers – may occur in patients with chronic viral hepatitis B or C, but in most of these cases SMA lack F-actin specificity [14,15,42,43]. From the clinical point of view, interferon-alpha administration is generally safe in most cases of viral hepatitis with ANA and/or SMA, although occasionally may provoke mild self-limited autoimmune disorders compared to viral hepatitis patients without ANA or SMA autoantibodies [44-46]. During immunosuppressive treatment, disappearance of ANA and/or SMA is observed in the majority of patients with AIH-1 [47]. However, autoantibody status is unable to predict immediate outcome after cessation of corticosteroid administration. Additionally, neither autoantibody titers at first diagnosis nor autoantibody behaviour in the time course of the disease are prognostic markers for AIH-1 [14,15,47]. These findings indicate that ANA and SMA are not involved in the pathogenesis of AIH-1 and furthermore, their determination is more of diagnostic than prognostic value [5,14,15,47].
3.2. Anti-neutrophil cytoplasmic autoantibodies (ANCA)
These autoantibodies are directed against cytoplasmic constituents of neutrophil granulocytes and monocytes. Classically, they are detected by IIF using ethanol-fixed granulocytes as substrate [48]. Using the above method, two major subtypes can be distinguished. ANCA showing a diffuse or granular cytoplasmic staining (c-ANCA) and ANCA characterized by a perinuclear-staining (p-ANCA). Both c-ANCA and p-ANCA are valuable diagnostic and prognostic markers in systemic vasculitides in particular Wegener's granulomatosis and microscopic polyangiitis, respectively [48,49]. Proteinase-3 has been identified as the major target-autoantigen of c-ANCA in cases with Wegener's granulomatosis, while myeloperoxidase is the documented autoantigen of p-ANCA in most patients with microscopic polyangiitis [48,49]. Since then, ANCA (in most cases of p-ANCA type) were detected in a high prevalence in other inflammatory disorders of unknown aetiology such as, inflammatory bowel disease (more frequently in ulcerative colitis than in Crohn's disease) [50,51] and primary sclerosing cholangitis (PSC), a liver disease that is frequently associated with ulcerative colitis [51,52].
Several recent studies however, have also documented the presence of high titers of p-ANCA in the sera of patients with AIH-1 (Fig. 5; prevalence range 40–96%) [53-58] and to a much lesser extent in PBC patients (prevalence range 0–39%) [54,58,59]. Occasionally, high titers of c-ANCA can be detected in AIH-1 (Dalekos GN, 2002, unpublished observations). In contrast, p-ANCA have not been detected in serum samples from patients with AIH-2 [57]. Low ANCA titers are detected infrequently in patients with alcoholic or chronic viral liver diseases [54,57,60]. However, a recent large study in 516 patients with hepatitis C virus (HCV) infection revealed the presence of ANCA in as high as 55.6% of patients [61]. Interestingly these investigators have shown that all HCV positive sera with ANCA had c-ANCA pattern on IIF and contained proteinase-3 specificity [61]. The clinical relevance of this finding remains to be determined. In patients with AIH-1, PBC or PSC the detection of ANCA appears to be associated with a more severe disease course or the presence of cirrhosis [54,62]. The latter suggestion however, was not confirmed by more recent studies [53,54].
Figure 5 Perinuclear staining of anti-neutrophil cytoplasmic autoantibodies (p-ANCA) by indirect immunofluoerescence on ethanol fixed human granulocytes (serum from an ANA negative patient with autoimmune hepatitis type 1). Original magnification 40×).
To determine the antigenic specificities of ANCA, antigen-specific enzyme linked immunosorbent assays (ELISAs) and Western blotting followed by immunodetection can be performed [14,63]. Using these techniques it became obvious that in AIH-1 the target-autoantigens recognized are multiple including cathepsin G, catalase, alpha-enolase, lactoferrin, actin and high mobility group (HMG) non-histone chromosomal proteins HMG1 and HMG2 [14,54,56,58,62-65]. However, the determination of antigenic specificities of ANCA seems to have limited clinical relevance in patients with AIH-1 [14,15,54,56].
In conclusion, the detection of ANCA may be a useful additional marker in searching for AIH-1, in particular in ANA/SMA/anti-LKM-1 negative cases of AIH. With the exception of a recent paper by Wu et al [61] the detection of ANCA is rather rare in chronic viral hepatitis [14,54,57,60]. The latter may prove useful in the differential diagnosis between patients with AIH and those with viral hepatitis who tested positive for ANA or SMA. Furthermore, since ANCA appear to be relatively rare in PBC [54,59], these autoantibodies may prove useful for distinguishing between genuine cases of AIH and cases of PBC with features overlapping with those of AIH [6]. However, due to the lack of specificity for the diagnosis of AIH and to its obscure role – if any – in AIH, their routine determination is not recommended [14,15].
3.3. Autoantibodies against the asialoglycoprotein receptor (anti-ASGP-R)
The asialoglycoprotein receptor (ASGP-R) is a liver-specific glycoprotein of the cell membrane. Its main function is the internalization of asialoglycoproteins by binding a terminal galactose residue to coated pits. Anti-ASGP-R autoantibodies are detected in 88% of patients with AIH (both types) [66,67]. However, these autoantibodies are also found in some patients with PBC, chronic viral hepatitis B and C and alcoholic liver disease although at lower frequency and lower titers [14,15,66,67].
The ASGP-R is preferentially expressed on the surface of periportal liver cells where piecemeal necrosis is found as a marker of severe inflammatory activity in patients with AIH [68]. This finding may suggest a possible immunopathogenetic involvement of anti-ASGP-R autoantibodies in AIH [69]. The general presumption is that target of potentially tissue-damaging autoreactions in AIH must be liver-specific and available to the immune system in vivo (e.g. expression on the surface of hepatocytes). So far, ASGP-R is the only target-autoantigen that has been positively identified and fulfils these criteria [68,69]. Additional support to this emerged from the determinations of anti-ASGPR autoantibodies in consecutive AIH patients. The levels of anti-ASGP-R autoantibodies vary according to the inflammatory activity of the disease. In addition, anti-ASGP-R antibody titers decreased significantly in response to immunosuppression, while they reappear when the disease has relapsed [66,70]. These autoantibodies may be diagnostically helpful when other autoantibodies are not detected and AIH is suspected. However, due to the belief that anti-ASGPR antibody represents a general marker of liver autoimmunity and the limitations in its detection (requires chemically purified ASGP-R, which is not yet widely available), its routine use is not generally recommended.
3.4. Antibodies against soluble liver antigens (anti-SLA) or to liver-pancreas antigen (anti-LP)
The anti-SLA autoantibodies were described for the first time in 1987 [20]. They cannot be detected by IIF on common substrate. A competitive ELISA or a radioimmunoassay usually detects these autoantibodies [20,32,71]. SLA is found in 100000 g supernatant of liver homogenate and represent a cytosolic protein which is neither organ nor species specific [72]. However, the highest concentrations are found in liver and kidney tissues. The anti-SLA autoantibodies are detected in patients with AIH alone or in combination with SMA and/or ANA [30-32,73]. As noted above, similarities in the clinical profile between patients with AIH-1 (ANA and/or SMA positive) and AIH patients with anti-SLA alone in addition with an approximately 30% seropositivity overlap between anti-SLA and SMA and/or ANA suggest that anti-SLA is rather an additional important marker for the diagnosis of AIH-1, than a marker of a third type of AIH [6,14,30-32].
A scientific group from Tuebingen, Germany described for the first time the anti-LP autoantibodies in 1981 [21]. The LP antigen was predominantly detected in the S100 supernatant of liver and pancreas homogenates, indicating that this antigen was a soluble protein. Until recently, anti-LP and anti-SLA autoantibodies were thought to be different [20-22]. However, Wies et al report [74] provides convincing evidence and confirms previous suggestions that anti-SLA and anti-LP are one and the same autoantibody (anti-SLA/LP). In addition, the same study demonstrated that the identified target-autoantigen of anti-SLA/LP autoantibodies (a 35–50 kDa protein) was neither cytokeratins 8 or 18 [71] nor glutathione-S-transferase isoenzyme [75]. The results from two independent groups [76,77] were similar with those found by Wies et al [74]. After screening of cDNA expression libraries they identified a previously unknown amino acid sequence, which presumably encodes a UGA-suppressor tRNA-associated protein, as the targen-autoantigen of anti-SLA/LP autoantibodies [76,77]. The UGA-suppressor serine tRNA-protein complex is likely to be involved in cotranslational selenocysteine incorporation in human cells [78]. It was then obvious that the identification of SLA/LP autoantigen would allow the establishment of a reliable, universally available diagnostic test for AIH but also it would provoke the investigation in the area of autoimmune liver diseases.
Regarding disease specificity, anti-SLA/LP autoantibodies have not been detected in patients with AIH-2, PBC, PSC, chronic viral hepatitis, alcoholic liver disease and non-hepatic autoimmune diseases by standardized ELISAs using reference autoantibody or recombinant antigen [20,32,73,79]. Ballot et al [32] also showed that these autoantibodies are different from anti-LC1. For these reasons, anti-SLA/LP has been considered as a valuable and specific diagnostic marker of AIH [31,32,73,74,76,77,79]. However, a recent study from the United Kingdom [80] has shown that anti-SLA/LP autoantibodies can also be detected in AIH-2 and in children with PSC. These investigators used eukaryotically expressed tRNP ((Ser) Sec)/SLA as target in a radioligand assay (RLA) which is well known as a more sensitive test than ELISAs and immunoblot due to its ability to identify antibodies directed to conformational epitopes [81-83]. Their novel findings need confirmation from other research groups and particularly to address whether anti-SLA/LP reactivity is also present in adult PSC. Recent data confirmed the previous finding that patients with anti-SLA/LP display a more severe course of AIH [79,80,84]. The latter suggest that anti-SLA/LP may be linked to the pathogenesis of the autoimmune process although the exact function and its role in autoimmunity are so far unclear [14,15]. From the clinical point of view however, this autoantibody may be helpful in an attempt to reduce the group of cryptogenic hepatitis and/or cirrhosis.
4. Detectable Autoantibodies in AIH-2
4.1. Autoantibodies against liver-kidney microsomes (anti-LKM)
Three types of anti-LKM autoantibodies have been identified [3,5,14,15,18,30,63,72,85]. The LKM type 1 autoantibody (anti-LKM-1) is the characteristic serologic marker for the diagnosis of AIH-2 [5,18,63,72]. These autoantibodies were first described by Rizzetto et al [86], using the IIF method on rodent liver and kidney sections. The characteristic features of anti-LKM-1 autoantibodies are the diffuse staining of cytoplasm of the entire liver lobule and the exclusive staining of the P3 portion of the proximal renal tubules (Fig. 6) [18]. Due to this staining pattern of kidney sections anti-LKM-1 can be easily distinguished from AMA, which stain proximal and distal renal tubules (Fig. 7). Western blots with hepatic and renal microsomes revealed a protein band at 50 kDa [5,14,15,63,72,87].
Figure 6 Antibodies against liver-kidney microsomes type 1 (anti-LKM-1) react to the proximal tubules of the rat kidney. The absence of reactivity against thedistal tubules of the rat kidney (see also Fig. 6B) and parietal cells of the rat stomach distinguishes anti-LKM-1 autoantibodies from antimitochondrial antibodies (original magnification 40×).
Figure 7 Antimitochondrial antibodies react to the proximal and distal tubules of the rat kidney (original magnification 40 ×). In these cases there is also reactivity to the parietal cells of the rat stomach.
The major target-autoantigen of anti-LKM-1 autoantibodies in AIH-2 has been identified as the cytochrome P450 2D6 (CYP2D6) [87-89]. It has been shown that anti-LKM-1 autoantibodies inhibit the enzymatic activity of CYP2D6 in vitro, but not in vivo [90]. Epitope mapping experiments of CYP2D6 autoantigen have defined at least four different linear epitopes [91,92]. The most immunodominant epitopes of CYP2D6 were amino acids 257–269 and 321–351, which are recognized in about 70% and 50% of AIH-2 cases, respectively [91,92]. Two infrequent epitopes consisting of amino acids 373–389 and 410–429 are also recognized by anti-LKM-1 in some cases [92]. Recently, Klein et al [93] and Kerkar et al [94] reported another immunodominant epitope of CYP2D6 (amino acids 193–212) recognized in about 70% and 93 % of AIH-2 patients, respectively. However, due to failure of inhibition of CYP2D6 enzymatic activity using epitope specific antibodies and since the absorption of the above linear epitopes was unable to absorb inhibitory anti-CYP2D6 autoantibodies, the existence of additional conformational epitopes on CYP2D6 autoantigen has been postulated [95].
It is noteworthy to state here that depending on the geographic origin, 0–7% of patients with chronic hepatitis C – irrespective of the HCV genotype – develop anti-LKM-1 autoantibodies [6,14,43,63,96,97]. Recently, two studies have shown a higher prevalence of anti-LKM autoantibodies (up to 10%) in a small number of children or adult patients with HCV infection [98,99]. As stated for AIH-2, CYP2D6 is the major target autoantigen recognized by anti-LKM-1 autoantibodies in HCV patients [14,15,81-83,88,92-96]. However, we and others have failed to document CYP2D6 as the major target autoantigen of anti-LKM antibodies in HCV/anti-LKM positive sera [98,99]. In addition, recently Miyakawa et al [100] identified CYP2E1 and CYP3A4 as target autoantigens of anti-LKM autoantibodies in two patients with anti-LKM-positive chronic hepatitis C. Taking together, these findings may further indicate the heterogeneous autoimmune reactions that might take place in anti-LKM positive patients with chronic hepatitis C.
The antigenic sites on CYP2D6 autoantigen recognized by anti-LKM-1 autoantibodies are different in AIH-2 and HCV/anti-LKM-1 positive cases [92-95,101-104]. For example, the major linear epitope of 257–269 amino acids, as well as the newly reported peptide of 193–212 amino acids are recognized in 70–93% of AIH-2 patients but only in 18–50% of HCV/anti-LKM-1 positive patients [83,93,94,101]. Additional support to the presence of conformation-dependent anti-LKM-1 autoantibodies in HCV/anti-LKM-1 positive serum samples has emerged from previous studies [99,102,105]. In the latter studies only about 30% of HCV/anti-LKM-1 positive sera reacted with 50 kDa component using Western blot assays, while additional bands at 59 kDa, 70 kDa and 80 kDa were detected [99,102,105]. However, even taking into account the above additional bands, no more than 45% of all sera tested reactive by Western blot. In contrast, a significant proportion of the previous negative sera tested positive for anti-LKM-1 using a specific competitive ELISA, while denaturation of the antigens prior to perform the ELISA resulted in complete loss of the signal [105].
Recently, the development of a more sensitive and specific assay for the detection of anti-LKM-1 autoantibodies was achieved [14,15]. This novel assay is a quantitative RLA based on immunoprecipitation using 35S-methionine-labelled CYP2D6 antigen obtained by in vitro transcription and translation synthesis [81-83,99,104]. Using this assay it was shown that the anti-LKM-1 titers do not differ significantly between AIH-2 and HCV/anti-LKM-1 positive patients [81-83]. The presence of anti-LKM-1 in some patients with HCV infection led to the proposal for a further division of AIH-2 into AIH-2a (younger, predominantly female patients without evidence of HCV infection) and AIH-2b (older, predominantly male patients with HCV infection) [13,106]. Nowadays however, after the marked improvements in the reliability and availability of tests for HCV detection such a subdivision of AIH-2 appears unreasonable and tends to be deleted. Actually, HCV/anti-LKM-1 positive patients represent cases of "true" HCV infection with autoimmune features [6,107].
From the clinical point of view, screening for anti-LKM autoantibodies is recommended before the initiation of interferon-alpha therapy in HCV patients and if found positive a careful monitoring appears reasonable because occasionally interferon-alpha may unmask, or provoke autoimmune hepatic reactions and even "true"AIH [6,43,104,108-110]. Dalekos et al [104] studied antibody titers and performed epitope mapping of LKM-1-positive sera from patients with chronic hepatitis C. Interestingly, a patient with a high LKM-1 titer and autoantibodies directed against an epitope of amino acids 257–269, which are preferentially recognized by patients with AIH-2, showed exacerbation of the disease under interferon-alpha treatment. In contrast to other patients with HCV infection, this patient further recognized a rarely detected epitope on the C-terminal third of the protein. These results suggest that determination and monitoring of CYP2D6 autoantibody titers by both IIF and the RLA in combination with epitope mapping of CYP2D6 in HCV/anti-LKM positive patients before the initiation of interferon-alpha treatment, might be helpful in an attempt to identify those patients at risk of developing undesired autoimmune reactions [104].
The mechanism(s) of the development and the pathogenic role of anti-LKM-1 autoantibodies in hepatocellular injury are still unclear. It has been suggested that viral infections by herpes simplex virus (HSV) and related viruses may trigger the autoantibody formation through molecular mimicry in at least some individuals with AIH-2 [91]. Manns et al [91] tested 26 LKM-positive sera using Western blot with partial sequences of recombinant CYP2D6. Eleven sera recognized a short minimal epitope of eight amino acids with the sequence DPAQPPRD. Twelve other clones recognized a larger epitope containing this eight-amino acid core sequence. The search of electronic databases revealed an interesting match of the minimal epitope with the primary structure of the immediate early protein IE 175 of HSV-1 now known as infected cell protein 4 (ICP4) of HSV-1 (Fig. 8). Sequence identity was present for the PAQPPR sequence. This hypothesis was further supported by a case study in a pair of identical twins [111]. In this study, one sister suffered from AIH-2 but the other one was healthy. Interestingly, only the sister suffering from AIH-2 was HSV positive, and her serum recognized the viral protein ICP4 in lysates of HSV-infected cells [111]. So far however, overall evidence for mimicry as a driving force of AIH is not convincing.
Figure 8 Linear B-cell epitopes on cytochrome P450 2D6 in autoimmune hepatitis type 2. The immunodominant epitope 257–269 aa shares sequence homology with the immediate early protein IE 175, a transcription factor of herpes simplex virus type 1 (now known as infected cell protein 4 or ICP4). Although this is an attractive model for the hypothesis of molecular mimicry, overall evidence for mimicry as a driving force of autoimmune hepatitis is not convincing.
Besides molecular mimicry, chemical modification of self-proteins and/or immunological cross-reactivity to homologous autoantigens may also provide potential triggers for autoimmune responses. The latter has been suggested by Choudhuri et al [112] who have shown that in AIH-2 patients the linear epitope 321–351 of CYP2D6 cross reacts with amino acids 33–51 of carboxypeptidase-H (the target autoantigen of islet cell autoantibodies in insulin dependent diabetes mellitus), as well as, with amino acids 307–325 of 21-hydroxylase (major target autoantigen in Addison's disease). These findings possibly indicate the presence of a common motif of CYP2D6, carboxypeptidase-H and 21-hydroxylase, which may contribute through a cross reactive immune response to the development of multiple endocrinopathies in the course of AIH-2. Additional support to this hypothesis emerged from two recent studies by Kerkar et al [94] and Bogdanos et al [113]. In the first study the authors were able to show the similarity and cross-reactivity between the immunodominant epitope 193–212 of CYP2D6 and homologues of two unrelated viruses (HCV 2977–2996 and CMV 121–140) [94]. In the second study the researchers investigated whether the immunodominant epitope 252–271 of CYP2D6 in anti-LKM-1 positive AIH-2 and homologues from the NS5B and E1 proteins of the HCV polyprotein and the ICP4 of HSV-1 are targets of humoral immune response in anti-LKM-1 positive and anti-LKM-1 negative HCV infected patients and furthermore whether this response is cross-reactive [113]. The hypothesis of molecular mimicry and cross-reactivity in LKM-1 production has not been addressed experimentally. The authors for the first time gave experimental support to the notion that molecular similarity between CYP2D6, HCV and HSV can result in LKM-1 production via a cross-reactive response in genetically susceptible individuals (interestingly only the HCV positive/LKM-1 positive patients with viral/self cross-reactivity possessed the HLA B51 allotype) [113]. Taking together, the above studies suggest that multiple exposure to viruses mimicking self may represent an important pathway to the development of autoimmunity [94,113].
Two possible mechanisms have been proposed for the involvement of anti-LKM-1 autoantibodies in the pathogenesis of liver injury. The first appears to be a direct binding of these autoantibodies to hepatocytes, leading to lysis of liver cells, while the second is associated with an anti-LKM-1 induction of activating liver-infiltrating T lymphocytes, which indicates the combination of B and T cell activity in the autoimmune process involved [114-117]. A prerequisite for both anti-LKM-1 production and the activation of pathogenetic mechanisms involved in liver injury, is the expression of CYP2D6 on the surface of the patients' hepatocytes. Under this context, Ma et al [118] showed that key residues of a major CYP2D6 epitope (316–327) are exposed on the surface of the molecule and may represent key targets for anti-CYP2D6 production. In addition, recent data provides convincing evidence that anti-LKM-1 autoantibodies recognize CYP2D6 exposed on the plasma membrane of hepatocytes from either AIH-2 or HCV/anti-LKM-1 positive patients [114,115] suggesting a pathogenetic role for these autoantibodies in hepatic tissue damage either in AIH-2 or in some cases of HCV/anti-LKM-1 positive patients [104,109,110,115].
So far, anti-LKM type 2 autoantibodies (anti-LKM-2) have been detected only in some cases of drug-induced hepatitis caused by tienilic acid [14,63]. The target autoantigen of anti-LKM-2 has been documented as the CYP2C9 [85]. A proposed mechanism for the induction of anti-LKM-2 could be the binding of an active metabolite of the drug to the CYP2C9 protein, which then becomes antigenic [14,63,72,85].
Anti-LKM type 3 autoantibodies (anti-LKM-3) alone or in combination with anti-LKM-1 are also detected in about 5–10% of patients with AIH-2 [16,119]. In contrast to anti-LKM-1 and anti-LKM-2 autoantibodies, which on immunofluorescence stain liver and kidney tissues only, with anti-LKM-3 additional fluorescence signals may be present with tissue from the pancreas, adrenal gland, thyroid, and stomach. Family 1 of UDP-glycuronosyltransferases (UGT1) is the main target autoantigen of anti-LKM-3 autoantibodies (molecular weight of 55 kDa) [119,120]. These autoantibodies were first described in about 13% of patients with chronic hepatitis D, but not in patients with chronic hepatitis B or C [121]. However, three recent reports have shown the presence of anti-LKM-3 autoantibodies in some patients with HCV infection [99,122,123]. These findings may further support the heterogeneous phenomenon of the HCV-induced autoimmunity.
4.2. Autoantibodies against liver cytosolic protein type 1 (anti-LC1)
In 1988 a second autoantibody marker of AIH-2 was recognized [19]. This autoantibody was found to react to a liver cytosolic protein. The autoantibody is organ specific but not species specific and was therefore called anti-LC1 [19]. The anti-LC1 autoantibodies are characterized by a cytoplasmic staining of the periportal hepatocytes when the IIF assay is used for their detection. The hepatocellular layer around the central veins is not stained [19,124]. These findings indicate that the target autoantigen of anti-LC1 autoantibodies is not uniformly distributed in rodent liver tissues. They are detected in about 30% of patients with AIH-2 [19,124] and in approximately 50% of all anti-LKM-1 positive cases [125]. It is noteworthy that the anti-LC1 autoantibodies proved to be the only serological marker in 10% of patients with AIH [19].
The detection of anti-LC1 autoantibodies by IIF is obscured due to the anti-LKM-1 pattern that frequently found in most of the anti-LC1 positive sera. For these reasons other techniques such as, the ouchterlony double diffusion, immunoblot or counter-immunoelectrophoresis are required for their detection [19,124-126]. By immunoblotting, anti-LC1 positive serum samples recognize a liver specific cytosolic protein of 58–62 kDa [124-126]. Recently the molecular target of anti-LC1 was identified as the formiminotransferase cyclodeaminase (FTCD) [127], which is a polymeric bifunctional enzyme involved in folate metabolism. However, another group demonstrated the arginninosuccinate lyase (ASL) as the target autoantigen of a weak precipitin line detected by the ouchterlony double diffusion assay in patients with autoimmune or viral hepatitis [128].
Anti-LC1 autoantibodies have been proposed as a more specific marker of AIH-2 than anti-LKM-1 autoantibodies, since in the original reports their presence was never associated with HCV infection [19,124]. However, a recent study by Lenzi et al [125] confirmed the above aspect only in the pediatric subset of their patients, while a substantial proportion of the adults with anti-LC1 autoantibodies had also markers of HCV infection. The significance of the association between anti-LC1 autoantibodies and HCV infection remains uncertain and has to be established [106,129]. In contrast to what has been found for anti-LKM-1, the titers of anti-LC1 autoantibodies appear to parallel with disease activity [130]. The latter may indicate a possible involvement of anti-LC1 in the pathogenesis of AIH-2. However, the clinical significance of anti-LC1 is not yet completely defined.
5. Detectable Autoantibodies in AIH in APECED
Chronic hepatitis as a disease component of APECED may develop in 10–18% of patients [14,15,23-25,28,29,63]. APECED appears to be caused by mutations in a recently identified gene, the autoimmune regulator gene (AIRE), and represents the only known autoimmune disease with a monogenetic mutation today [26,27,131]. It is interesting that patients with AIH in the absence of APECED do not display mutations of the AIRE gene and are therefore genetically distinct from patients with AIH as a component of APECED [132].
Similar to AIH-2, hepatitis in APECED is associated with autoantibodies directed against cytochrome P450 proteins. In a large study with APECED patients, a typical LKM staining pattern and a predominant staining of the perivenous hepatocytes in the absence of staining of the kidney were observed [23]. The latter pattern is due to autoantibodies called liver microsomal autoantibodies (anti-LM). In this study each of anti-LKM and anti-LM antibodies were found in 8% of the patients [23]. These findings indicate that two or more different microsomal antigens are hepatic target-autoantigens in APECED.
Indeed, screening of APECED sera with recombinant antigens using Western blots has shown reactivity against four different hepatic cytochromes P450: CYP1A1, CYP1A2, CYP2A6 and CYP2B6 [23-25,133]. CYP1A1, CYP2A6 and CYP2B6 are expressed both in liver and in kidney resulting to an LKM staining pattern, while CYP1A2 is not expressed in the kidney leading to the LM staining. Among the four autoantibodies anti-CYP2A6 were detected with the highest prevalence in a Finnish APECED patients group (15.6%), while anti-CYP1A2 were found in 6.3% [23]. These results were confirmed by quantitative immunoprecipitation assays with recombinant 35S-labeled CYP1A2 and CYP2A6.
Contrary to a previous work in Sardinian patients with APECED [25], the detection of anti-CYP2A6 autoantibodies in a larger group of Finnish patients was not associated with the presence or absence of hepatitis, while anti-CYP1A2 autoantibodies were detected only in APECED patients with hepatitis [23]. These findings indicate that anti-CYP1A2 is a specific marker for AIH as a component of APECED, albeit with a low sensitivity [23,24]. Anti-CYP2A6 autoantibodies may be used as an indicator for APECED, if they are present in a patient with AIH. In parallel with the above conclusions is the anti-LKM/LM detection by IIF in about 50% of patients with AIH as part of the APECED and in only 11% of APECED patients without hepatitis [23]. The same study showed that the prevalence of ANA detection in APECED patients was high (22%) but irrespective of the presence or absence of hepatitis. Therefore ANA are not useful laboratory markers for AIH in APECED [23]. To the contrary, none of the patients' sera tested positive for anti-SLA, anti-CYP2D6 or anti-FTCD autoantibodies, which are specific markers of AIH-1 and AIH-2 [23]. On the other hand, CYP1A2 and CYP2A6 could not be identified as hepatic autoantigens in the disease control groups consisting of patients with idiopathic AIH or patients with autoimmune rheumatic diseases [23]. These findings indicate that idiopathic AIH and AIH in APECED are characterized by different molecular targets of autoimmunity, which do not overlap. Therefore, AIH and hepatitis as part of the APECED may be distinguished on the basis of differences in autoantibody profile (Tables 4 and 5).
Table 4 Detectable autoantibodies in AIH-1, AIH-2 and AIH as part of APECED
AIH-1 or AIH-2 AIH in APECED
ANA, SMA, ANCA, anti-ASGP-R anti-SLA/LP (molecular target: UGA suppressor tRNA-associated protein), anti-LKM-1 (molecular target: CYP2D6), anti-LKM-3 (molecular target: UGT1), anti-LC1 (molecular target: FTCD) ANA, anti-LC (molecular target: unknown), anti-LKM (molecular targets: CYP2A6, CYP1A1 and CYP2B6), anti-LM (specific autoantibody; molecular target: CYP1A2)
Abbreviations are the same as shown in the text.
Table 5 Differential diagnosis of chronic liver diseases according to the presence or absence of autoantibodies against molecularly defined autoantigens of cytochrome P450 complex using the radioligand assay.
Anti-CYP2D6 Anti-CYP2A6 Anti-CYP1A2 Chronic liver disease
+ - - AIH-2 (94–100%), HCV (0–10%)
- + - HCV, APECED with or without hepatitis
- - + AIH in APECED, drug induced hepatitis
+ + - HCV (0–7%)
- + + AIH in APECED
Abbreviations are the same as shown in the text.
Dalekos et al [134] using the sensitive quantitative RLA reported for the first time the presence of anti-CYP2A6 autoantibodies in about 2% of HCV-positive sera in general and in 7.5% of LKM-1-positive/HCV-positive sera. The latter further supports the low specificity of this autoantibody as a marker for AIH in APECED. Interestingly, anti-CYP2A6 autoantibodies were not detected in patients with AIH-2 who exhibit high titers of anti-LKM-1 autoantibodies [134]. The clinical relevance of this finding in HCV infection remains to be determined.
Anti-LM autoantibodies are first described in dihydralazine-induced hepatitis [135]. The major target autoantigen of anti-LM in both conditions (hepatitis as part of the APECED and drug-induced hepatitis) has been documented as the CYP1A2 [23-25,133,135]. In cases of dihydralazine-induced hepatitis the production of anti-LM autoantibodies has been attributed to adduct formation of CYP1A2 with an activated metabolite of the drug [136]. By contrast, in APECED patients no relationship between CYP1A2 and drug usage is known. In addition, it is not known whether in APECED patients a close monitoring of anti-LM may lead to early, or even prophylactic, treatment of hepatitis as a new disease component. Evidence that autoantibodies may be found before the clinical and/or laboratory manifestation of a new disease component in APECED comes from adrenal and ovarian insufficiencies, where the respective autoantibodies are detected 2–3 years before the clinical presentation of the autoimmune components [137].
Another hepatic autoantigen in APECED, the aromatic-L-amino acid decarboxylase (AADC) has also been identified recently [133,138]. This enzyme is expressed in the liver cytosol and was originally described as a β-cell autoantigen [133]. The prevalence of anti-AADC autoantibodies is significantly increased in APECED patients with vitiligo (88%) and hepatitis (92%) [5,14,29]. So far, anti-AADC autoantibodies have only been reported in APECED and their role in AIH and vitiligo as disease components of APECED deserve further investigation.
6. Concluding Remarks
In clinical practice the recognition of AIH is of great importance since most of the patients respond favorably to antiinflammatory and immunosuppressive treatment. In addition, recent novel findings dealing with the bone marrow hemopoietic progenitor cells and bone marrow stromal cells of patients with AIH suggests alternative therapeutic options even in refractory cases [139]. Diagnostic criteria for this disease have been codified recently [6]. These include descriptive criteria and scoring system based on clinical, serologic and histologic features of AIH (Table 1), which contribute substantially to the differential diagnosis of the disease from other forms of chronic hepatitis associated with autoimmune phenomena (Table 6). The discrimination between AIH and HCV infection is of particular importance, since the immunosuppression used in the former can deteriorate liver disease in HCV patients, while interferon-alpha treatment used in HCV infection may lead to exacerbation of AIH [104,108-110].
Table 6 Differential Diagnosis of Autoimmune Hepatitis.
Other autoimmune liver diseases
- Overlap syndromes
- Primary biliary cirrhosis
- Primary sclerosing cholangitis
Chronic viral hepatitis
- Chronic hepatitis B with or without hepatitis delta
- Chronic hepatitis C
- Chronic hepatitis non A to G
Cholangiopathy due to human immunodeficiency virus infection
Alcoholic liver disease
Drug-induced hepatitis
Non-alcoholic steatohepatitis
Granulomatous hepatitis
Hemochromatosis
α1-antithrypsin deficiency
Wilson's disease
Systemic lupus erythematosus
The detection of non-organ specific autoantibodies remains the hallmark of AIH. A step by step diagnostic application of autoantibody tests is mandatory for the evaluation of acute or chronic hepatitis of unknown cause. ANA, SMA and anti-LKM-1 autoantibodies should be first tested in patients with acute or chronic elevation of aminotransferases when virologic tests are negative and there is no current or past history for drug or alcohol abuse. Determination of ANCA, which occur in up to 90% of patients with AIH-1, may be useful in the identification of individuals who are seronegative for the above conventional autoantibody markers but should be kept in mind that this autoantibody lacks specificity. Many target autoantigens of the non-organ specific autoantibodies have been identified, but the latter has not led to the characterization of specific subpopulations of patients or changes in the treatment strategies. In addition, most of the non-organ specific autoantibodies do not seem to be involved in the pathogenesis of liver injury in AIH. Anti-LKM-1 autoantibodies could be an exception to the above aspect since recent data have demonstrated the expression of CYP2D6 on the surface of hepatocytes, while AIH-2 has not been observed in individuals who are deficient for CYP2D6. These findings provide arguments for an antigen-driven autoimmune process. It is possible that mutations in the autoantigen itself can lead to alterations in the three dimensional structure of the antigen, which induces autoimmunity.
Antibodies directed against liver-related antigens have had similar limitations. Anti-ASGP-R and anti-LC1 autoantibodies appear to correlate with disease severity and response to treatment suggesting a pathogenetic role to the hepatocellular damage. In general however, autoantibodies should not be used as a tool for monitoring of treatment or to predict AIH activity and outcome. Anti-SLA/LP autoantibodies have been considered as valuable and specific markers for the diagnosis of AIH-1. However, a recent study has shown that anti-SLA/LP autoantibodies can also be detected in AIH-2 and in children with PSC. Irrespective of the disease specificity of this marker, it is obvious that testing for anti-SLA/LP will help to reduce the group of cryptogenic liver disease, by recognizing previously misdiagnosed patients with AIH who were seronegative for ANA, SMA or anti-LKM-1.
In APECED, autoantibodies are directed against specific cytochrome P450 enzymes (e.g. CYP1A2, CYP2A6, CYP21, CYP17, and CYP11A1), that are expressed in organs affected by the disease process. These observations argue against the idea that antibodies against cytochrome P450 complex are simply epiphenomena secondary to tissue damage and that they have no relation to the etiology and pathogenesis of APECED.
It is not known what triggers autoimmunity in AIH. The hypothesis that different causes may lead to loss of tolerance against the same molecular target autoantigen seems attractive. For instance, CYP1A2 is the molecular target in dihydralazine-induced hepatitis and AIH as a component of APECED, CYP2D6 in AIH-2 and in some patients with HCV infection, CYP2A6 in APECED and in a proportion of patients with HCV infection and UGT1 in some cases of AIH-2 and chronic hepatitis D or C.
Research protocols in order to define AIH pathogenesis, disease susceptibility, determinants of disease severity, and to understand the epidemiology of AIH are future challenges in the investigational and clinical arena of this disease [139-141].
Competing interest
The author(s) declare that they have no competing interests.
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| 15679907 | PMC544946 | CC BY | 2021-01-04 16:37:48 | no | J Autoimmune Dis. 2004 Oct 15; 1:2 | utf-8 | J Autoimmune Dis | 2,004 | 10.1186/1740-2557-1-2 | oa_comm |
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J Autoimmune DisJournal of Autoimmune Diseases1740-2557BioMed Central London 1740-2557-1-31567991810.1186/1740-2557-1-3ResearchInsulin expressing hepatocytes not destroyed in transgenic NOD mice Tabiin Muhammad T [email protected] Christopher P [email protected] Grant [email protected] Bernard E [email protected] Diabetes Transplant Unit, Prince of Wales Hospital, The University of New South Wales, Sydney, Australia2 Joslin Diabetes Centre, Harvard Medical School, Boston, Massachusetts, USA3 Walter Eliza Hall Institute of Medical Research, Melbourne, Australia2004 8 11 2004 1 3 3 24 8 2004 8 11 2004 Copyright © 2004 Tabiin et al; licensee BioMed Central Ltd.2004Tabiin 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 liver has been suggested as a suitable target organ for gene therapy of Type 1 diabetes. However, the fundamental issue whether insulin-secreting hepatocytes in vivo will be destroyed by the autoimmune processes that kill pancreatic β cells has not been fully addressed. It is possible that the insulin secreting liver cells will be destroyed by the immune system because hepatocytes express major histocompatibility complex (MHC) class I molecules and exhibit constitutive Fas expression; moreover the liver has antigen presenting activity. Together with previous reports that proinsulin is a possible autoantigen in the development of Type 1 diabetes, the autoimmune destruction of insulin producing liver cells is a distinct possibility.
Methods
To address this question, transgenic Non-Obese Diabetic (NOD) mice which express insulin in the liver were made using the Phosphoenolpyruvate Carboxykinase (PEPCK) promoter to drive the mouse insulin I gene (Ins).
Results
The liver cells were found to possess preproinsulin mRNA, translate (pro)insulin in vivo and release it when exposed to 100 nmol/l glucagon in vitro. The amount of insulin produced was however significantly lower than that produced by the pancreas. The transgenic PEPCK-Ins NOD mice became diabetic at 20–25 weeks of age, with blood glucose levels of 24.1 ± 1.7 mmol/l. Haematoxylin and eosin staining of liver sections from these transgenic NOD PEPCK-Ins mice revealed the absence of an infiltrate of immune cells, a feature that characterised the pancreatic islets of these mice.
Conclusions
These data show that hepatocytes induced to produce (pro)insulin in NOD mice are not destroyed by an ongoing autoimmune response; furthermore the expression of (pro)insulin in hepatocytes is insufficient to prevent development of diabetes in NOD mice. These results support the use of liver cells as a potential therapy for type 1 diabetes. However it is possible that a certain threshold level of (pro)insulin production might have to be reached to trigger the autoimmune response.
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Background
Genetic alteration of non-pancreatic cells in a diabetic person to synthesise, store and secrete insulin in the same manner as a pancreatic β cell is a potential therapy of type 1 diabetes. The hepatocyte has been suggested as a suitable target cell for such gene therapy [1-9]. Such cells made in vitro are capable of synthesizing and storing pro(insulin) and can secrete this peptide in response to a physiological challenge with glucose [1,2]. Moreover, when transplanted into diabetic mice these cells can lower blood glucose levels to the normal range [2]. Further, Ferber et al. [9] has recently showed that adenovirus-mediated in vivo transfer of the PDX-1 transgene to the mouse liver results in the conversion of a hepatocyte subpopulation to the beta cell phenotype. Ferber also showed that this population of trans-differentiated liver cells was induced to produce the prohormone convertases PC1/3 and PC2 leading to complete processing of proinsulin. The amount of insulin produced was sufficient to ameliorate streptozotocin-induced hyperglycaemia in the mice.
For this gene therapy approach to be viable, the insulin producing liver cells must not be destroyed by the immune system or else this could lead to liver damage. This fundamental question has not been addressed by previous studies with (pro)insulin producing hepatocytes as diabetes in these models was induced chemically rather than by autoimmune means [6-9]. The autoimmune destruction of insulin-producing hepatocytes is a possibility since hepatocytes express major histocompatibility complex (MHC) class I molecules [10], and constitutively express Fas [11], moreover the liver cells have antigen presenting activity [12] and are attacked in autoimmune diseases such as chronic active hepatitis. Furthermore (pro)insulin appears to be an autoantigen in the development of type 1 diabetes [13,14]. Evidence for this comes from the presence insulin and Glutamic Acid Decarboxylase (GAD) autoantibodies in the sera of people with prediabetes [13]. It also comes from studies that show that diabetes can be adoptively transferred to normoglycaemic mice by the introduction of insulin-specific T cell clones [14]. These data taken together suggest that hepatocytes which produce (pro)insulin might be targeted and destroyed by the same autoimmune processes responsible for the destruction of pancreatic β cells.
To address this question in vivo, transgenic Non-Obese Diabetic (NOD) mice that express insulin in the liver were created using the Phosphoenolpyruvate Carboxykinase (PEPCK) promoter [15] to direct expression of the mouse insulin I gene. This promoter had previously been used to create a transgenic C57BL/6 human insulin secreting mouse line with hepatic insulin expression [7]. However these mice do not develop autoimmune diabetes and would be unsatisfactory to address the issue of insulin autoantigenicity.
The liver of the transgenic PEPCK-Ins NOD mice were characterised with respect to insulin mRNA transcription, (pro)insulin content and (pro)insulin release. The blood sugar levels of the animals were monitored and the livers of the animals were analysed for any evidence of immune cell infiltration.
Methods
Materials
The PEPCK promoter was generously donated by Dr R. W. Hanson (Case Western Reserve University, Cleveland, Ohio, USA). DNA modifying enzymes and competent bacteria for transformation were purchased from Promega (Madison, Wisconsin, USA). RPMI 1640, α-MEM and fetal bovine serum (FBS) were purchased from Trace Biosciences (Castle Hill, Sydney, Australia). Hybond-N+ nylon membrane for the Southern Blot and ribonuclease protection assay (RPA) was from Amersham International (Bucks, UK). Human insulin and rat insulin standards for the in-house radioimmunoassay (RIA) were obtained from Novo Nordisk (Bagsvaerd, Denmark).
Transient transfection of HEPG2 cells
The transfection of the HEP G2 cells with the PEPCK-Ins transgene was carried out using Lipofectamine (GibcoBRL, Madison, Wisconsin, USA). Cells were trypsin harvested and seeded 24 hr prior to transfection at a density of 200,000 cells/well in 24 well tissue culture plates (Becton Dickinson, New Jersey, USA) to achieve 60–80% confluence at the time of transfection. 10μl of Lipofectamine was added to 300μl of serum free media and incubated at room temperature for 30 minutes. After the incubation, 300μl of DNA solution (3–4μg) was added and the lipid:DNA complex solution incubated at room temperature for 15 minutes. The cells were washed once with 1 ml of serum free medium prior to the transfer of 300μl of the lipid:DNA complex solutions to the cells. The cells were then incubated for 5 h at 37°C in an air incubator. The DNA/lipid complex media was removed and replaced with 2 mL of α-MEM 10% FBS which contains Antimycotic and Antibiotic solution (10μL/mL) (Sigma, St Louis, Missouri, USA). In addition, for the PEPCK-Ins transgene, a final concentration of 100 nM glucagon was added to the media to upregulate the PEPCK promoter. The cells were incubated at 37°C in an air incubator for 48–60 hr. After the incubation, the conditioned culture medium was collected and assayed for mouse (pro)insulin.
Insulin Radioimmunoassay (RIA)
(Pro)insulin was measured by an in-house RIA using rat insulin standard (Novo Nordisk Laboratories, Bagsvaerd, Denmark). The lowest value on the rat insulin standard curve used in the assay was 0.25 ng/ml. The intra and interassay coefficients of variance were <5% and 10% respectively.
Generation of transgenic mice
To direct the expression of the mouse insulin I gene to the liver of the transgenic mice, the 5' flanking sequences (-460 bp to +73 bp) of the rat PEPCK gene [15] were used to drive the expression of the genomic mouse insulin I gene (-25 bp to +557 bp) [16]. The construct was digested with Xba I and Pst I (Fig 1A) and the PEPCK-Ins transgene was isolated from the agarose gel using the Supelco GenElute Spin Column (Sigma, St Louis, Missouri, USA). Standard procedures were followed to generate transgenic mice [17]. Fertilized mouse eggs were flushed from the oviducts of superovulated NOD mice 6–8 h after ovulation. Male pronuclei of the fertilized eggs were injected with 2pl of the DNA solution (2 ng/μl) and the embryos that divide to the 2 cell stage were implanted in the oviducts of pseudopregnant mice. After birth the animals were tested for the presence of the transgene by PCR and Southern blot of the DNA from tail tip samples taken after weaning at 3 weeks of age.
Figure 1 A-C. The PEPCK-Ins transgene. Schematic representation (A). Example of PCR screening of PEPCK-Ins transgenic mice (B). Lanes 1–5 and lanes 10–14 are PCR reactions with genomic DNA extracted from mice that developed from microinjected mouse eggs. Primers specific for the PEPCK-Ins transgene were used for lanes 1–9 (30 cycles) while primers specific for mouse insulin 1 were used for lanes 10–18 (30 cycles). Lanes 6 and 15 are PCR negative controls (no DNA added). Lanes 7 and 16 are PCR reactions to demonstrate the specificity of the primers used (wild type NOD mouse DNA added). Lanes 8, 9, 17 and 18 are half copy spiked and plasmid controls respectively (positive controls). Example of Southern Blotting for the PEPCK-Ins transgene (C). Lane 1 One copy spiked sample (100 ng Balb/c genomic DNA + 102fg PEPCK-Ins plasmid), lane 2 genomic DNA from tail tip of F2 PEPCK-Ins mouse, lanes 3 and 4 are DNA from pups which died before weaning and lanes 5–7 are DNA from still born pups. Each lane was loaded with 15μg of genomic DNA that was digested with Xba I and Pst I to release the transgene.
Treatment of the animals
The mice were fed ad libitum with a standard diet and kept under a light-dark cycle of 12 h in compliance with the animal ethics of our institution. The facilities used to breed and maintain the mice were specific pathogen free, with air passaged through a HEPA filter. The transgenic PEPCK-Ins NOD mice and the wild type littermates were monitored for natural development of diabetes. The diabetic PEPCK-Ins transgenic NOD mice were maintained by daily insulin injections and sacrificed 4 weeks after they became diabetic.
Blood glucose measurements of mice
Blood glucose levels of the wild type as well as the transgenic NOD mice were performed using the Medisense Precision QID Blood Glucose Sensor System (Bedford, Massachusetts, USA). The mice were bled by pricking the tail vein and 5μl of the blood placed on to the Precision Plus blood glucose electrode. A mouse was classified as being diabetic if a reading of ≥ 15 mmol/l was obtained on more than one occasion.
Insulin mRNA analysis
The total RNA from liver and pancreas was isolated using TRIzol (Life Technologies, Grand Island, New York, USA) [18]. The detection of mouse insulin I mRNA by RT-PCR was performed using Superscript II RNase H- Reverse Transcriptase from GibcoBRL (Grand Island, New York, USA) to synthesize the first strand cDNA as recommended by the manufacturer. The cDNA sample was then used as a template in PCR amplification (30 cycles) using primers specific for mouse insulin I mRNA. The sequence of primers used were:
(Forward) TAA CCC CCA GCC CTT AGT GAC CAG CTA TAA
(Reverse) AAA GTT TTA TTC ATT GCA GAG GGG TGG GGC
The PCR products were run on a 1.2% Tris Acetate EDTA agarose gel, photographed and analysed using the Gel Doc 1000 system (Biorad, Hercules, California, USA).
Insulin mRNA in the samples was also visualised using the ribonuclease protection assay (RPA) method. The plasmid used as the template to transcribe the riboprobe for mouse insulin I was a generous gift from Chirgwin J. M. (San Antonio, Texas, USA). The Digoxigenin (DIG) labelled insulin riboprobe was synthesized by the in vitro transcription method using the DIG RNA labeling kit (SP6/T7) from Boehringer Mannheim (Mannheim, Germany), which utilises DIG-dUTP, according to the manufacturer's recommendations.
Hybridisation and RNase treatment were performed using the RPA II kit from Ambion according to the manufacturer's recommendations. Next the samples were loaded and run on a 6% polyacrylamide/8 M urea denaturing mini-gel in Tris borate EDTA buffer. The RNA bands on the gel were transferred onto a nylon membrane by using an electro-gel transfer apparatus and the membrane was fixed by using an UV cross linker (Hybaid, UK).
For the visualisation of the RNA bands on the nylon membrane, the DIG Wash and Block Buffer Set and the DIG Chemiluminescent Detection Kit, both of which were purchased from Boehringer Mannheim, were used. The membrane was then exposed to a standard Kodak X-ray film for 30 minutes. The film was subsequently analysed using the Gel Doc 1000 system.
Organ culture
Pancreatic and hepatic tissue organ cultures were performed using organ culture dishes from Becton Dickinson (New Jersey, USA) [19]. Briefly, the organs were taken from mice, and an aliquot of 10–30 mg removed from each organ. The tissue was then diced into 1 mm3 explants and spread on a filter paper placed on a sterile wire grid in the inner well of the organ culture dish such that the tissue was exposed to air above and the RPMI 1640 10% FBS medium below. Sterile saline was placed in the outer well to maintain a humidified environment. The tissue in the organ culture dishes were incubated for 24 hr in a humidified 5% CO2 and air incubator at 37°C. Culture supernatant from all media changes of respective wells were pooled separately and kept at -20°C until the RIA was performed. The results were expressed per mg of tissue.
(Pro)insulin content
(Pro)insulin was extracted by homogenising a weighed sample of liver or pancreas in acid-ethanol (solution of 0.18 mol/l HCl in 70% ethanol) and incubating overnight at 4°C. The next day, the samples were spun down and the supernatant removed. The (pro)insulin content was determined by RIA and the results expressed per mg tissue.
In situ hybridisation of (pro)insulin mRNA
To detect the transcription expression of (pro)insulin, in situ hybridisation (ISH) was performed on pancreatic and liver tissues of diabetic transgenic PEPCK-Ins NOD mice and diabetic wild type NOD mice using a modification of a previously described method (20). Briefly, 4μm thick paraffin sections were deparaffinized with xylene, ethanol and air dried. The sections in 0.01 mol/l citric buffer, pH 6.0 were then treated with microwave irradiation for 10 min. Following this procedure the sections were treated with proteinase K (Invitrogen, Carlsbad, CA, USA) 1μg/ml for 15 min at 37°C. They were washed 3 times with phosphate buffered saline for 5 min and dried with ethanol. The sections were then hybridized with [35]-S-labelled riboprobes (1μg/ml) overnight at 57°C. The [35]-S-labelled riboprobes were synthesized by in vitro transcription (SP6/T7) incorporation of [35]-S-dUTP using RNA polymerase (Roche, Mannheim, Germany). The template used was mouse insulin I cDNA that was generously donated by Chirgwin JM (San Antonio, Texas, USA). The hybridisation buffer consisted of a 25μl hybridisation cocktail (labelled riboprobe 500,000 cpm/section, 50% formamide, 0.1% SDS, 0.1% sodium thiosulfate, 0.1 mol/l DTT, 0.3 mol/l NaCl, 20 mmol/l Tris-HCl [pH 7.5], 2 mmol/l EDTA, 20% dextran sulphate, 0.02% sheared salmon sperm DNA, 0.1% total yeast RNA, 0.02% yeast tRNA, 1 × Denhardt's solution). After hybridisation, the slides were soaked in 2X standard saline citrate (SSC), rinsed with deionised water and then treated with RNase A (20μg/ml) for 30 min at room temperature. High stringency washes were then performed on the slides with 2XSSC followed by 0.2XSSC. The slides were then dried by ethanol, air dried for 20 min and the results viewed by NBT2 emulsion autoradiography (Kodak, Rochester, New York). For controls, sense-probe instead of anti-sense was used for hybridisation. The photos were taken using the Olympus IX70 microscope (Melville, New York) with a dark field condenser.
Histochemistry
Sections of the liver and pancreas from the transgenic mice were washed twice in Phosphate Buffered Saline (PBS), fixed in 10% formalin overnight and embedded into paraffin. Consecutive 5μm sections were cut and placed on poly-L-lysine-coated slides. After dewaxing and serial alcohol rehydration, the tissue sections were treated with H2O2, and then incubated in PBS containing 10% goat serum or 1% BSA for 20 minutes at room temperature to block nonspecific binding.
For insulin staining, primary insulin antibody (Dako Corporation, Via Real, Carpinteria, California, USA) at 1:1250 was added and the sections were incubated overnight. The next morning the sections were washed in PBS and incubated with anti-guinea pig IgG (1:400) at room temperature for 20 minutes. Thereafter the sections were washed again with PBS, and biotinylated anti-rabbit / mouse antibody added to the sections for 15 minutes followed by streptavidin-peroxidase conjugate for a further 15 minutes. Finally, the sections were treated with substrate-chromogen AEC. A standard concentration (0.1%) of haematoxylin was added as a counterstain. The primary antibody was omitted for the negative control. The haematoxylin and eosin staining of sections was performed automatically by the Jung Autostainer XL machine (Leica, New Jersey, USA).
Statistical analysis
The log rank-test was used to determine whether the occurrence of diabetes in the transgenic mice was significantly different from the wild type NOD mice. The non-parametric t-test (Mann-Whitney U test) was used to determine whether the blood glucose levels of the diabetic transgenic and wild type NOD mice were significantly different.
Results
Transient transfection of HEP G2 with PEPCK-Ins
To confirm that the PEPCK-Ins transgene (Figure 1A) was functional, 3–4μg of the transgene was transfected into the human hepatoma cell line HEP G2. After the transfection, the cells were incubated in α-MEM supplemented with 10% FBS. Glucagon was added to upregulate the PEPCK promoter expression at a final concentration of 100 nmol/l for 24 hr. At the end of the incubation period, the conditioned culture medium was positive for rodent insulin at a concentration of 0.90 ± 0.04 ng/200,000 HEP G2 cells (n = 8).
Generation of transgenic mice
From a series of two microinjections, 43 mice were obtained of which 4 were positive for the PEPCK-Ins transgene when analysed by PCR (Figure 1B) and Southern blot (Figure 1C). Two of the 4 transgenic founder mice were found to have established germline transmission of the transgene. From the Southern blot analysis, these mice have only a single copy of the transgene (Figure 1C).
Transcription of insulin I mRNA in the liver
Of the two lines only one was found to transcribe insulin mRNA in the liver, as determined by mouse insulin I RT-PCR (Figure 2A) and mouse insulin I Ribonuclease Protection Assay (RPA) (Figure 2B). No insulin mRNA was detected in the second line despite documented germline transmission of the transgene. The transgenic mice appeared healthy and active, and were normoglycaemic. The blood glucose levels were 6.0 ± 1.1 mmol/l (n = 20) as compared to 6.5 ± 0.5 (n = 20) for wild type littermates.
Figure 2 A-B. Mouse insulin 1 mRNA. RT-PCR (A). Lanes 1A-6A are RT-PCR reactions using primers specific for mouse insulin 1 mRNA (30 cycles). Lanes 1B-6B are RT-PCR reactions using primers specific for mouse GAPDH mRNA (30 cycles). Lanes 1–4 are total RNA from the liver of progeny from founder T645-18, lane 5 is total RNA from the liver of progeny from founder T647-2 and lane 6 total RNA from the pancreas of progeny from founder T645-18. RPA for mouse insulin 1 mRNA (B). Lane 1 pancreatic total RNA, lane 2 liver total RNA from T647-2 progeny and lanes 3–7 liver total RNA from T645-18 progeny.
Production of (pro)insulin from liver cells
The ability of the liver cells from PEPCK-Ins positive transgenic mice to produce (pro)insulin was examined on liver removed from mice that had been sacrificed. Ideally production of (pro)insulin from the liver would be sought in vivo. This was not possible since any (pro)insulin released from the liver would be indistinguishable from that secreted from pancreatic β cells. Two methods were used to analyse if (pro)insulin was produced from liver tissue removed from the mice. The liver was both extracted for its hormonal content, and placed in organ culture with analysis of the conditioned culture medium for (pro)insulin. In both cases, (pro)insulin was measurable. The (pro)insulin content was 0.5 ± 1 ng/mg (n = 5), which is equivalent to 8% of the (pro)insulin content of the pancreas (Table 1) on a weight to weight basis. Organ culture of explants in the presence of 100 nM glucagon resulted in release of 3.4 ± 1 ng/mg (pro)insulin per day, which is 23% of that produced by cultured pancreatic explants. No (pro)insulin could be found either in liver extracts or from cultured liver explants taken from wild type NOD mice. These results show that there was translation of (pro)insulin from preproinsulin mRNA in the liver of transgenic mice. The amount of (pro)insulin present in the liver cells was too small for it to be detected immunohistochemically (data not shown).
Table 1 (Pro)insulin content of and release from pancreatic and hepatic tissue of transgenic and wild type NOD mice.
PANCREAS LIVER
F2 progeny (Pro)insulin content (ng/mg) (Pro)insulin secretion (ng/mg tissue/ day) (Pro)insulin content (ng/mg) (Pro)insulin release in the presence of 100 nmol/l glucagon (ng/mg tissue/ day)
Normoglycaemic transgenic (n = 5) 6.3 ± 0.8 15.8 ± 1.6 0.5 ± 0.1 3.4 ± 1.0
Normoglycaemic wild type (n = 3) 6.3 ± 1.0 15.2 ± 2.3 ND ND
Diabetic transgenic (n = 5) 0.5 ± 0.1 (n = 3; 2 ND) ND (n = 3) 0.6 ± 0.2 (n = 5) 3.6 ± 0.3 (n = 3)
ND – not detectable (<0.025 ng/mg tissue)
Data expressed as X ± SD.
In situ hybridisation of (pro)insulin mRNA
In situ hybridisation confirmed the presence of (pro)insulin mRNA in the pancreatic and liver sections of diabetic PEPCK-Ins transgenic mice (Figure 3A and 3C) but not in the liver section of the wild type NOD mice (Figure 3E). The (pro)insulin mRNA was localised to the hepatocytes of the diabetic PEPCK-Ins transgenic mice which appear to be evenly distributed throughout the liver section (Figure 3C). The sense control sections were negative (Figure 3B,3D and 3E).
Figure 3 A-F. In-situ hybridisation. Pancreatic islets from a diabetic transgenic mouse illustrating insulitis, (A) antisense and (B) sense. Transgenic liver from a diabetic transgenic mouse (C) antisense and (D) sense. Wild type liver from a diabetic NOD mouse (E) antisense and (F) sense.
Occurrence of diabetes in transgenic mice
One out of 14 males (7%) and four out of 25 females (16%) of the F1 and F2 progeny of the PEPCK-Ins transgenic NOD mice became diabetic (Figure 4). The age at which this occurred was 20–25 weeks. Among the wild type NOD mice in our colony, 5 males out of 45 (11%) and 22 out of 51 females (43%) became diabetic (Figure 4), at 19–28 weeks of age. The mice were followed for a total of 15 months but no other transgenic mice became diabetic while 55% of the NOD females became diabetic. The incidence of diabetes in the transgenic PEPCK-Ins NOD females (16% diabetic at 30 weeks) differs significantly from the incidence of diabetes in the wild-type NOD females (43% diabetic at 30 weeks) as determined by the log-rank test (chi squared value 16.1, P < 0.001). The overall incidence of diabetes in the transgenic PEPCK-Ins NOD mice (13%) was also significantly lower compared to the wild type (28%) NOD mice as determined by the log-rank test (chi squared value 18.7, P < 0.001). The blood glucose levels of the diabetic transgenic NOD PEPCK-Ins mice (24.8 ± 1.9 mmol/l) were also significantly lower than those of the diabetic wild type NOD mice (>33 ± 2.1 mmol/l) as determined by the Mann Whitney U test, U = 32 and P = 0.004.
Figure 4 Incidence of diabetes in F1 and F2 PEPCK-Ins transgenic NOD mice versus wild type F1 and F2 NOD mice. PEPCK-Ins NOD males total n = 14, PEPCK-Ins NOD females n = 25, wild type NOD males n = 45 and wild type NOD females n = 51.
The livers of the diabetic transgenic mice were examined histologically to determine if there was cellular infiltration and destruction of hepatocytes. This was not so, the liver having a normal architecture with no evidence of necrosis (Figure 5). In contrast, there was a cellular infiltrate in the islets of the diabetic (Figure 6A) but not normoglycaemic transgenic mice (Figure 6B) Immunohistochemical staining for (pro)insulin showed a reduced number of β cells (Figure 6C) compared to normoglycaemic littermates (Figure 6D).
Figure 5 Staining of the liver of diabetic transgenic PEPCK-Ins mice with haematoxylin and eosin. (Black Bar = 20μm).
Figure 6 A-D. Haematoxylin and eosin, and insulin staining of pancreatic sections from transgenic PEPCK-Ins NOD mice. H & E staining of a pancreatic islet from a diabetic transgenic mouse illustrating insulitis (A), and from a normoglycaemic transgenic mouse (B). Insulin staining of a pancreatic islet from a diabetic transgenic mouse showing few remaining β cells (C) and from a normoglycaemic transgenic mice (D). Black Bar = 20μm for A-C, and 40μm for D.
Next, the (pro)insulin content of the liver of the diabetic mice was determined. This was similar to that from the liver of normoglycaemic transgenic mice (Table 1). (Pro)insulin release from cultures of liver cells was measured, and again found to be similar to that from the liver cells of non-diabetic transgenic mice (Table 1). However, as expected, the (pro)insulin content of the pancreas of diabetic transgenic mice was lower than that of normoglycaemic transgenic or wild type mice (Table 1). Indeed, the level was so low as to be comparable to that seen in the liver of either normoglycaemic or diabetic transgenic mice. The amount of (pro)insulin released from pancreatic explants from the diabetic transgenic mice was too low to be detected.
Discussion
At the end of the study, 5 heterozygous transgenic PEPCK-Ins NOD mice had become diabetic. Nevertheless, preproinsulin mRNA was localised to the hepatocytes by in situ hybridisation and was detected in total RNA extracts of the livers of these transgenic mice by both RT-PCR and RPA. (Pro)insulin was detected in acid ethanol extracts of the livers from the transgenic mice by RIA. Furthermore, (pro)insulin was released from explants of transgenic liver placed in organ culture. These results indicate that the liver cells from these transgenic PEPCK-Ins NOD mice did synthesise and produce (pro)insulin.
Histological examination of the livers of the diabetic transgenic mice showed no infiltration of the liver by immune cells even though insulitis was observed in the pancreatic sections. This indicates that hepatic insulin production did not cause the development of tolerance to insulin in these PEPCK-Ins transgenic NOD mice. The lack of infiltration of immune cells in the livers of the diabetic transgenic mice also suggests that the mouse insulin I mRNA transcribing hepatocytes were not targeted or destroyed by the immune system. In addition, as the transgenic mice were sacrificed 4 weeks after the onset of diabetes, this reduces the possibility of delayed destruction of the (pro)insulin expressing hepatocytes. During this time, hepatic insulin expression was at least equal to that found in the pancreas. At the apparently low levels of transgene expression, these results suggest that (pro)insulin is not targeted by the immune system in this transgenic PEPCK-Ins mouse model superimposed on the autoimmune diabetes model of the NOD mouse. Likewise, Lipes et al reported that when pituitary cells in transgenic NOD mice were made to produce insulin, the cells were not targeted by the immune system even though the pancreatic β cells were. No cellular infiltrate also was shown when the pituitary cells that produced (pro)insulin were taken outside the blood brain barrier and transplanted beneath the renal capsule of NOD mice [21]. Our data and those of Lipes et al suggest that autoimmune destruction of (pro)insulin producing cells in the NOD mouse is specific to the islets.
The blood glucose levels of the diabetic transgenic NOD PEPCK-Ins mice (24.8 ± 1.9 mmol/l) were significantly lower (P = 0.004) than those of the diabetic wild type NOD mice (>33 ± 2.1 mmol/l). This is consistent with some (pro)insulin being released from the liver. These results are in agreement with those of Valera et al in which the majority of their high copy number PEPCK human insulin C57BL/6 mice also became diabetic when injected with streptozotocin despite the constitutive release of human (pro)insulin from the livers of the mice (7). Similarly, the transgenic mice which Valera et al made diabetic by streptozotocin injections also had lower blood glucose levels compared to the diabetic wild type NOD mice suggesting that the constitutive hepatic insulin release lowered the blood glucose levels of the diabetic mice but not to normal. This lack of therapeutic effect could be due to the fact that the PEPCK promoter is induced by glucagon and cyclic AMP. The high glucose levels in the diabetic transgenic mice might have resulted in the inhibition of endogenous glucagon release and thus shutting down (pro)insulin production in the liver.
However it is possible that in some of our transgenic mice, enough (pro)insulin was produced to prevent diabetes. This would explain the significantly lower incidence of diabetes in the transgenic PEPCK-Ins (13%) compared to the wild type (28%) NOD mice (P < 0.001). One possibility is a protective effect of insulin, whether by allowing exhausted beta cells to rest or by altering the makeup of the T cells in the pancreas destroying the β cells there. The first reason was the basis for the large North American trial in pre-diabetic people in the late 90's with small doses of parenteral insulin [22]. The second reason was the basis for the large North American trial in pre-diabetic people in the very late 90's with oral insulin [23].
It is possible that a critical amount of (pro)insulin needs to be produced for an autoimmune effect to be observed. The (pro)insulin content of the transgenic liver cells in normoglycaemic mice was much lower than that of a pancreatic β cell and was also lower than that in the liver of PEPCK-human insulin C57BL/6 transgenic mice produced by Valera et al [7]. (Pro)insulin was not detected immunohistochemically in the liver of our PEPCK-Ins transgenic mice, whereas it was in the liver of Valera's transgenic mice. Attempts by us to increase production of (pro)insulin by producing homozygous mice failed, with all mice being stillborn (Figure 1C). This might be because of transient upregulation of the PEPCK gene at birth [24], resulting in increased production of (pro)insulin that caused hypoglycaemia. Alternatively, it could be due to a transgene integration effect where the transgene disrupted a vital gene; with a double copy deletion being lethal.
Another possible explanation for the lack of an autoimmune effect is that presentation of insulin peptides by hepatocytes might be limited by the ability of the liver cells to cleave proinsulin. The enzymes responsible for this in the β cell, prohormone convertases (PC) 1/3 and PC2, are absent from normal liver cells, but the endopeptidase furin is present [25]. Cleavage of proinsulin by furin however would require genetic modification of the peptide [8].
In summary, we have shown that transgenic NOD mice that produce (pro)insulin in their liver do not develop cellular infiltration of their liver when autoimmune destruction of pancreatic β cells occur. Furthermore the expression of (pro)insulin in hepatocytes is insufficient to prevent development of diabetes in NOD mice. These results offer hope that eventually liver cells, or a subpopulation of them, may be of value as a therapy for type 1 diabetes.
Acknowledgements
Funding for this project was obtained from a project grant from the Juvenile Diabetes Research Foundation (JDRF) and from an National Health Medical Research Council – JDRF Special Program Grant (GM). We would like to thank Dr Alan Baxter for constructive comments on the design of the experiments and Ms Lindy Williams for her assistance with the insulin immunohistochemical staining.
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| 15679918 | PMC544947 | CC BY | 2021-01-04 16:37:48 | no | J Autoimmune Dis. 2004 Nov 8; 1:3 | utf-8 | J Autoimmune Dis | 2,004 | 10.1186/1740-2557-1-3 | oa_comm |
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J Neuroengineering RehabilJournal of NeuroEngineering and Rehabilitation1743-0003BioMed Central London 1743-0003-1-11567991510.1186/1743-0003-1-1EditorialJNER: a forum to discuss how neuroscience and biomedical engineering are reshaping physical medicine & rehabilitation Bonato Paolo [email protected] Department of Physical Medicine & Rehabilitation, Harvard Medical School, Spaulding Rehabilitation Hospital, 125 Nashua Street, Boston MA 02114, USA2 The Harvard MIT Division of Health Sciences and Technology, Cambridge, MA, USA2004 13 10 2004 1 1 1 15 9 2004 13 10 2004 Copyright © 2004 Bonato; licensee BioMed Central Ltd.2004Bonato; 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.
Advances in neuroscience and biomedical engineering deeply affect the clinical practice of physical medicine & rehabilitation. New research findings and engineering tools are continuously made available that have the potential of dramatically enhancing the ability of clinicians to design effective rehabilitation interventions. This quickly evolving research field is difficult to track because related literature appears in a wide range of scientific journals. There is a need for a scientific journal that offers to its readership a forum at the intersection of neuroscience, biomedical engineering, and physical medicine & rehabilitation. The Journal of NeuroEngineering and Rehabilitation (JNER) is intended to fill this gap and foster cross-fertilizations among these disciplines. By making readily available to clinicians selected studies with potential impact on physical medicine & rehabilitation, JNER is anticipated to foster the development of novel and more effective rehabilitation strategies. Conversely, by presenting clinical problems to a readership of neuroscientists and engineers, JNER is expected to generate innovative work in neuroscience and biomedical engineering with future applications to physical medicine & rehabilitation. JNER will leverage on Open Access as a means to guarantee that its content is readily available to scientists, clinicians, and the general public thus promoting scientific and technological advances that are relevant to rehabilitation. JNER is an Open Access initiative. Open Access assures dissemination to the widest possible audience and is seen by many as essential for publicly funded research. BioMed Central offers an outstanding platform to make JNER possible and allow neuroscientists, biomedical engineers, and clinicians to see their work published in a timely manner and thus make an immediate impact in the field of rehabilitation. JNER will focus on innovative work with higher likelihood of a dramatic impact on rehabilitation. Thus, priority will be given to outstanding and visionary scientific reports, i.e. those proposing exceptionally innovative concepts with great potential in the field.
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A new journal for a quickly evolving research field
During the past decade, we have witnessed profound changes in physical medicine & rehabilitation originated by advances in neuroscience and biomedical engineering. For example, imaging and neurological assessment methods have dramatically improved the management of patients with motor impairments; robotics and artificial muscle research have generated revolutionary concepts in orthotics and prosthetics; and advances in cortical recordings and the understanding of central nervous system mechanisms have changed the way clinicians look at movement disorders. These techniques and others have brought about, and will continue to give rise in the future to, dramatic advances in physical medicine & rehabilitation.
As advances in neuroscience and biomedical engineering continue to generate new techniques, with tremendous impact in the field of physical medicine & rehabilitation, it becomes apparent that there is an urgent need for establishing an outlet for the intersection of these three research fields. Journal of NeuroEngineering and Rehabilitation (JNER) aims to provide such an outlet, hosting the introduction of new methods and the discussion of their clinical implications, and offering an opportunity to publish, in a timely manner, articles relevant to the cross-fertilization of neuroscience, biomedical engineering, and physical medicine & rehabilitation.
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Open Access will increasingly become an accepted way to disseminate information to the scientific community and the public at large. By becoming part of the movement for Open Access, JNER will contribute to make the latest advances in neuroscience and biomedical engineering, which have the potential to impact on the clinical practice of physical medicine & rehabilitation, readily available to scientists, clinicians, and the general public. Because of its inherent interdisciplinary nature, JNER will foster further advances in the field thanks to the cross-fertilization among science, technology, and clinical practice. Science and technology are expected to offer new tools to design clinical interventions and, vice versa, clinical problems are anticipated to foster basic research in neuroscience and the development of new technologies. Besides, increased awareness of the way science and technology can improve clinical outcomes will lead to better quality of healthcare in rehabilitation. Changes currently occurring in this field are so dramatic that we expect, in a few years, that modernized rehabilitation inpatient and outpatient units will be completely different from what is the state-of-the-art today. For instance, we envision that continuous monitoring of patient status will be performed via miniature, wireless, wearable sensors which not only allow clinicians to monitor vital signs, but also track motor activities and provide a means to analyze motor patterns associated with recovery. Furthermore, robotic devices will be used to enhance physical therapy, ad hoc protocols will be designed for each patient, and augmented and virtual reality tools will enhance rehabilitation by becoming part of routine exercise protocols.
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Open Access to outstanding and visionary scientific reports appears to be a tremendous tool to increase the speed at which clinical practice changes as a result of advances in neuroscience and biomedical engineering. By prioritizing outstanding and visionary publications, JNER intends to provide a forum for ideas and concepts that could make a difference in physical medicine & rehabilitation by innovating the design of clinical interventions.
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The first articles published in the journal demonstrate the commitment of JNER to high quality, prioritizing visionary work, and focusing on research that has the potential of a great impact on physical medicine & rehabilitation. Forthcoming articles will further prove such commitment. Topics of interest to come in the next few months are virtual and augmented reality in rehabilitation, wearable technology in rehabilitation, methods for the analysis of movement, and robotics applied to rehabilitation. These are all topics of great relevance for the research at the intersection of neuroscience, biomedical engineering, and physical medicine & rehabilitation.
Special thanks to the authors of the first articles published in JNER as well as to the authors who submit their manuscripts in the future and support our journal and the Open Access initiative. Members of the editorial board and reviewers have done some excellent work; special thanks to them and the Managing Editor, Sara Midwood, for their contributions to JNER.
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Journal of NeuroEngineering and Rehabilitation, Editorial Board
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PubMed Central
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Velterop J Should scholarly societies embrace Open Access (or is it the kiss of death)? Learned Publishing 2003 16 167 169 10.1087/095315103322110932
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Tan-Torres Edejer T Disseminating health information in developing countries: the role of the internet BMJ 2000 321 797 800 11009519 10.1136/bmj.321.7264.797
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| 15679915 | PMC544948 | CC BY | 2021-01-04 16:37:38 | no | J Neuroengineering Rehabil. 2004 Oct 13; 1:1 | utf-8 | J Neuroeng Rehabil | 2,004 | 10.1186/1743-0003-1-1 | oa_comm |
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J Neuroengineering RehabilJournal of NeuroEngineering and Rehabilitation1743-0003BioMed Central London 1743-0003-1-21567991010.1186/1743-0003-1-2ResearchTime and frequency domain methods for quantifying common modulation of motor unit firing patterns Myers Lance J [email protected] Zeynep [email protected] Madeleine M [email protected] Rehabilitation Institute of Chicago, Sensory Motor Performance Program, 345 East Superior St, Chicago, Illinois, 60611, USA2 Department of Physical Medicine and Rehabilitation, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA2004 14 10 2004 1 2 2 30 8 2004 14 10 2004 Copyright © 2004 Myers et al; licensee BioMed Central Ltd.2004Myers 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 investigations of the human motor system, two approaches are generally employed toward the identification of common modulating drives from motor unit recordings. One is a frequency domain method and uses the coherence function to determine the degree of linear correlation between each frequency component of the signals. The other is a time domain method that has been developed to determine the strength of low frequency common modulations between motor unit spike trains, often referred to in the literature as 'common drive'.
Methods
The relationships between these methods are systematically explored using both mathematical and experimental procedures. A mathematical derivation is presented that shows the theoretical relationship between both time and frequency domain techniques. Multiple recordings from concurrent activities of pairs of motor units are studied and linear regressions are performed between time and frequency domain estimates (for different time domain window sizes) to assess their equivalence.
Results
Analytically, it may be demonstrated that under the theoretical condition of a narrowband point frequency, the two relations are equivalent. However practical situations deviate from this ideal condition. The correlation between the two techniques varies with time domain moving average window length and for window lengths of 200 ms, 400 ms and 800 ms, the r2 regression statistics (p < 0.05) are 0.56, 0.81 and 0.80 respectively.
Conclusions
Although theoretically equivalent and experimentally well correlated there are a number of minor discrepancies between the two techniques that are explored. The time domain technique is preferred for short data segments and is better able to quantify the strength of a broad band drive into a single index. The frequency domain measures are more encompassing, providing a complete description of all oscillatory inputs and are better suited to quantifying narrow ranges of descending input into a single index. In general the physiological question at hand should dictate which technique is best suited.
coherencecommon drivemotor unit dischargedescending drive
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Introduction
Common oscillations in neurophysiological activity in the human motor system have been well documented. During voluntary muscle contraction, the human central nervous system drives motor neurons at a range of frequencies which cause common modulations in the firings of these neurons. These drives are reviewed in [1] and [2] where they are summarized into four broad frequency ranges: (1) A low frequency drive at around 1–3 Hz (2) A neurogenic component of physiological tremor that occurs between 5–12 Hz and is likely to have both spinal and supraspinal components. (3) A corticospinal drive in the beta (15–30 Hz) range (4) A corticospinal drive in the low gamma (30–60 Hz) range, that increases in importance with stronger contractions and is called the Piper rhythm.
There are two distinct approaches toward the identification of these drives. The majority of the literature has examined common modulation to motor units using frequency domain methods. This methodology was first introduced by Rosenberg and colleagues [3] and applied by Farmer and colleagues [4] who used coherence analysis to identify both a significant low frequency and beta-band association between motor unit firings in the 1–12 Hz and 15–30 Hz frequency ranges respectively. Subsequently, coherence analysis has become an established technique to study bivariate motor system measurements and a number of works have used this to investigate corticomuscular interactions [5-8,1]; tremor [9]; aging [10]; oscillatory drive in Parkinson's disease [11,12]; dystonia [13]; stroke [14]; and cortical myoclonus [15].
A separate body of literature has focused specifically on the low frequency common drive. This drive was first identified by De Luca and colleagues [16] who demonstrated that the firing rates of concurrently active motor units (MUs) were modulated in a highly interdependent manner. They low-pass filtered the impulse trains corresponding to MU firing times to obtain the time-varying mean firing rates which they high-pass filtered at 0.75 Hz. They then performed a time domain cross-correlation analysis between pairs of zero-mean signals representing the fluctuations in mean firing rates. Peaks occurring near the zero lag location in the normalized cross correlations implied that those firings rates were essentially simultaneously modulated with virtually no time delay. This phenomenon was termed 'common drive' to indicate a common excitation to the motor neuron pool that results in concurrent fluctuations in the firing rates of motor units from the same pool. A number of subsequent studies have utilized this technique to investigate the relationship of this drive to handedness [17-19]; different proprioceptive conditions [20]; exercise [21]; task and disease [22]; and aging [23]. These works have established this time domain method as an accepted means of quantifying the common low frequency modulation of MU firings.
In a recent review [1], it was suggested that the low frequency common drive first identified by De Luca and colleagues [16] using time domain methods is essentially the same low frequency drive as detected by Farmer and colleagues [4] using frequency domain methods. In this paper we explore the relationship between the two techniques using mathematical and experimental approaches.
Analytic methods
Frequency domain methods: Coherence
The coherence between two zero-mean stationary random processes x1 (t) and x2 (t), at frequency f, is defined as:
where (f) is the cross spectral density and (f) and (f) are the auto spectral density functions of x1 (t) and x2 (t) respectively. The coherence function is a complex quantity and its squared magnitude provides a bounded measure of linear association between the two series, taking on a value of 1 for a perfect linear relationship and a value of 0 to indicate that the series are uncorrelated. In practice, we are often limited to a single time-limited realization of each random process and hence it is necessary to estimate the magnitude squared coherence, , by windowing the time series to obtain multiple sections as follows:
where * denotes complex conjugation, N is the number of data segments employed and X1n (f) and X2n (f) are the discrete Fourier transforms of the nth data segments of x1 (t) and x2 (t). This estimate is biased and its probability density function for non-weighted and non-overlapping windows has been analytically determined [24]. This may be used to derive the value of the estimated coherence, with a particular probability of occurrence, α, that would be obtained when the true value is zero. Any value exceeding this level is considered to be unlikely to be a false indication of coherence with (α × 100) % confidence. This confidence level is given by [24,3]
Eα = 1 - (1 - α)1/(N-1) (3)
The resolution of the coherence estimate is determined from the inverse of the length of the windowed sections, i.e., for a 2 s window, the coherence resolution will be 0.5 Hz. Figure 1 depicts a typical coherence plot computed for the spike trains of two MU's and the associated 95% confidence level. The coherence plots reveal the bandwidth and values of significant coherence for the given resolution. Results from coherence analyses are usually quantified in terms of either the peak value and its frequency or the frequency range of significant coherence. In Figure 1 there is significant coherence between 0.5 and 3.5 Hz and the peak value of coherence is 0.46 and occurs at 1.5 Hz.
Figure 1 Example of a magnitude squared coherence plot. Magnitude coherence between two motor unit spike trains recorded from the FDI muscle. The dashed horizontal line indicates significance at the 95% confidence level. Significant coherence occurs between 0.5 and 3.5 Hz with the peak coherence of 0.46 occurring at 1.5 Hz.
Time domain methods: Cross correlation
The cross correlation between two zero-mean stationary random processes x1 (t) and x2 (t) is defined as:
where E [·] is the estimation operator. Assuming ergodicity, for single time-limited realizations of each random process, this is determined using the integral:
where * denotes complex conjugation and τ is the time lag between the signals. The Fourier transform of the cross correlation function, defines the cross-spectrum, (f). Cross correlation functions are unbounded measures and are typically normalized by the values of the autocorrelations at zero lag to bound the estimate between -1 and 1. The autocorrelation functions are the time domain equivalent of the auto power spectra and their value at zero lag represents the total energy in the signal. The normalized and bounded measure is known as the cross correlation coefficient, , which provides a measure of the linear association between the two signals at a given time lag and is given by:
The original method employed by De Luca and colleagues [16] represents the time series as a binary pulse train with ones corresponding to the firing times of the MUs and zeros comprising the remainder of the signal. A moving average window is then used to smooth these binary signals, which is analogous to filtering the time-series with a low-pass filter. These smoothed signals are depicted in figure 2a. A high-pass filter is then used to remove the mean bias of the signal as 'shown in figure 2b. The filtered signals are subsequently cross correlated and an index obtained from the peak value of the normalized cross correlation function within a specified lag window. Here we term this index the time domain common drive coefficient.
Figure 2 Example of the construction of a low frequency common drive plot. A low-pass, moving average Hanning window filter of length 400 ms was applied to two motor unit spike trains recorded from the FDI muscle. (a) A 5 s epoch of the time-varying smoothed firing rates; (b) the high-pass filtered version of the smoothed firing rates shown in (a); and (c) the low frequency common drive coefficient function between two motor unit spike trains. This results in an effective pass band of 0–5 Hz. The peak of the signal is 0.75 and occurs at a lag of 3.5 ms.
Figure 2c depicts the normalized cross correlation function for the same two motor unit spike trains used in Figure 1 obtained using a moving average Hanning window of 400 ms duration and high pass filtering at 0.75 Hz. The function is displayed for lags up to ± 400 ms and the peak of the signal indicated at a lag of 3.5 ms. The time domain common drive coefficient is measured as 0.75.
Relationship between cross correlation and coherence
The cross correlation coefficient is related to coherence using a similar analysis to Gardner [25] as follows:
We begin with the real and stationary signals x1 (t) and x2 (t), where x2 (t) = αx1 (t - τ0) + n (t) is a scaled and time-delayed version of x1 (t), with additive uncorrelated, zero mean noise, n (t). The cross-correlation function is given by
since the cross correlation with the noise is zero everywhere. The cross spectrum is given by
Assume that the signals y1 (t) and y2 (t) result from passing the signals x1 (t) and x2 (t) respectively through a tunable narrowband bandpass filter with transfer function denoted by H (f):
where and Δ are the center frequency and bandwidth of the ideal bandpass filter. The cross-correlation function between filtered signals y1 (t) and y2 (t) is given by:
where (f) is the cross power spectral density function of y1 (t) and y2 (t).
The cross power spectrum may be written in terms of its magnitude and phase, . Since for stationary, real signals, the autocorrelation is real and even and hence, (f) is real, the phase of the cross spectrum is given by (equation 8):
Thus replacing (f) with |H (f)|2 (f) in equation 10 we get
since is real for real x1 (t) and x2 (t).
Similarly for the autocorrelation functions we get
and
Thus as Δ → 0 we obtain the expression for the normalized cross correlation function as:
The peak of the cross-correlation function occurs at the time delay, τ = τ0. Thus
Thus we see that the peak of the normalized cross-correlation function between two signals after ideally bandpass filtering to contain a single frequency, is identical to the magnitude of the coherence function of the original signals at the frequency of the filter. The phase of the coherence function is the same as the phase of the cross-spectrum and provides the time delay.
For a less ideal filter that spans several frequencies the relationship is less precise and may be derived as follows:
Let W (f) be the new filter transfer function and thus the normalized cross-correlation function is:
where f1 and f2 are the cut-off frequencies of the filter. Thus when multiple frequencies are present, this may be thought of as taking the weighted summation of the cross-correlation functions at each frequency present and normalizing this by the product of the weighted summations of the autocorrelations across all frequencies present. The more narrow band the filter used, the more similar the time domain correlation and frequency domain magnitude coherence measures. As the filter encompasses a greater range of frequencies, measures from the two methods will increasingly deviate.
The low frequency time domain method employed by De Luca and colleagues [16] utilized a moving Hanning window as a low pass filter. The cut-off frequency of the filter is dependent on the time constant of the filter which is typically 400 ms [16,21] but values up to 0.95 s have also been used [26]. However different window lengths will modify the relationship between this time domain measure and the coherence function.
The effect of varying window length may be illustrated by obtaining an expression for the filter transfer function. The equation for the Hanning window is given as:
where τ is the length of the window. The discrete Fourier transform of this is given as (Kay, 1988):
where
Figure 3 depicts the transfer function power spectrum (|W (f)|2) for τ = 200, 400 and 800 ms. The figure clearly demonstrates that as the length of the analysis window decreases, the bandwidth of the filter increases. Therefore the only information that can be ascertained with shorter windows is that the frequency of the common modulating input lies somewhere within the frequency range specified by the window. Longer windows result in a better correlation with coherence values at lower single frequencies (close to zero), while shorter windows lump into a single value a weighted expression of the coherence values in the frequency range which they span.
Figure 3 Magnitude squared spectra of Hanning window filters. Magnitude spectra of the transfer functions of Hanning window filters for three different time constants, τ = 200 ms (dotted line), τ = 400 ms (dashed line) and τ = 800 ms (solid line). As the time constant increases the bandwidth of the filter decreases and its magnitude increases.
Experimental methods
In this section we demonstrate the relationship between time and frequency domain based methods to estimate the common modulating drive using empirical data. The methods are applied to data collected during isometric contractions of the First Dorsal Interosseous muscle at 20% of maximal effort. Two contractions where the activities of 4 and 5 MUs were identified yielded a total of 16 pairs of coactive MUs. The periods of concurrent activity of these MU pairs ranged between 30 s to 1 minute and were further divided into pairs of non-overlapping 10 s intervals resulting in a total of 50 pairs of 10 s long spike trains. Each method was applied to these spike train pairs and the correlation between the results yielded by the two methods were investigated as discussed below.
The time domain method was used to estimate low frequency common drive according to the method described by De Luca and colleagues [16]. Three different time domain estimates were formed by smoothing the spike trains using Hanning windows of length 200, 400 and 800 ms respectively. These smoothed firing rate signals were then digitally high pass filtered with a low frequency cut-off of 0.75 Hz using a third order Butterworth filter to remove the mean bias discharge rates. The cross correlation coefficient function of these high pass filtered records was then obtained and the peak value of this function within ± 50 ms of the zero time lag was recorded and termed the time domain common drive coefficient.
The coherence analyses were performed in a similar manner to the procedure of Rosenberg and colleagues [3] for point process data. The spike trains were represented as binary pulse trains with ones corresponding to the firing times of the MU's and zeros comprising the remainder of the signal. Fourier transforms of these trains were obtained for each appropriately windowed section and then averaged according to equation (2). However where Rosenberg and colleagues [3] do not use overlapping or tapered data windows, we used overlapping, tapered Hanning windows of 2048 ms to optimize the variance and bias of the estimate. With any non-parametric spectral estimation technique, there is a trade-off between the variance and both the bias and resolution of the estimation. A window size of 2048 ms, gives a frequency resolution of 0.49 Hz, which is adequate to discriminate frequencies for our purposes. However, when analyzing 10 s of data using 2048 ms non-overlapping windows, only 5 different records are available and this small number of records will increase the variance of the estimate. Furthermore rectangular windows introduce an estimation bias due to the effect of their sidelobes. These concerns may be reduced by using the Welch periodogram method which uses tapered windows (to reduce spectral leakage and therefore the estimation bias) and overlapping windows (to increase the total number of windows and hence reduce the variance). The minimum variance for this method is obtained using an overlap of 62.5% [27]. The frequency corresponding to the first zero-crossing of the Hanning filter was obtained according to equation (20) and the peak value of the coherence in the range between 0.75 Hz and this frequency was recorded.
A linear regression between the time domain common drive coefficients and corresponding frequency domain peak coherence values was performed to determine whether a linear relationship between the two indices existed. The regression r2 values are reported at a significance level of p < 0.05.
Results and discussion
Figure 4a,4b,4c displays the regression between the low frequency time domain common drive coefficients for Hanning windows of length 200, 400 and 800 ms and peak low frequency coherence. All regressions are significant at p < 0.05 and the r2 statistics are 0.56, 0.81 and 0.80 respectively. A unitary slope line is displayed in the figure and this describes the theoretical relationship between the two indices. These results indicate that for the larger 400 ms and 800 ms windows, the time domain method is more closely correlated with the coherence estimate, with the 400 ms window yielding a marginally better fit. The data for the smaller 200 ms window exhibits a consistent bias, with the coherence estimate larger than the time domain common drive estimate, whilst the 400 ms and 800 ms windowed data are more evenly distributed around the unitary slope line, indicating less bias. There are a number of possible factors that could contribute to the observed mismatches between the two methods.
Figure 4 Regression plots for low frequency common drive time versus frequency domain techniques. Regression plots for low frequency common drive time versus frequency domain techniques. Three different moving average Hanning windows were used to low pass filter the time series for the time domain method. The time constants for the filters are as follows: (a) τ = 200 ms, (b) τ = 400 ms, (c) τ = 800 ms. All regressions are significant at p < 0.05 and the r2 statistics are (a) 0.56, (b) 0.81 and (c) 0.80. The unitary slop line is indicated in the figures as a dashed line and represents the ideal mathematical relationship.
As demonstrated in Figure 2, the cross-correlation peak can occur at lags slightly different than zero. A time delay or misalignment has been shown to introduce a bias into the coherence estimate that is proportional to the delay and coherence magnitude and inversely proportional to the FFT epoch duration [24]. However for delays of the order of magnitude of ± 50 ms and FFT lengths of approximately 2 s, this type of bias is very small and unlikely to account for the observed differences between the time and frequency domain estimates.
The use of a short duration window in the time domain method results in the inclusion of multiple frequencies in the time domain correlation estimation according to equation (18). The bandwidths of descending oscillatory drives may be variable. Thus when the descending drive occupies a narrow bandwidth and the time domain window includes a greater range of frequencies than this bandwidth, this will bias the time domain estimate to be lower than the peak coherence value as is the case in figure 4a. Alternatively should the drive span a broader bandwidth, the time domain measure would encompass all the correlated frequencies into a single value and would thus be different than the value obtained from any single peak coherence frequency. This idea is illustrated in Figure 5 where a typical coherence plot is displayed. Superimposed on this are vertical lines representing various moving average filter cut-off frequencies. The 0.75 Hz high pass cut-off frequency is also displayed. Thus from the figure we see that in this case the coherence occupies a fairly broad bandwidth from 1–5 Hz, peaking at 1.5 Hz. The cut-off frequency of the 200 ms filter is approximately 10 Hz and thus the time domain estimate will include coherence values at all these frequencies which would make it significantly different from the peak coherence. The 400 ms and 800 ms windows would better correlate with the peak coherence frequencies and the 400 ms window would provide a better overall index encompassing the full bandwidth of the drive. However, if the middle peak at 8 Hz were stronger and actually the main peak, the wider time windows would miss it altogether. This emphasizes the importance of a priori knowledge in choosing the appropriate time windows in the time-domain based method. Therefore in summary, the time domain measure is more effective in quantifying a range of frequencies into a single index and the peak coherence estimate is better at representing the coherence at any single frequency.
Figure 5 Magnitude squared coherence between two motor unit spike trains recorded from the FDI muscle. Magnitude coherence between two motor unit spike trains recorded from the FDI muscle. The vertical dotted lines from left to right represent the cut-off frequencies of the 0.75 high-pass filter, and the 800 ms, 400 ms and 200 ms moving average low-pass filters. The peak coherence occurs at 1.5 Hz.
Coherence estimates are typically formed from data records of around 1–5 minutes in length [4,28,29] or from pooled coherence measures of repeated trial measurements [30]. This increases the number of non-overlapping windows in the calculation, thereby reducing the variance of the coherence estimate. Non-overlapping, rectangular windows are traditionally preferred due to the clear relationship with significance levels. Overlapping, tapered windows will allow coherence to be estimated from shorter data segments and parametric techniques, in particular multivariate autoregressive (MAR) methods are suggested for the analysis of very short duration data segments [31]. When using short records of data (<5 s), the coherence estimates are likely to be significantly biased. However, the time domain method is more robust for such short data lengths and would therefore be preferred in these situations.
The time domain method uses a high pass filter to remove the mean bias from the smoothed signals, whereas the frequency domain coherence method simply subtracts the mean component of the signal prior to forming the estimate. Although similar, these two methods are not identical and may further explain some of the variation between the time and frequency domain techniques. A further possibility is to employ a low order polynomial detrending technique instead of high pass filtering or subtracting the mean. In general, a visual examination of the smoothed firing rate signals would indicate whether this would be necessary.
It is straight forward to quantify any time delay using the time domain technique. Although this is also possible with the frequency domain technique, this delay information is incorporated in the phase of the estimate and is therefore 2π periodic and would thus yield the same result for integer multiples of delay. For significant coherence present over a band of frequencies, Mima and colleagues [32] suggest a constant phase shift plus constant time delay regression model to compute time delays from coherence estimates. However for narrow band descending drives, the time domain technique provides a clearer estimate of any time delay.
It should be noted that the time domain technique may be generalized to cover any arbitrary frequency. This would necessitate bandpass filtering the signals to the frequency range of interest, removing the mean trends and then evaluating the cross-correlation function. Although this requires a priori knowledge of the drive bandwidth, this method would then be able to provide a single index to quantify a particular bandwidth.
Conclusions
Two separate bodies of literature offer techniques to estimate band limited common oscillatory input to motor neurons. These techniques are based in either the time or in the frequency domain. Indices derived from both measurement techniques are well correlated with each other and in the theoretical limit, the techniques are shown to be mathematically equivalent. However, for practical purposes there are a number of minor discrepancies which may favour the use of one particular method for a given application.
The time domain method offers greater resolution in time (the latency of the correlations are easily revealed) at the expense of the requirement of a priori knowledge of the bandwidth of the common modulating drive. On the other hand the frequency domain technique reveals information regarding the frequency distribution of the common modulating drives but it is more difficult to obtain accurate estimates of the coherence with short signal lengths as well as of the exact delay.
Time domain methods of estimation are preferred for short data segments and are well suited to quantifying the strength of a broad band drive into a single index. This proves useful in quantitative, comparitive analyses of the common behavior of MUs such as statistical tests that investigate the effects of aging or disease. Frequency domain measures tend to be more encompassing as they provide a complete description of all common oscillatory inputs. This facilitates qualitative analysis of distribution of coherence across frequencies and hence leads to a better understanding of the nature of the common inputs. They are well suited for estimation from large data segments, that may be assumed to be stationary, and are better able to quantify narrow ranges of descending inputs into a single index. Thus the selection of one technique over another should be dictated by the nature of the physiological question to be addressed.
Acknowledgements
The authors gratefully acknowledge the financial support of the Falk Medical Research Trust.
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| 15679910 | PMC544949 | CC BY | 2021-01-04 16:37:38 | no | J Neuroengineering Rehabil. 2004 Oct 14; 1:2 | utf-8 | J Neuroeng Rehabil | 2,004 | 10.1186/1743-0003-1-2 | oa_comm |
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J Neuroengineering RehabilJournal of NeuroEngineering and Rehabilitation1743-0003BioMed Central London 1743-0003-1-31567991210.1186/1743-0003-1-3ResearchThumb force deficit after lower median nerve block Li Zong-Ming [email protected] Daniel A [email protected] Robert J [email protected] Hand Research Laboratory, Departments of Orthopaedic Surgery and Bioengineering, University of Pittsburgh, PA 15213 USA2004 19 10 2004 1 3 3 30 8 2004 19 10 2004 Copyright © 2004 Li et al; licensee BioMed Central Ltd.2004Li 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.
Purpose
The purpose of this study was to characterize thumb motor dysfunction resulting from simulated lower median nerve lesions at the wrist.
Methods
Bupivacaine hydrochloride was injected into the carpal tunnel of six healthy subjects to locally anesthetize the median nerve. Motor function was subsequently evaluated by measuring maximal force production in all directions within the transverse plane perpendicular to the longitudinal axis of the thumb. Force envelopes were constructed using these measured multidirectional forces.
Results
Blockage of the median nerve resulted in decreased force magnitudes and thus smaller force envelopes. The average force decrease around the force envelope was 27.9%. A maximum decrease of 42.4% occurred in a direction combining abduction and slight flexion, while a minimum decrease of 10.5% occurred in a direction combining adduction and slight flexion. Relative decreases in adduction, extension, abduction, and flexion were 17.3%, 21.2%, 41.2% and 33.5%, respectively. Areas enclosed by pre- and post-block force envelopes were 20628 ± 7747 N.N, and 10700 ± 4474 N.N, respectively, representing an average decrease of 48.1%. Relative decreases in the adduction, extension, abduction, and flexion quadrant areas were 31.5%, 42.3%, 60.9%, and 52.3%, respectively.
Conclusion
Lower median nerve lesion, simulated by a nerve block at the wrist, compromise normal motor function of the thumb. A median nerve block results in force deficits in all directions, with the most severe impairment in abduction and flexion. From our results, such a means of motor function assessment can potentially be applied to functionally evaluate peripheral neuropathies.
ThumbHandForceMedian nerve block
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Introduction
The thumb has unique anatomical and biomechanical characteristics that are required to perform many manipulative tasks. Thumb motor dysfunction resulting from neuromuscular and musculoskeletal pathologies severely hinders the performance of these daily tasks. Clinical treatment, prevention protocols, and rehabilitation efficacy requires a thorough understanding of thumb motor capabilities, as well as its associated functional deficit. Investigations of underlying pathological mechanism of the thumb help advance clinical treatments such as tendon transfers [1], functional electrical stimulation [2] and plasticity suppression [3].
Measurement of strength during maximum voluntary contraction is a simple and direct means of assessing neuromuscular function. Popular instruments used for quantitative assessment of thumb strength are pinch dynamometers. The pinch output, however, provides limited information about thumb motor function in that it offers a single generic force in one specific direction. Each muscle/tendon within the thumb has a distinct anatomical origin and insertion, suggesting its external force potential in a particular direction [4-6]. Hence, evaluation of strengths in multiple directions offers insight concerning the motor capacity of individual muscles. Force production of a digit has been measured in various directions such as flexion/extension [7,8], abduction/adduction [9-14], or in combined directions [15,16]. Bourbonnais et al. developed an apparatus to measure thumb force production in eight directions in the transverse plane of the thumb and investigated force dependence on the direction of effort [15]. Yokogawa and Hara measured index fingertip forces in various directions within the flexion/extension plane [8]. Recently, we developed experimental apparatuses to measure multi-directional forces of a digit in its transverse plane [17-19]. From these multi-directional forces we constructed force envelopes representative of the characteristic force output pattern of a digit [17-19].
Disorders resulting from traumatic injuries to and various diseases of these nerves are common in clinical practice. Clinical manifestations of hand dysfunction are distinctive depending on the nerve involved. For example, thenar atrophy is a major clinical observation affecting thumb function at the later stages of compression neuropathy of the median nerve. Several studies have been conducted to investigate the effects of simulated peripheral neuropathies using local anesthetization [5,16,20,21]. Kozin et al. [21] studied the effects of median and ulnar nerve blocks on grip and pinch strength and showed significant decreases following nerve blockage [21]. Boatright and Kiebzak [20] investigated the effects of median nerve block on thumb abduction strength. Kaufman et al. [5] measured isometric thumb forces in eight directions together with electromyographic signals of thumb muscles after block of the median nerve. Labosky and Waggy [22] studied the strength related to grip, pinch, thumb adduction, thumb abduction, and finger flexion after radial nerve block [22]. Kuxhaus studied the three dimensional feasible force set at the thumb-tip before and after ulnar nerve block and reported this to be a reproducible and sensitive means to detect impairment.
The purpose of this study was to utilize our developed apparatus and protocols to investigate the effects of lower median nerve lesion on thumb motor function. The lesion was simulated by blocking the median nerve at the wrist using an anesthetic. We hypothesized that a median nerve block would cause (1) a decrease in force production, which would be direction-dependent with the most severe reduction in the abduction direction, and (2) a decrease in the force envelope area and force quadrant area, with the greatest decrease in the abduction quadrant.
Methods
Subjects
Six healthy male subjects (mean age: 26.9 ± 5.1 years) participated in this study. The subjects had no previous history of neuromuscular or musculoskeletal disorders of the upper extremities. Each subject signed an informed consent form approved by the Institutional Review Board prior to participating in the experiment.
Median nerve block
Injections were performed under aseptic conditions while the subjects sat with the forearm supinated and the wrist slightly extended. After the skin at the palmer area of the wrist was cleaned with alcohol, 4 mL of 0.5% bupivacaine hydrochloride (Astra Pharmaceuticals, Westborough, MA, USA) was injected into the carpal tunnel with a sterile 25-gauge short-bevel needle. The needle was inserted through the transverse carpal ligament in line with the radial border of the fourth digit slightly ulnar to the palmaris longus tendon at the level of the distal wrist crease. Forty minutes was allowed for the median nerve block to reach complete effectiveness [23] and was verified using the Semmes-Weinstein monofilament test. The average monofilament score was 2.85 across the five digits before nerve block. About 40 minutes after nerve injection, little sensory impairment occurred in the ulnar distribution (score = 3.22), while the sensory score in the median distribution was greater than 6.15. The effects of nerve block lasted more than 6 hours with all subjects regaining normal hand function within 12 hours.
Testing apparatus
The experimental apparatus was designed and constructed to measure maximum voluntary contraction forces of any digit at any point along the digit. Force application was possible in any direction within the transverse plane of the longitudinal axis of the digit. The apparatus consisted of position control accessories, a force transducer, and a custom fitted aluminum ring attached to the transducer (Figure 1A,1B). The transducer (Mini40, ATI Industrial Automation, NC, USA), capable of measuring 6 degrees of freedom forces and moments, was attached to a mounting clamp via an aluminum adapter plate while the aluminum ring was secured to the tool side of the transducer using a custom adapter. The ring served as a connection anchor for the transducer and the digit. The force transducer and ring attachment were positioned in a desired orientation using an aluminum slide rail, tubing, and lockable mounting clamps (80/20 Inc., Columbia City, IN, USA). The slide rail was secured to an aluminum base plate. Foam padded wooden blocks with two locking straps secured the arm to the base plate.
Figure 1 Schematic of experimental setup to measure thumb force production in the transverse plane. (A) 3D view. (B) Side view with hand and thumb in place. Thumb extension and flexion occur in parallel with the palm, and abduction and adduction occur in a plane perpendicular to the palm with abduction moves away from the palm. (C) Visual guide for circumferential force production.
The analog outputs from the transducer were digitized using a 16-bit analog-to-digital converter (PCI-6031, National Instruments, TX, USA). The X (abduction/adduction) and Y (flexion/extension) force components in the transverse plane were displayed on the screen while the subject performed a force production task. The resolutions of the force transducer in its axial (flexion/extension) and horizontal (abduction/adduction) directions were 0.16 N and 0.08 N, respectively. A personal computer equipped with LabVIEW (National Instrument, TX, USA) was used for force data acquisition, display, and processing.
Experimental procedures
Each subject was tested before and after median nerve block. The nerve block procedures were performed immediately after the completion of the first testing session. Post-block testing started after the verification of complete median nerve block, approximately 40 minutes after the injection. During each test, the subject was seated in a chair adjacent to the testing station modified with a wooden board to align their back vertically throughout the trials. The subjects rested their forearm on padded wooden blocks positioning their shoulder in approximately 60° of frontal plane abduction. Nylon straps fitted with plastic snap locking mechanisms secured the forearm and minimized the intervention of the elbow and shoulder during thumb force application. Subjects grasped a vertical dowel secured to the distal end of the wooden blocks in a midprone position. Formable thermoplastic braces were used to fix the elbow in 90° of flexion, and the wrist in 20° of extension and 0° of ulnar deviation. A metallic brace was used to fix the interphalangeal joint of the thumb in full extension. The aluminum ring was placed around the middle of the proximal phalanx and oriented to accommodate comfortable thumb position with the metacarpophalangeal joint flexed approximately 15°. Prior to testing, a line was drawn on the proximal phalange at the midpoint between the interphalangeal and metacarpophalangeal joints. The alignment of the ring with the circumferential line standardized the location of force application within and between subjects. As force application was at the middle of the proximal phalanx, mechanical action pertains to both the metacarpophalangeal and carpometacarpal joints. We chose the terminology of flexion/extension and adduction/adduction based on the mechanical action with respect to the metacarpophalangeal joint. With the thumb in the ring (Figure 1B), extension and flexion occurred in parallel with the palm, and abduction and adduction occurred in a plane perpendicular to the palm.
Each subject performed 15 circumferential MVC trials with randomized starting directions (Figure 1C). The subject was allotted 15 seconds to complete each circumferential trial, and was instructed to use the entire time allotted to traverse the perimeter of the ring once. A dot generated on the computer screen was programmed to traverse a circle within 15 seconds to provide the subject with directional feedback of their force application. Subjects were given 60 seconds of rest between each circumferential trial. Each subject was familiarized with the task with a few practice trials. Data were collected from each subject at 100 samples per second producing a total of 22,500 pairs of force components from the 15 circumferential trials. Our previous study [19] indicated that the testing protocol did not cause noticeable fatigue.
Force envelope and quadrants
Data from multiple circumferential trials were accumulated to construct a force envelope. The procedures to generate a force envelope were as follows:
Cartesian force coordinates (Xi, Yi) were transformed into polar coordinates (Rα, α), where Rα was the force magnitude at an angular position α. Each α was rounded to the nearest integer ranging from 0 to 359 degrees.
The maximum, Fα, was determined from a string of N data points along each radial line defined by α. At the completion of the 15 trials, there were, on average, N = 63 data points on each radial line of α based on the distribution off the 22,500 data points around 360°.
A moving average with an interval of 10° was applied to the maximal series data Fα (α = 0, 1, 2,..., 359) to obtain filtered maximal forces. These forces formed a force envelope.
The area formed by a force envelope was divided into adduction-extension, extension-abduction, abduction-flexion, and flexion-adduction quadrants by radial lines oriented at 0°, 90°, 180°, and 270° A quadrant force was represented using the mean magnitude of the forces in that quadrant. The areas of the entire envelope and each quadrant were calculated by summing the areas of individual arc sections formed by the polar coordinates of the force envelope. (Figure 2).
Figure 2 Division of force envelope into extension, abduction, flexion, and adduction quadrants.
Statistical Analyses
One- and two-factor repeated measures analyses of variance (ANOVA) were used to analyze outcome measures. The independent variables were testing SESSION (n = 2, i.e., pre- and post-block), force DIRECTION (n = 16), and force QUADRANT (n = 4), with SESSION as a repeated variable. Dependent variables were directional force, individual quadrant area and force envelope area. Statistical analyses were performed using SPSS 11 (SPSS Inc., Illinois) with statistical significance set at α = 0.05.
Results
Force envelope and directional forces
Figure 3 shows the force envelopes produced by each subject (A to F) before and after median nerve block. The post-block force envelope was inside the pre-block envelope for each subject, indicating a decrease in force magnitude in all directions after nerve block. Figure 4 shows the average pre- and post-block force envelope across all subjects. Force magnitudes were significantly reduced after nerve block (p < 0.001) resulting in significantly smaller force envelopes. The average decrease across all directions was 27.9%. A maximum decrease of 42.4% occurred at 199°, corresponding to a combined direction of abduction and slight flexion, while a minimum decrease of 10.5% occurred at 328° corresponding to a combined direction of adduction and slight flexion. Relative decreases at 0° (adduction), 90° (extension), 180° (abduction), and 270° (flexion) directions were 17.3%, 21.2%, 41.2% and 33.5%, respectively.
Figure 3 Force envelopes before and after median nerve block of subjects A, B, C, D, E, and F. For each subject, the inner envelope represents post-block results.
Figure 4 Average force envelopes produced by the thumb before and after median nerve block.
A single force in each quadrant was represented using the mean magnitude of the forces in that quadrant (see description in the Methods). The average quadrant forces were significantly decreased after nerve block (p < 0.001; Figure 5). The amount of decrease was also different between quadrants (p < 0.005). Relative decreases in mean quadrant forces were 24.5%, 38.7%, 32.1%, and 18.1% for extension, abduction, flexion, and adduction, respectively. The maximal decreases in mean quadrant force, 38.7%, occurred in the abduction quadrant.
Figure 5 Average force magnitude, N, in individual quadrants. The percentage values denote the percent decreases of post-block forces relative to pre-block forces.
Force envelope areas and quadrant areas
Areas enclosed by the post-block envelopes were significantly smaller than the pre-block envelopes (p < 0.001; Figure 4). Post-block force envelope area, 10700 ± 4474 N.N, was 51.9% of pre-block force envelope area, 20628 ± 7747 N.N. Quadrant area decreased significantly (p < 0.001; Figure 6). The maximal percentage decrease in area after nerve block was 60.9% in the abduction quadrant, followed by a 52.3% area decrease in the flexion quadrant.
Figure 6 Area (N-N) of individual force quadrants, and percentage decrease after nerve block. The percentage values denote the percent decrease of post-block quadrant areas.
Discussion
In this study we simulated a lower median nerve lesion and evaluated the resultant thumb motor function deficit. Our internal control via pre- and post-block design offered a particular advantage of investigating the mechanical role of muscles innervated by a targeted nerve. The testing and analytical methods employed have provided advanced quantification of thumb motor function. The results have confirmed our initial hypotheses that greatest force decreases occurred in directions related to abduction, and that the post-block thumb force envelope area was smaller than the pre-block force envelope area.
Preferential force attenuation in the quadrants of abduction and flexion after median nerve block are in agreement with anatomical and neuromuscular features of the thumb. The median nerve innervates the abductor pollicis brevis, the opponens pollicis and superficial head of the flexor pollicis brevis, all of which contribute to the abduction and flexion of the thumb [4]; therefore, denervation of these muscles after median nerve block would cause the greatest force deficit related to median nerve function [5]. Additionally, as force application moved towards adduction, the force deficit decreased as neuromuscular control shifted from the median nerve to the ulnar nerve via the first dorsal interosseous and adductor pollicis brevis. Force deficit in extension was also comparably small as extension forces are mainly produced by the extensors pollicis brevis and longus originating in the forearm.
Our reported force decreases following a median nerve block (40.9% in abduction, 34.1% in flexion) were smaller than those reported in the literature. Kozin et al. [21] reported a 60% decrease in pinch strength after a median nerve block using mepivicaine hydrochloride [21]. Boatright and Kiebzak [20] reported an approximate 70% decrease in thumb abduction strength after median nerve block using Lidocaine [20]. Kaufman et al. [5] stated that a median nerve block with Lidocaine almost completely diminished force production in the abduction direction [5]. The discrepancy may be due to the anesthetic used and strength testing method. Although the sensory block appeared to be complete for each method, the motor capabilities of the muscles associated with the median nerve might or might not be completely eliminated. Such a result is largely dependent on a particular anesthetic, its concentration and dosage, as well as the efficacy of the injection technique at immersing the nerve. The methods of strength testing may also help explain the different magnitudes of strength deficit after the nerve block. All previous results were based on forces obtained in discrete direction(s), and focused exertions, while the current study utilized a method of force production in a continuous, circumferential and dynamic manner. Furthermore, thumb motor performance can be maintained despite the absence of certain individual muscles. For example, Britto and Elliot reported that the loss of abductor pollicis longus and extensor pollicis brevis in their two patients did not show functional compromise of strength and grip strength [24]. In a broader sense, the neuromuscular system has remarkable capabilities to accomplish the same motor function goal using different effectors and different goals using the same effectors, a phenomenon so called "motor equivalence" [25].
An unexpected finding from this study was that the force deficit occurred in all directions (Figure 4). In other words, the median nerve block caused reduced force production by those muscles not associated with the median nerve. Several potential explanations exist to describe such a phenomenon. First, the injection into the carpal tunnel at the wrist, although localized, potentially diffused into the intrinsic fascia of the hand partially compromising function of the ulnar nerve, which innervates the adductor pollicis. Although Semmes-Weinstein monofilament testing confirmed the continued sensation of the digits within the ulnar nerve distribution, it is not inconceivable that the injection could have contaminated the ulnar innervated muscles, the first dorsal interosseous and deep head of the flexor pollicis brevis [20]. Secondly, thumb force in any direction is produced by synergistic activation of the many intrinsic muscles, and as a result, the muscular deficiency associated with one direction may hinder the force production in other directions by other muscles [5,22]. For example, Kaufman et al. demonstrated that thumb muscles not innervated by the median nerve displayed lower electromyographical activation and shifted the direction of maximum activation after a median nerve block [5]. Labosky and Waggy showed that a radial nerve block caused a 53% decrease in thumb abduction strength because of the lack of stabilization of the radial innervated extensor muscles [22]. Consequently, deficiency of median innervated muscles inherently limits force production in other directions as neuromuscular switching is necessary to produce force in changing directions.
The median innervated muscles are the dominant abductors of the thumb metacarpophalangeal and carpometacarpal joint. The more than 50% residual abduction force found in this study suggests that the injection did not totally block the motor function of these muscles, even though a complete sensory loss was verified. This concurs with clinical observations of median compression neuropathy. Individuals with carpal tunnel syndrome complain of sensory dysfunction early in the disease process (at the beginning), while motor signs of thenar wasting and thumb weakness occur as the disease advances. The concept that the motor deficit is more resistant to peripheral median neuropathy than sensory loss has been well documented [23,26,27]. Butterworth et al. studied the temporal effects on sensory and motor blockade after injection of bupivacaine or mepivacaine, and found that sensory loss was complete but about a 20% compound motor action potential remained after 40 minutes [23].
In conclusion, we have incorporated a method for assessing thumb motor deficit based on strength measurement with a standard local anesthetic to investigate the effects of a simulated median neuropathy on thumb motor function. Median nerve block results in force deficits in all directions, with the most severe impairment in abduction and flexion. Future endeavors using this methodology can potentially further elucidate underlying pathomechanisms of peripheral neuropathies in all digits of the hand.
Acknowledgements
This work was partially supported by the Aircast Foundation and the Whitaker Foundation. The authors thank Robert A. Kaufmann for helping perform anesthetic injections.
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| 15679912 | PMC544950 | CC BY | 2021-01-04 16:37:38 | no | J Neuroengineering Rehabil. 2004 Oct 19; 1:3 | utf-8 | J Neuroeng Rehabil | 2,004 | 10.1186/1743-0003-1-3 | oa_comm |
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J Neuroengineering RehabilJournal of NeuroEngineering and Rehabilitation1743-0003BioMed Central London 1743-0003-1-41567991310.1186/1743-0003-1-4ResearchInfluence of passive leg movements on blood circulation on the tilt table in healthy adults Czell David [email protected] Reinhard [email protected] Rüdiger [email protected] Stephen [email protected] Gery [email protected] Volker [email protected] Spinal Cord Injury Center, Balgrist University Hospital, Zurich, Switzerland2 Orthopaedic Hospital of Heidelberg University, Department II, Heidelberg, Germany3 Hocoma AG, Medical engineering, Volketswil, Switzerland2004 25 10 2004 1 4 4 30 8 2004 25 10 2004 Copyright © 2004 Czell et al; licensee BioMed Central Ltd.2004Czell 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
One problem in the mobilization of patients with neurological diseases, such as spinal cord injury, is the circulatory collapse that occurs while changing from supine to vertical position because of the missing venous pump due to paralyzed leg muscles. Therefore, a tilt table with integrated stepping device (tilt stepper) was developed, which allows passive stepping movements for performing locomotion training in an early state of rehabilitation. The aim of this pilot study was to investigate if passive stepping and cycling movements of the legs during tilt table training could stabilize blood circulation and prevent neurally-mediated syncope in healthy young adults.
Methods
In the first experiment, healthy subjects were tested on a traditional tilt table. Subjects who had a syncope or near-syncope in this condition underwent a second trial on the tilt stepper. In the second experiment, a group of healthy subjects was investigated on a traditional tilt table, the second group on the tilt ergometer, a device that allows cycling movements during tilt table training. We used the chi-square test to compare the occurrence of near-syncope/syncope in both groups (tilt table/tilt stepper and tilt table/tilt ergometer) and ANOVA to compare the blood pressure and heart rate between the groups at the four time intervals (supine, at 2 minutes, at 6 minutes and end of head-up tilt).
Results
Separate chi-square tests performed for each experiment showed significant differences in the occurrence of near syncope or syncope based on the device used. Comparison of the two groups (tilt stepper/ tilt table) in experiment one (ANOVA) showed that blood pressure was significantly higher at the end of head-up tilt on the tilt stepper and on the tilt table there was a greater increase in heart rate (2 minutes after head-up tilt). Comparison of the two groups (tilt ergometer/tilt table) in experiment 2 (ANOVA) showed that blood pressure was significantly higher on the tilt ergometer at the end of head-up tilt and on the tilt table the increase in heart rate was significantly larger (at 6 min and end of head-up tilt).
Conclusions
Stabilization of blood circulation and prevention of benign syncope can be achieved by passive leg movement during a tilt table test in healthy adults.
tilt steppertilt tableblood circulationsyncopenear-syncopevertical mobilization
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Background
Several studies have confirmed that lack of movement leads quickly to profound negative physiological and biochemical changes in all organs and systems of the body [1-5]. It is important for patients suffering from diseases such as stroke, spinal cord and traumatic brain injury to be mobilized at an early state of rehabilitation [6]. As these patients are bedridden, their lower limbs are mainly mobilized through manual therapy or with cycling ergometers. Patients with spinal cord injuries are disposed to the occurrence of circulatory collapse when changing from a horizontal to a vertical position because of the lack of sympathetic activity and the missing contractions of leg muscles in the lower extremities that normally act as muscle pumps [7,8]. This instability of the circulatory system occurs at an early stage of rehabilitation and leads to delayed functional training of these patients. In a chronic phase, an overactivity of the spinal sympathetic system could take place, which can lead to vasoconstriction and hypertension [9].
Head-up tilt table testing has been used for over 50 years by physiologists and physicians for many purposes. This includes the study of the human body's heart rate and blood pressure adaptations to changes in position, for modeling responses to hemorrhage, as a technique for evaluating of orthostatic hypotension, as a method to study hemodynamic and neuroendocrine responses in congestive heart failure, autonomic dysfunction and hypertension, as well as a tool for drug research [7,10-14]. It also has become a useful device in the mobilization of spinal cord and traumatic brain injured patients, as well as in patients suffering from stroke [15]. The key feature of a tilt table is the continuously adjustable position of a patient from horizontal to vertical. This represents an orthostatic challenge, because blood pools in the lower extremities, with the danger that in susceptible individuals vasovagal syncope could occur within approximately 20 minutes. The afferent end of this reflex pathway may be mediated by left ventricular or right atrium mechanoreceptors that are activated during vigorous contraction around under-filled chambers, in a situation similar to severe hemorrhage. Information from these mechano-receptors travels along vagal afferent C fibers to the brainstem, which mediates the efferent response consisting of withdrawal of sympathetic vasomotor tone and by the vagal system [16,17].
In addition to the traditional tilt table, a novel apparatus with stepping device (tilt stepper) was developed in 1998 at the research department of the Paraplegia Centre of the Balgrist University Hospital in Zurich, Switzerland in collaboration with the Department Orthopaedic II of the Orthopaedic Hospital of Heidelberg to enable a mobilization with stabilized circulation and to begin with a locomotion training in an early state of rehabilitation.
In the tilt stepper, the patient is strapped by a safety belt to the tilt table while the legs are moved passively in a physiological stepping pattern (Figure 1). The inclination can be continuously adjusted from a horizontal to a vertical position. The distribution of the blood correlates directly to the sinus of the angle of inclination. Between 30 and 60 degrees this angle is linear [18]. For inclines larger than 60 degrees, there is a plateau of hemodynamic effects. For the present study, we choose an angle of 75 degrees because in previous studies it has been shown that syncope was more likely to occur at an angle over 60 degrees [18]. To investigate if there is a difference between passive stepping and passive cycling leg movements, we also used a tilt table with an ergometer device (tilt ergometer, Figure 3).
Figure 1 The tilt table with stepping device (tilt stepper)
Figure 3 The tilt table with ergometer device (tilt ergometer)
There are only a few studies that have investigated how passive movement of the legs during a tilt table treatment affects circulation. In these studies, either functional electrical stimulation of the leg muscles [19-21] was used, or patients were placed in sitting positions on a cycle ergometer [22]. The results of these studies suggest that passive movements of the legs could stabilize blood circulation. There have also been studies which have utilized a tilt table with passively moving legs. However, in these studies only patients with recurrent vasovagal syncopes were enrolled, the syncope was pharmacologically provoked [5,23-30].
The aim of our experiments was to investigate if passive stepping and cycling movements of the legs during a tilt table test can stabilize blood circulation and prevent neurally mediated syncope in healthy young adults.
Methods
Participants
With the permission from the local Ethics Committee and the informed consent of the volunteers, the response of the blood circulation was analyzed in healthy subjects. The exclusion criteria included: recurrent syncope or near-syncope in clinical history, regular medication, abuse of nicotine or alcohol, cardiovascular or neurological diseases, acute or chronic infections, psychiatric disorder and body mass index <18 or >25. All subjects underwent a physical investigation and an ECG was completed one week before the experiment.
In the first experiment, we examined 12 healthy young adults (age 24 ± 5 years) on a traditional tilt table. The subjects, who had syncope or near-syncope were treated on the tilt stepper after a waiting period of 4 weeks. Syncope was defined as a transient loss of consciousness associated with a loss of postural tone. Near-syncope was defined as the appearance of pallor, nausea, light-headedness, diaphoresis or blurred vision. Both conditions were associated with the following hemodynamic changes: a decrease in systolic blood pressure > 60% from baseline values or an absolute value < 80 mmHg (vasodepressor response) and/or a decrease in heart rate > 30 % form the baseline value or an absolute value < 40 beats/min (cardio-inhibitory response) [31].
In the second experiment, we enrolled 42 healthy subjects (age 27 ± 4 years). They were randomized into two groups: group I (23 subjects) was put on a traditional tilt table, while the Group II (19 subjects) on a tilt ergometer. The age of the subjects was restricted to below 35 years, because the cardiovascular response is strongly dependent on age [32].
Procedures
The aim of the first experiment was to investigate if the blood circulation could be stabilized in people who have a disposition for an "early" appearance of a neurally-mediated syncope on a traditional tilt table. The appearance of a neurally-mediated syncope is physiological and it may occur in all subjects. The interpersonal difference lies in the duration that the subject can be in standing posture until syncope or near-syncope occurs. A decrease of the systolic blood pressure up to maximal 15 mmHg and/or an increase in heart rate up to 20 bpm during the first 6 minutes are considered as a normal reaction to compensate the change in the position of the body [31].
The blood pressure was non-invasively measured with a tonometric blood pressure device. Subjects who suffered a syncope or near-syncope during the first session on the traditional tilt table were in the second session treated on the tilt stepper. In the second experiment, we investigated the effect of passively induced movements on circulation by a cycle ergometer on a tilt table. We enrolled 42 subjects: 23 on a traditional tilt table and 19 on the tilt ergometer.
In both experiments, after 15 minutes of rest the subjects were tilted head-upright at a 75° angle and were returned to the supine position if a syncope or near-syncope occurred or after completion at 30 minutes. Heart rate and blood pressure were measured continuously and non-invasively. Head-up tilt tests were performed in the morning in a dim room. All subjects were instructed to fast overnight and relax the muscles of their lower limbs during the trials. This was monitored with an EMG-measurement on legs (Mm. biceps femoris, rectus femoris, gastrocnemii, tibialis ant.), randomly tested in the first experiment and regularly tested during the second experiment. The EMG signals were amplified and transferred to a personal computer. They were recorded by a data acquisition tool (SolEasy by Aleasolution GmbH, Zurich, CH).
Tilt table with stepping device (Tilt stepper)
The tilt stepper is a traditional tilt table (Gymna, Belgium) combined with an integrated leg drive that allows a passive movement of the lower extremities (Figures 1 and 2)
Figure 2 The tilt stepper: generation of leg movements
The leg drive that is connected to the thigh by a cuff induces a hip flexion or extension movement. As the feet of the patient are fixed to footplates, the knee is also flexed or extended, respectively. In those phases where the hip and knee joints are extended, the leg pushes down a spring-dampened footplate, which is then again pushed against a foot spring that is mounted within these plates. This footplate generates a loading force on the foot sole of the patient during extension. Applying this cycle of flexion and extension in an alternating way leads to physiological kinetics of the generated motion. A special mechanism is mounted under the hip joint and allows for adjustment of hip extension up to 20°.
Depending on the blood circulation condition of the patient, the device can be tilted to different angles up to a vertical position. This makes it possible for the patient to become accustomed, step-by-step, to the upright position in combination with passive leg movements. The speed of the alternating stepping movements and the range of motion of hip/knee joints can be adjusted by a control panel. The basic construction consists of a linear drive (Parker-Hannifin, Germany), with a precision ball screw that is driven by a synchronous motor via toothed belt (maximum speed 450 mm/sec, maximum force 1400 N, maximum torque of 400 Nm at the hip joint). The movement frequencies range from 0.2 to 0.5 Hz (i.e. one cycle of flexion and extension takes between 2 and 5 sec).
To secure subjects on the tilt table during experiments, fixation with a special harness was used during all experiments (Figure 1).
The tilt table with ergometer (Tilt ergometer)
The tilt ergometer consists of a traditional tilt table with an additional ergometer device (Tera Joy Germany) that allows a passive cycling movement of the lower extremities. From a technical point of view, the tilt ergometer construction is simpler than the tilt stepper, but it generates a non-physiological motion concerning gait phase related forces on the foot sole. The cycle frequency was between 0.2–0.5 Hz.
Recordings and Measurement
Blood pressure was measured continuously and non-invasively by a Colin CBM-7000 (Hayashi, Komaki City, Japan). The Colin CBM – 7000 is a tonometric blood pressure device that allows measuring beat-to-beat blood pressure (systolic, mean, diastolic), continuous arterial blood pressure waveform, beat-to-beat and continuous electrocardiography.
Statistical analysis
In both experiments we used the chi-square test to compare the occurrence of near-syncope/syncope in both groups (tilt table/tilt stepper and tilt table/tilt ergometer). We performed 2 by 4 repeated measures ANOVAs with 2 between factors (device group – namely tilt table vs. tilt stepper or tilt table vs. tilt ergometer), 4 within factors (time – supine, 2 minutes, 6 minutes and end of head-up tilt) and in their interaction (groups × time) for blood pressure and heart rate. Pairwise comparisons were made with the t-test with additional Bonferroni's correction.
Results
In the first experiment, 7 of 12 subjects (58%) had a syncope or near-syncope on the traditional tilt table. There was an obvious increase in heart rate in the first 6 minutes after changing the position from supine to upright. None of these 7 subjects had a syncope or near-syncope during the treatment session on the tilt stepper 4 weeks later. Comparing the occurrence of near syncope/syncope in both sessions with the chi-square test, there was a significant difference ((χ2 (1.1) = 6.465, p = 0.011). Table 1 gives a short overview of these results. The same subjects, who collapsed on the traditional tilt table, did not have syncope or near-syncope while treated on the tilt stepper.
Table 1 Occurrence of near-syncope and syncope in experiment one (tilt stepper)
no syncope near-syncope syncope
traditional tilt table [n = 12] 5 (42%) 5 (42%) 2 (18%)
tilt stepper [n = 7] 7 (100%) 0 (0%) 0 (0%)
In the ANOVA for repeated measures there were no significant differences for blood pressure within each group (time 4 levels: supine, 2, 6 and end of head-up tilt; F (1,6) = 4.66, p < 0.0743), but there were significant differences between groups (two levels: tilt stepper and tilt table; F (3,33) = 6.33, p < 0.0016)) and in the interactions (F(3,18) = 7.24, p < 0.0022). The blood pressure differs between the two treatments at the end of head – up tilt (p < 0.0029), but not at 2 minutes (p < 1.000) and at 6 minutes (p < 1.000) (Pairwise comparisons with the t-test and additional Bonferroni's correction). However, there could be shown a trend for a higher blood pressure at 2 minutes and at 6 minutes after head-up tilt in the group treated in the tilt stepper.
There were significant differences for heart rate within each group (time 4 levels: supine, 2, 6 and end of head-up tilt; F (1, 6) = 12.17, p < 0.0130) between groups (two levels; F (3, 33) = 21.16, p < 0.0001) and in the interactions (F (3, 18) = 8.68, p < 0.0009). For the group treated on the traditional tilt table, pairwise comparisons with the t-test with additional Bonferroni's correction showed a significantly higher heart rate at 2 minutes (p < 0.0.0060), but no significant differences at 6 minutes (p < 0.2051) and at the end of head-up tilt (p < 1.000).
In the second experiment, 13 of 23 subjects (57 %) who were on the traditional tilt table had syncope (3) or near-syncope (10). None of the 19 subjects who were on the tilt ergometer had syncope but 4 subjects had near-syncope (21%). Comparing the occurrence of near syncope/syncope in both sessions with the chi-square test (χ2 (1.1) = 5.443) there was a significant difference (p = 0.021) (Table 2).
Table 2 Mean blood pressure and heart Rate +/- SE during 75° head-up tilt on the tilt-stepper
during supine position 2-min after head-up tilt 6-min after head up tilt end of head-up tilt
mean blood pressure [mmHg]
traditional tilt table [n = 12] 90 +/- 4 95 +/- 4 94 +/- 4 80 +/- 3*
tilt stepper [n = 7] 89 +/- 4 93 +/- 5 97 +/- 2 95 +/- 6*
heart rate [beats/min]
traditional tilt table [n = 12] 65 +/- 5 80 +/- 5* 78 +/- 4 65 +/- 5
tilt stepper [n = 7] 61 +/- 3 69 +/- 3* 71 +/- 3 71 +/- 5
* p < 0.05 (compared with ANOVA for repeated measures)
In the ANOVA for repeated measures, there were significant differences for blood pressure within each group (time 4 levels: supine, 2, 6 and end of head-up tilt; F (1,6) = 34.43, p < 0.0001) between groups (two levels; F (3,33) = 13.42, p < 0.0001)) and in the interactions (F(3,18) = 10.95, p < 0.0001). Pairwise comparisons with the t-test (additional Bonferroni's correction) showed no significant differences at 2 minutes (p < 0.5221) and at 6 minutes (p < 0.4429) but a significant difference at the end of head – up tilt (p < 0.0001). However, there could be shown a trend for a higher blood pressure at 2 minutes and at 6 minutes after head-up tilt in the group treated on the tilt ergometer. There were significant differences for heart rate within each group (time 4 levels: supine, 2, 6 and end of head-up tilt; F (1,6) = 12.17, p < 0.0130), between groups (two levels; F (3,33) = 21.16, p < 0.0001), and in the interactions (F(3,18) = 8.68, p < 0.0009). Pairwise comparisons with the t-test (additional Bonferroni's correction) showed no significant differences at 2 minutes (p < 0.3317), but a significantly higher heart rate in the group treated on the tilt table at 6 minutes (p < 0.0007) and at the end of head – up tilt (p < 0.0002).
All subjects on the tilt stepper and tilt ergometer completed 30 minutes of head-up tilt. The duration of the head-up tilt was different in the group on the traditional tilt table, as an abrupt decrease of blood pressure or symptoms of near-syncope occurred.
In the head-up tilt position the subject stands on the footplates on the tilt stepper, whereas in the tilt ergometer the harness holds the whole body weight. The subjects who were investigated on the tilt stepper felt comfortable during the whole experiment, whereas the subjects examined on the tilt ergometer in experiment two complained of discomfort. The subjects on the tilt ergometer experienced more discomfort because of the perception of no lower limb support. These statements were subjective; no standardized assessment instrument was used to measure the comfort.
Tables 3 and 4 and Figures 4 and 5 provide an overview about the response of the blood pressure of subjects tested on the traditional tilt table (with and without syncope, n = 12 in experiment one and n = 23 in experiment two) and subjects with passive leg movements during the tilt table test on the tilt stepper (n = 7) and tilt ergometer n = 19).
Table 3 Occurrence of near-syncope and syncope in experiment two (tilt ergometer)
no syncope syncope near-syncope
traditional tilt table [n = 23] 10 (43%) 3(14%) 10 (43%)
tilt ergometer [n = 19] 15 (79%) 0 (0%) 4 (21%)
Table 4 Mean blood pressure and heart rate +/- SE during 75° head-up tilt on the tilt ergometer
during supine position 2-min after head-up tilt 6-min after head-up tilt end of head-up tilt
mean blood pressure [mmHg]
traditional tilt table [n = 23] 92 +/- 4 92 +/- 6 90 +/- 5 80 +/- 4*
tilt ergometer [n = 19] 91 +/- 5 96 +/- 3 95 +/- 2 93 +/- 4*
heart rate [beats/min]
traditional tilt table [n = 23] 64 +/- 5 79 +/- 5 82 +/- 3* 78 +/- 5*
tilt ergometer [n = 19] 65 +/- 3 74 +/- 4 73 +/- 5* 68 +/- 4*
* p < 0.05 (compared with ANOVA for repeated measures)
Figure 4 Blood pressure +/- SE during 75° the tilt table and tilt stepper test
Figure 5 Blood pressure +/- SE during 75° the tilt table and tilt ergometer test
In Figures 6 and 7, recordings illustrating the development of systolic and diastolic blood pressure and heart rate for one subject with syncope (Figure 6) and another subject without syncope (Figure 7) during the tilt table test are shown. The observed progression of blood pressure and heart rate of the subject who had syncope is typical for a neurally-mediated syncope, because of the sudden decrease of systolic and diastolic blood pressure combined with bradycardia more than 20 minutes after head-up tilt. Also typical is the increase in heart rate observed in the first 6 minutes after head-up tilt. All subjects treated on the tilt table had this benign form of syncope and showed a similar blood pressure and heart rate progression during the tilt table test.
Figure 6 Typical recordings illustrating a subject with syncope. RF = M. rectus femoris, BF = M. biceps femoris, TA = M. tibialis anterior, GM = M. gastrocnemius
Figure 7 Typical recordings illustrating a subject without syncope. RF = M. rectus femoris, BF = M. biceps femoris, TA = M. tibialis anterior, GM = M. gastrocnemius
Figure 7 is a good example for the normal progression of blood pressure and heart rate during a tilt table test. 2 minutes after head-up tilt there is a slight decrease of systolic and diastolic blood pressure and a slight increase of heart rate, a physiological mechanism of compensation for the change of position (supine to head-up tilt).
Figure 8 is an example for the EMG activity in the right leg during the tilt stepper test, and Figure 9 during the tilt table test. It becomes obvious that there is no active muscle activity. The ups and downs in the curve of the muscle gastrocnemius on the tilt stepper are from the passive movements.
Figure 8 Muscle activity during the tilt table and tilt stepper test
Figure 9 Muscle activity during the tilt table and tilt stepper test
Discussion
The tilt table is an apparatus that has become an important part in the evaluation of patients with unexplained syncope or loss of consciousness [14,24,33]. It has also proven useful for circulatory training of patients suffering from several neurological diseases. However, the treatment is limited by the occurrence of circulatory collapse [16]. Both hypotension and bradicardia leading to syncope during tilt tests are also common events in healthy persons. These responses are considered to be part of a reflex response triggered by a sympathetic-induced hypercontraction of an almost empty left ventricular chamber [34]. In both experiments there were no recurrent syncope or near-syncope in the clinical history of the subjects and the ECG did not show any abnormities. For these reasons, and because of the development of the heart rate and blood pressure in our experiments, the occurring syncopes and near-syncopes that occurred ought to be benign and so called neurally-mediated syncopes or vasovagal syncopes. It is a physiological form of syncope that can occur in healthy persons. Some persons have the disposition of suffering a neurally-mediated syncope earlier than others [35]. This benign form of syncope can be differentiated from malignant syncopes, like the hyperadrenergic orthostatic hypotension (decrease of blood pressure and increase of heart rate), hypoadrenergic orthostatic hypotension (decrease of blood pressure without an increase of heart rate) and postural tachycardia syndrome (massive increase of heart rate without decrease in blood pressure) by recording heart rate and blood pressure [31].
Although the tilt table has become an accepted diagnostic tool, there are no comparable studies with the tilt table in which the effect of passive leg motion on circulation have been investigated.
The aim of these two experiments was to investigate if passive leg movements during head-up tilt can prevent syncope. The data in the present study show a stabilizing effect on the blood circulation and this study suggests that there is an effect on preventing neurally-mediated syncope by both devices. In the first experiment, none of the subjects who had syncope/near-syncope on the traditional tilt table had syncope/near-syncope four weeks later on the tilt stepper. In the second experiment, only 4 subjects who were treated on the tilt ergometer had near-syncope. In both experiments the increase of heart rate was larger in the group tested on the traditional tilt table. A correlation between heart rate and appearance of syncope was described [16,36]. An increase in heart rate > 18 bpm during the first minutes after changing position from supine to upright leads to syncope, with a sensitivity of 90% and a specification of 100%. Consequently, the positive effect of passive leg movement on heart rate is obvious. Heart rate and blood pressure give an indication of the sympathetic activity, which is activated on the tilt table [37,38]. This increased sympathetic activity stimulates mechano-receptors in the ventricle, which leads to an activation of the vagus nerve and a reflexive decrease of sympathetic activity. The vagus activity leads to bradycardia and vasodilatation: the Bezold-Jarisch-Reflex [36]. We suggest that the sympathetic activity becomes reduced by the tilt stepper, preventing this vicious cycle that leads to a vasovagal syncope. This has to be proved in further studies by an intra-arterial catecholamine measurement.
In the first experiment we treated the same subject twice on a tilt table. It cannot be excluded that an adaptation to the orthostatic change occurred in these subjects. However, there was an interval of four weeks between the first treatment on the traditional tilt table and the second treatment on the tilt stepper. Therefore, a training effect or an effect of habituation, such as described in another study in which patients suffering from syncope were treated each day over 6 weeks, seems to be unrealistic [39].
The results in both experiments indicate that blood circulation can be stabilized by passive leg movements. However, the movements of the two devices used in these experiments are very different: on the tilt stepper there are stepping like movements and the legs can be loaded during extension and unloaded in flexion. In the tilt ergometer, the movements are the other way round. There might be more afferent input from the load receptors in the tilt stepper compared to the tilt ergometer. For example it could be shown that the load moments acting about the bilateral hip, knee and ankle joint axes during cycling are found to be generally lower than those induced during normal level walking [10] and concluded that afferent input from hip joints, in combination with that from load receptors during walking, plays a crucial role in the generation of locomotor activity in the isolated human spinal cord [1]. Also, the range of motion is adjustable in the tilt stepper, so that the extent of flexion and extension can be increased or decreased depending on the condition of the patient. Therefore, the tilt stepper may be more effective in activating a locomotion pattern. In addition, both devices might help to decrease spasticity [40] and serve to prevent osteoporosis [41]. Although these effects were not part of our current investigation, some of these issues could be proven in trials with treadmill training in the rehabilitation of patients with stroke, spinal cord and traumatic brain injury [18,39]. Thus, we plan to use the tilt stepper in further studies to investigate if it leads to a stabilization of blood circulation, prevention of neurally mediated syncope in an early state of rehabilitation, decrease in spasticity, prophylaxis of osteoporosis and activation of the locomotion pattern generator of patients suffering from neurological diseases. This in turn may lead to a better outcome and quality of life for the patient.
In conclusion, we could show that both passive cycle and stepping movements of the legs during head-up tilt testing can stabilize blood circulation and prevent syncope in young healthy people. In further studies, we aim to investigate if the tilt stepper could become a helpful device for patients suffering from neurological diseases.
Acknowledgements
We thank Miriam Hiestand, Monika Stüssi and Daniel Salzmann for data collecting support.
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| 15679913 | PMC544951 | CC BY | 2021-01-04 16:37:39 | no | J Neuroengineering Rehabil. 2004 Oct 25; 1:4 | utf-8 | J Neuroeng Rehabil | 2,004 | 10.1186/1743-0003-1-4 | oa_comm |
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J Neuroengineering RehabilJournal of NeuroEngineering and Rehabilitation1743-0003BioMed Central London 1743-0003-1-51567991610.1186/1743-0003-1-5ResearchRehabilitation robotics: pilot trial of a spatial extension for MIT-Manus Krebs Hermano I [email protected] Mark [email protected] Stephen P [email protected] Miranda J [email protected] Antonio [email protected] Michael [email protected] Daniel [email protected] Bruce T [email protected] Neville [email protected] Massachusetts Institute of Technology, Mechanical Engineering Department, Cambridge, MA, USA2 Weill Medical College of Cornell University, Department Neurology and Neuroscience, New York, NY, USA3 Burke Medical Research Institute, White Plains, NY, USA4 Imperial College, London, UK5 Interactive Motion Technologies, Inc., Cambridge, MA, USA6 Massachusetts Institute of Technology, Brain and Cognitive Sciences, Cambridge, MA, USA2004 26 10 2004 1 5 5 30 8 2004 26 10 2004 Copyright © 2004 Krebs et al; licensee BioMed Central Ltd.2004Krebs 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
Previous results with the planar robot MIT-MANUS demonstrated positive benefits in trials with over 250 stroke patients. Consistent with motor learning, the positive effects did not generalize to other muscle groups or limb segments. Therefore we are designing a new class of robots to exercise other muscle groups or limb segments. This paper presents basic engineering aspects of a novel robotic module that extends our approach to anti-gravity movements out of the horizontal plane and a pilot study with 10 outpatients. Patients were trained during the initial six-weeks with the planar module (i.e., performance-based training limited to horizontal movements with gravity compensation). This training was followed by six-weeks of robotic therapy that focused on performing vertical arm movements against gravity. The 12-week protocol includes three one-hour robot therapy sessions per week (total 36 robot treatment sessions).
Results
Pilot study demonstrated that the protocol was safe and well tolerated with no patient presenting any adverse effect. Consistent with our past experience with persons with chronic strokes, there was a statistically significant reduction in tone measurement from admission to discharge of performance-based planar robot therapy and we have not observed increases in muscle tone or spasticity during the anti-gravity training protocol. Pilot results showed also a reduction in shoulder-elbow impairment following planar horizontal training. Furthermore, it suggested an additional reduction in shoulder-elbow impairment following the anti-gravity training.
Conclusion
Our clinical experiments have focused on a fundamental question of whether task specific robotic training influences brain recovery. To date several studies demonstrate that in mature and damaged nervous systems, nurture indeed has an effect on nature. The improved recovery is most pronounced in the trained limb segments. We have now embarked on experiments that test whether we can continue to influence recovery, long after the acute insult, with a novel class of spatial robotic devices. This pilot results support the pursuit of further clinical trials to test efficacy and the pursuit of optimal therapy following brain injury.
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Background
Rather than using robotics as an assistive technology for a disabled individual, our research focus is on the development and application of robotics as a therapy aid, and in particular a tool for therapists. We foresee robots and computers as supporting and enhancing the productivity of clinicians in their efforts to facilitate a disabled individual's functional motor recovery. To that end, we deployed our first robot, MIT-MANUS (see figure 1), at the Burke Rehabilitation Hospital, White Plains, NY in 1994 [1]. In the last ten years, MIT-MANUS class robots have been in daily operation delivering therapy to over 250 stroke patients. Hospitals presently operating one or more MIT-MANUS class robots include Burke (NY), Spaulding (MA), Rhode Island (RI), Osaka Prefectural (Japan) and Helen Hayes (NY) Rehabilitation Hospitals, and the Baltimore (MD) and Cleveland (OH) Veterans Administration Medical Centers.
Figure 1 Stroke Inpatient during Therapy at the Burke Rehabilitation Hospital (White Plains, NY). Therapy is being conducted with a commercial version of MIT-MANUS (Interactive Motion Technologies, Inc., Cambridge, MA).
Most of the work to date has focused on the fundamental question of whether task specific training affects motor outcome and positively influences brain recovery. These efforts directly confront the overwhelming task of reversing the effects of natural injury where lesion size, type and location profoundly determine outcome, and applying controlled conditions in environment and training – nurture – to exploit the ability of the mature nervous system to learn, adapt and change.
Through our work with MIT-MANUS, providing task specific training for patients' with moderate to severe hemiparesis, we have gathered convincing evidence (summarized below) that nurture has a significant impact in speeding motor recovery of the paretic shoulder and elbow, and that robot therapy is effective in delivering the necessary exercise. This recovery is most pronounced in the trained muscle groups and limb segments. Encouraged by these positive results, we have expanded our project to develop a family of novel, modular robots, designed to be used independently or together to rehabilitate other muscle groups and limb segments. This paper describes two different implementations of a new module developed at MIT that expands the capabilities of MIT-MANUS to include motion in a three-dimensional workspace. We will present both implementations, the basic engineering differences between these modules, and pilot clinical results from their use with stroke patients.
Proof-of-Concept
Volpe [2] reported the composite results of robotic training with 96 consecutive stroke inpatients admitted to Burke who met inclusion criteria and consented to participate [3-7,2]. Patients were randomly assigned to either an experimental or control group and although the patient groups were comparable on all initial clinical evaluation measures, the robot-trained group demonstrated significantly greater motor improvement (higher mean interval change ± standard error measurement) than the control group on the Motor Status and Motor Power scores for shoulder and elbow (see Table 1). In fact, the robot-trained group improved twice as much as the control group in these measures. These gains were specific to motions of the shoulder and elbow, the focus of the robot training. There were no significant between-group differences in the mean change scores for wrist and hand function. Similar results were gathered in patients who have had a paralyzed upper extremity after stroke for at least one year [8-10] (See Table 1).
Table 1 Mean interval change in impairment and disability (significance p < 0.05).
Between Group Comparisons: Final Evaluation Minus Initial Evaluation Robot Trained (N = 55) Control (N = 41) P-Value
Impairment Measures (±sem)
Motor Power (MP) 4.1 ± 0.4 2.2 ± 0.3 <0.01
Motor Status shoulder/elbow (MS-se) 8.6 ± 0.8 3.8 ± 0.5 <0.01
Motor Status Wrist/Hand (MS/wh) 4.1 ± 1.1 2.6 ± 0.8 NS
Description of Robots
Modularity and Integration Potential
MIT's experience with well over 250 stroke patients has reinforced the importance of one of our core design specifications: Modularity. From the outset we believed that modularity is essential to success in robotic therapy, particularly in extending the approach to patients suffering from distinct afflictions. Consider, for example, patients undergoing surgery at the wrist (e.g., Colles Fracture) who might not require a device that manipulates the payload of all the degrees-of-freedom (DOF) of the arm. Therapy for these patients requires only the wrist robot [11-13]. Conversely there will be patients for whom different modules must be coupled to deliver therapy and carry the payload of the human arm. Presently MIT has deployed four modules into the clinic (Burke Rehabilitation): a planar 2-dof active module; vertical 1-dof active module; wrist 3-dof active module; and 1-dof passive grasp module.
Features common to all modules
All of our robot modules are specifically designed and built for clinical rehabilitation applications. Unlike most industrial robots, they are configured for safe, stable, and compliant operation in close physical contact with humans. This is achieved using backdrivable hardware and impedance control, a key feature of the robot control system. Each active module can move, guide or perturb movements of a patient's limb and can record motions and mechanical quantities such as the position, velocity, and forces applied.
The most profound engineering challenge specific to this family of robots is achieving the dual goals of high force production capability and backdrivability. Each module must be capable of generating sufficient force to move a patient's limb, but it must also itself be easily movable by an elderly or frail patient. Backdrivability is essential in keeping the patient engaged in the task and in allowing him to observe his successful and unsuccessful attempts at motion. Backdrivable hardware also improves the performance of systems controlled by impedance controllers. Achieving backdrivability and high force production, together in a single machine, is often difficult and becomes more so when the robot geometry is more complex.
The robot control system is an impedance controller that modulates the way the robot reacts to mechanical perturbation from a patient or clinician and ensures a gentle compliant behavior. Impedance control refers to using a control system (actuators, sensors and computer) to impose a desired behavior at a specified port of interaction with a robot, in this case the attachment of the robot to the patient's hand. Conceived in the early 1980's by one of the co-authors [14], it has been applied successfully in numerous robot applications that involve human-motor interaction. Impedance control has been extensively adopted by other robotics researchers concerned with human-machine interaction. In rehabilitation robotics impedance control has been successfully implemented in MIT-MANUS since its clinical debut in 1994. For robots interacting with the human, the most important feature of the controller is that its stability is extremely robust to the uncertainties due to physical contact [14,15]. The stability of most robot controllers is vulnerable when contacting objects with unknown dynamics. In contrast, dynamic interaction with highly variable and poorly characterized objects (to wit, neurologically impaired patients) will not de-stabilize the impedance controller above; even inadvertent contact with points other than the robot end-effector will not de-stabilize the controller. This is essential for safe operation in a clinical context.
Planar 2-dof robot MIT-MANUS
The MIT-MANUS project was initiated in 1989 with support from the National Science Foundation. MIT-MANUS has been in daily operation since 1994 delivering therapy to stroke patients at the Burke Rehabilitation Hospital. This robot has been extensively described in the literature [4] (See Figure 1). MIT-MANUS is a planar module which provides two translational degrees-of-freedom for elbow and forearm motion. The 2-dof module is portable (390 N) and consists of a direct-drive five bar-linkage SCARA (Selective Compliance Assembly Robot Arm). This configuration was selected because of its unique characteristics of low impedance on the horizontal plane and almost infinite impedance on the vertical axis. These allow a direct-drive backdrivable robot to easily carry the weight of the patient's arm. The mechanism is driven by brushless motors rated to 9.65 Nm of continuous stall torque with 16-bit virtual absolute encoders for position and velocity measurements (higher torques can be produced for limited periods of time). Redundant velocity sensing may be provided by DC-tachometers with a sensitivity of 1.8 V/rad/sec. A six degrees-of-freedom force sensor is mounted on the robot end-effector. The robot control architecture is implemented in a standard personal computer with 16-bit A/D and D/A I/O cards, as well as a DIO card with 32 digital lines. Besides its primary control function, this computer displays the task to both the operator and the subject or patient via dedicated monitors. Custom-made hand holders connect a patient's upper limb to the robot end-effector.
The selected design created a highly backdrivable robot capable of delivering therapy in a workspace of 15" by 18" with an end-point anisotropy of 2:1 ratio (2/3 < I < 4/3 Kg; 56.7 < static friction < 113.4 grams) and achievable impedances between 0 and 8 N/mm. Note that the static friction is significantly below the just noticeable difference (JNF) for force, which is 7% of the reference force. The robot maximum achievable impedance is above the human perception of 4.2 N/mm for a "virtual wall." The robot is capable of delivering forces up to 45 N although the robot target design aimed at a force of 28 N, which corresponds to the arm strength during elbow extension for a weak woman in seated position [16].
Vertical 1-dof Novel Robot
Following the successful clinical trials of MIT-MANUS, a 1-dof module to provide vertical motion and force was conceived and built. The primary goal of this module is to bring the benefits of planar therapy on MIT-MANUS to spatial arm movements, including movement against gravity. The module can be used independently or mounted to MIT-MANUS for movement in a limited spatial workspace. The module can permit free motion of the patient's arm, or can provide partial or full assistance or resistance as the patient moves against gravity. Because the vertical module moves with the endpoint of the planar module when the two are integrated, overall module mass is an important design concern in addition to on-axis mass and friction. Two embodiments are described below.
Screw-driven module
One prototype of the 1-dof module was completed at MIT late 2000 and is shown alongside a test stand in Figure 2. A second clone was completed at MIT and deployed in the clinic (Burke Rehabilitation Hospital), where it is presently collecting pilot data with stroke patients. The module incorporates a custom-made "rollnut" and a custom-made screw with a linear guide system. Significant effort was engaged in the design of the screw transmission, which provides an efficient conversion of rotary to linear motion designed to eliminate nearly all-sliding friction in favor of rolling contact. Its low friction provides an intrinsically back-drivable design. The bracket mounted to the rollnut allows the attachment of different interfaces. Incorporated into the design are therapists' suggestions that functional reaching movements often occur in a range of motion close to shoulder scaption. Thus, the robotic therapy games that use the spatial robot focus on movements within the 45° to 65° range of shoulder abduction and from 30° to 90° of shoulder elevation or flexion. A Gripmate is used to hold the patient's hand in place.
Figure 2 Constant-Velocity Friction Experiments (0.5 to 50 mm/sec). Photo shows alpha-prototype. The mean friction force was 20.075 ± 1.056 N.
This prototype has been fully characterized at MIT [17,18] (Figures 4 and 5). In comparison to MIT-MANUS, the vertical module has a greater effective endpoint mass and friction, though the resulting system is still back-drivable. In order to partially compensate for this increased impedance, force-feedback is incorporated into the impedance controller, resulting in a substantial reduction in friction, down to approximately 3 N, and mass, to approximately 1 kg. This improvement is illustrated in Figure 3. The module is capable of providing well over the force specification of 65 N in the upward direction (20 N estimate of patient's arm weigh) and 45 N in the downward direction, and can achieve stiffness in excess of 10 N/mm, far greater than the values generally used for therapy.
Figure 4 Graduates from Planar Robot Protocol Receiving Additional Vertical Anti-Gravity Training at the Burke Rehabilitation Hospital (White Plains, NY). The robot is sufficiently backdrivable to be lifted with the tip of the little finger.
Figure 5 Graduates from Planar Robot Protocol Receiving Additional Vertical Anti-Gravity Training at the Burke Rehabilitation Hospital (White Plains, NY). The robot is sufficiently backdrivable to be lifted with the tip of the little finger.
Figure 3 The graph shows force versus position with spring behavior commanded (heavy dot). PD controller alone (solid), PD controller with force feedback, Kf = 5 (dashed). Qualitatively, the roughly 3 N of friction force is almost imperceptible.
Linear direct-drive module
The screw-driven prototype has proven very successful both in standalone operation and mounted at the end of the planar module enabling spatial movement therapy in the clinic with compliant and stable behavior. However recent changes in linear motor technology have created the potential to achieve similar outcomes with effective vertical endpoint inertia comparable to the planar MIT-MANUS and much lower friction, without the need for force feedback control. The main change is complete enclosure of the magnets within the motor forcer. While this does not increase the magnetic field strength, it dramatically increases the line integral and concatenates magnetic lines.
The practical advantages of converting the spatial system to direct-drive linear motors would be a significant reduction of friction and elimination of backlash. This simplification would also carry through to the control system and controller, as well as affording a reduction in the system's overall dimensions and weight. To determine if the expected friction levels are realistic, we tested Copley Control ThrustTube TB2504. Figures 6 ,7, 8 shows our experimental results characterizing the static friction for the TB series and the force vs current relationship. Figures 9 and 10 shows the commercial implementation of the novel module (Interactive Motion Technologies, Inc., Cambridge, MA). The novel module allows 19.4" of linear range of motion and it is capable of moving the desired target maximum endpoint force of 65 N upward and 45 N downward. This new module achieves significant reductions in friction and inertia to 25% and 76% of the lead-screw prototype.
Figure 6 Characterization of TB2504. Plot shows the force versus current curve.
Figure 7 Characterization of TB2504. Plot shows the static friction and cogging.
Figure 8 Characterization of TB2504.
Figure 9 Vertical 1-dof Module Using Electrical Linear Technology. This commercial version of MIT's module can be operated in standalone fashion or integrated to the planar MIT-MANUS to allow spatial movements. Note that in the standalone fashion it can be operated at any angle to the horizontal and vertical planes with adjustable handle positions.
Figure 10 Vertical 1-dof Module Using Electrical Linear Technology. This commercial version of MIT's module can be operated in standalone fashion or integrated to the planar MIT-MANUS to allow spatial movements. Note that in the standalone fashion it can be operated at any angle to the horizontal and vertical planes with adjustable handle positions.
Pilot Clinical Trials with the Anti-gravity Module
To test the novel vertical module we conducted a pilot study to analyze whether additional anti-gravity training further improves motor outcomes for "graduates" of the planar robot-assisted protocol.
In-/Exclusion Criteria
Outpatients were included in the study if they met the following criteria: a) first single focal unilateral lesion with diagnosis verified by brain imaging (MRI or CT scans) that occurred at least 6 months prior; b) cognitive function sufficient to understand the experiments and follow instructions (Mini-Mental Status Score of 22 and higher or interview for aphasic subjects); c) Motor Power score ≥1/5 or ≤3/5 (neither hemiplegic nor fully recovered motor function in the 14 muscles of the shoulder and elbow); d) informed written consent to participate in the study. Patients were excluded from the study if they have a fixed contracture deformity in the affected limb that limited pain-free range of motion. We have found severe tendon contractures around the rotator cuff particularly, in patients with complete hemiplegia for longer than 6 months after stroke. It is reasonable to expect that robotic training for the upper limb would not have an impact on a fixed contracture deformity. Trials commenced only after baseline assessment across three consecutive evaluations, 2 weeks apart, shows a stable condition in three motor impairment scales (F-M, MSS, MP). Our rationale for administering multiple baseline evaluations is based on an interesting "Hawthorne effect" that we observed in previous subjects [19,20]. Between first and second pre-treatment evaluations, some subjects have shown a remarkable improvement in clinical impairment scores. We speculate that the anticipated participation in a research study may contribute to a significant change in life routines.
Demographics
Ten (10) community dwelling volunteers who have suffered a single stroke at least 6 months prior to enrollment were enrolled in the pilot protocol. The mean group age was 62 ± 4.3 years old (mean ± sem) with the onset of the stroke occurring 50 ± 8.9 months (mean ± sem) prior to enrollment. Table 2 summarizes admission status of volunteers (See Table 2).
Table 2 Data on the Ten (10) Community Dwelling Stroke Volunteers
Age Handed Lesion foci Lesion side Months stroke Fugl-Meyer adm (/66)
59 Left AVM hem. Left 16.5 11
53 Ambi Intracerebral bleed Left 88.5 18
44 Right Carotid dissection Left 36 11
63 Right Cerebral embolism, subcortical Left 58 9
82 Right Cerebral embolism, subcortical Left 69 9
72 Right Carotid endarectomy Right 24 24
41 Right cortical/subcortical and basal ganglia stroke Right 96 17
72 Right cortical/subcortical stroke Right 47.5 9
57 Right Cerebral embolism, subcortical Right 48 30
77 Right cerebral embolism, mixed Right 16 34
Description of Protocol
Patients were trained during the initial six-weeks with the planar module (i.e., training limited to horizontal movements with gravity compensation as in past studies). This training was followed by six-weeks of robotic therapy that focused on performing vertical arm movements against gravity. The 12-week protocol includes three one-hour robot therapy sessions per week (total 36 robot treatment sessions) (Figure 9).
Figure 11 Movement Component Training. The circular display in front of the subject represents the workspace of the planar 6-weeks trial. The component training added 6 additional weeks training movements over two vertical lines.
For shoulder-and-elbow planar therapy, the center of the workspace was located in front of the subject at the body midline with the shoulder elevation at 30° with the elbow slighted flexed. The point-to-point movements started at the workspace center and extended in eight different directions of the compass (Figure 11). A one-hour session included two batches of 20 repetitions of point-to-point movements. The protocol incorporated a novel performance-based adaptive algorithm [21], which encouraged subjects to initiate movement with their hemiparetic arm.
Just as in the planar study [10], the anti-gravity robotic protocols consisted of visually evoked and visually guided point-to-point movements to different targets (along two vertical lines) with some robotic therapy games providing assistance and others visual feedback only. The protocol incorporated therapists' suggestions: a) robot therapy should focus on encouraging subjects to initiate movement against gravity with their hemiparetic arm beginning in a position of slight shoulder flexion (elevation) and scaption; b) functional reaching movements often occur in a range of motion close to shoulder scaption; c) no support should be provided at the elbow; and d) the visual display should be kept simple, since more complex displays proved to be difficult for our historical pool of stroke survivors to follow. Thus the robotic therapy protocols with the spatial robot focused on movements within the 45° to 65° range of shoulder abduction and between 30° to 90° of shoulder elevation or flexion. This sector is considered "safe" for the shoulder joint because it prevents gleno-humeral impingement that may occur when attempting to elevate the paretic limb to higher levels of shoulder elevation; The one-hour session included three batches of 20 repetitions of point-to-point movements. The first batch only provided visual feedback to a repetitive sequence of targets, while the second and third batches assisted the subject if needed to reach the target.
Clinical Assessment Scales
In this pilot, standard clinical evaluations included the upper extremity sub-test of the Fugl-Meyer Assessment (FM, maximum score = 66) from which we derived a Fugl-Meyer score for shoulder/elbow coordination (FM-SE, 42 out of 66); a more comprehensive evaluation of motor power or strength in 14 different muscle groups of the shoulder and elbow, using the MRC Motor Power score (MP, out of 70); and the Motor Status Score which is divided into two subscales, one for shoulder and elbow movements (MS-SE, maximum score = 40), and a second for wrist and hand abilities (MS-WH, maximum score = 42) [22,3,24,6,7]. The Modified Ashworth Scale (Bohannon, 1987) provides a measure of tone. The Fugl-Meyer test is a widely accepted measure of impairment in sensorimotor and functional grasp abilities [24]. To complement the Fugl-Meyer, we developed the Motor Status to further quantify discrete and functional movements in the upper limb. The MS-SE and MS-WH scales expand the F-M and have met the standards for inter rater reliability, significant intra-class correlation coefficients and internal item consistency [23,25].
Results
We have completed the study with ten (10) outpatients. Nine (9) outpatients who completed the planar training protocol at the Burke Rehabilitation Hospital were enrolled for an additional six-weeks with training three sessions per week on the vertical module. One (1) naïve outpatient completed only the six-week training on the vertical module (no previous exposure to the planar training). Table 3 and 4 shows the results for the shoulder-and-elbow subcomponent of the upper extremity Fugl-Meyer scale, the Motor Status Score, Motor Power, and Modified Ashworth scale.
Table 3 Anti-Gravity Vertical Module Pilot Study. Results from nine (9) outpatients that continued for an additional 6 weeks of training in the vertical module robotic unit. Statistical tests showed that outcomes at discharge from planar robot protocol were distinct from admission (B vs. A), and there was a trend favoring further improvement when comparing discharge from anti-gravity protocol with discharge from the planar protocol (C vs. B). Our protocol was safe and did not increase tone.
Timeline N = 9 A – Admission B – Discharge from planar robot protocol C – Discharge from anti-gravity protocol
F-M s/e (/42) 12.7 ± 1.6 14.8 ± 2.0 (p = 0.03, S) 17.0 ± 1.9 (p = 0.19, NS)
MSS s/e (/40) 18.1 ± 1.9 19.9 ± 2.0 (p = 0.01, S) 21.5 ± 1.8 (p = 0.29, NS)
MP 26.5 ± 3.5 33.3 ± 3.6 (p < 0.01, S) 38.8 ± 2.4 (p = 0.07, NS)
Ashworth 8.0 ± 1.4 4.9 ± 0.99 (p < 0.03, S) 4.4 ± 1.01 (p = 0.67, NS)
Table 4 Anti-Gravity Vertical Module Pilot Study. Results from one naive (1) outpatient that trained for 6 weeks in the vertical module robotic unit (no prior robot exposure).
Timeline N = 1 B – Admission to anti-gravity protocol C – Discharge from anti-gravity protocol
F-M s/e (/42) 24.0 27.0
MSS s/e (/40) 21.8 31.0
MP 35.0 43.0
Ashworth 4 1
Results of this pilot suggested that the vertical protocol was safe and well tolerated by the patients with no patient presenting any adverse effect (e.g., shoulder pain). Furthermore, pilot results suggested an additional reduction in shoulder-elbow impairment following the anti-gravity vertical training. In fact, for these 9 patients the reduction in impairment during the vertical training phase was comparable to the reduction during the planar phase (5.2% for the vertical training vs 5.0% for the planar training of the shoulder-elbow subcomponent of the Fugl-Meyer scale – albeit without achieving statistical significance). As mentioned earlier, we reliably incorporated therapists' feedback during the design phase of the protocol and have not observed increases in muscle tone or spasticity, as indicated by the Modified Ashworth scale, during the anti-gravity training protocol (actually the trend in 8 out of 9 patients is towards a decrease in tone). Note also that consistent with our past experience with the persons with chronic strokes, there was a statistically significant reduction in tone measurement from admission to discharge of performance-based planar robot therapy.
While the benefits from the additional anti-gravity therapy did not achieve statistical significance, this is likely due to the small sample size (9 outpatients). We anticipate that a modest increase of sample size will demonstrate statistical improvement for the shoulder and elbow of the anti-gravity training across the three clinical scales and we plan to commence trials shortly (See Table 3 and Table 4).
Conclusions
Our clinical experiments have focused on a fundamental question of whether task specific robotic training influences brain recovery. To date several studies demonstrate that in mature and damaged nervous systems, nurture indeed has an effect on nature. The improved recovery is most pronounced in the trained limb segments (i.e. shoulder and elbow). We have now embarked on experiments that will test whether we can continue to influence recovery, long after the acute insult, with a novel class of robotic devices.
It is broadly accepted that outcome measures do not significantly change in persons with chronic motor impairments more than six months from stroke onset. However, recent task specific training programs have resulted in improved motor performance in persons with chronic stroke. For example, our trials with persons with chronic stroke-related impairments showed that planar robotic training contributed to statistically significant improvements in motor abilities [8-10]. If so, further robot training of stroke survivors who were enrolled in the planar robot trials might result in additional performance improvements when a distinct training protocol is provided.
Our pilot results and novel robotic modules provide a proof of concept that supports our engineering efforts, as well as further clinical trials to test efficacy. We found that the protocol was safe and well tolerated by the patients with no patient presenting any adverse effect (e.g., shoulder pain). Furthermore, the pilot results suggested an additional reduction in shoulder-elbow impairment following the anti-gravity vertical training without detrimental changes in muscle tone or spasticity. Therefore we plan to investigate in detail the effect of training each movement component in isolation versus integrated spatial movement, and study its impact on disability. We expect that this will bring us closer to our ultimate goal, efficient delivery of optimal therapy, personalized to serve the individual's needs.
Acknowledgements
This work was supported by a grant from the Burke Medical Research Institute. Dr. Makiyama and Sandmann were supported by National Institute of Child Health and Development of N.I.H., Grant 1 R43 HD42900-01.
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| 15679916 | PMC544952 | CC BY | 2021-01-04 16:37:38 | no | J Neuroengineering Rehabil. 2004 Oct 26; 1:5 | utf-8 | J Neuroeng Rehabil | 2,004 | 10.1186/1743-0003-1-5 | oa_comm |
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J Neuroengineering RehabilJournal of NeuroEngineering and Rehabilitation1743-0003BioMed Central London 1743-0003-1-61567991410.1186/1743-0003-1-6ResearchA swimming robot actuated by living muscle tissue Herr Hugh [email protected] Robert G [email protected] Media Laboratory and the Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA2 Department of Biomedical Engineering, University of North Carolina at Chapel Hill, NC 27599, USA2004 28 10 2004 1 6 6 10 9 2004 28 10 2004 Copyright © 2004 Herr and Dennis; licensee BioMed Central Ltd.2004Herr and Dennis; 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.
Biomechatronics is the integration of biological components with artificial devices, in which the biological component confers a significant functional capability to the system, and the artificial component provides specific cellular and tissue interfaces that promote the maintenance and functional adaptation of the biological component. Based upon functional performance, muscle is potentially an excellent mechanical actuator, but the larger challenge of developing muscle-actuated, biomechatronic devices poses many scientific and engineering challenges. As a demonstratory proof of concept, we designed, built, and characterized a swimming robot actuated by two explanted frog semitendinosus muscles and controlled by an embedded microcontroller. Using open loop stimulation protocols, the robot performed basic swimming maneuvers such as starting, stopping, turning (turning radius ~400 mm) and straight-line swimming (max speed >1/3 body lengths/second). A broad spectrum antibiotic/antimycotic ringer solution surrounded the muscle actuators for long term maintenance, ex vivo. The robot swam for a total of 4 hours over a 42 hour lifespan (10% duty cycle) before its velocity degraded below 75% of its maximum. The development of functional biomechatronic prototypes with integrated musculoskeletal tissues is the first critical step toward the long term objective of controllable, adaptive and robust biomechatronic robots and prostheses.
Biomechatronicsbionicscyberneticshybrid roboticsmuscle actuatorsskeletal musclemuscle organ culturefunctional electrical stimulation
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Background
Many technological barriers exist for the implementation of life-like mobility in robotic and prosthetic systems. Included among these barriers are (1) the availability of high-energy density storage media, (2) the availability of adequate muscle-like actuators, and (3) the availability of biologically inspired sensory technologies. As a possible resolution to these challenges, we consider in this investigation the use of living muscle tissue as a viable actuator for synthetic devices.
Although important research has been conducted to advance a synthetic actuator technology with muscle-like properties, engineering science has not yet produced a motor system that can mimic the contractility, energetics, scalability and plasticity of living muscle tissue [1,2]. Muscle has several important advantages in addition to favorable dynamic characteristics [1-6]. In its function as a motor, muscle acts to provide positive mechanical work at a considerable aerobic transduction efficiency, or 1000 Joules of work per gram of glucose consumed [7]. It is a "smart material", having integrated sensors for the detection of displacement and rate of displacement (muscle spindles) as well as force (Golgi tendon organs). It can repair itself when damaged, and can functionally adapt to an increase in the demands of the environment by undergoing hypertrophic and hyperplastic growth [8] as well as fiber type transformations [9-12]. Muscle has integrated series-elastic components, which are thought to give rise to many of the "life-like" characteristics of animal movement [13], and the fuel that it consumes is a renewable resource, while the waste products produced are environmentally compatible.
In this investigation, we examine the feasibility of using animal-derived muscle as an actuator for artificial devices in the millimeter to centimeter size scale. Perhaps researchers in the past did not consider muscle tissue a viable mechanical actuator because of tissue maintenance and control difficulties. The objectives of this study are to identify, and to begin to address, the many technical challenges related to maintaining and controlling explanted muscle tissues in the context of a robotic platform. To this end, we construct a hybrid swimming robot comprising a synthetic elastomeric tail actuated by a single pair of whole muscle explants from frog semitendinosus muscle. We anticipate that basic swimming maneuvers such as straight-line swimming and turning can be performed by alternately modulating electrical signals to each muscle actuator across two electrode pairs, one on each muscle near the neuromotor junction. We further anticipate that a multi-day robotic maintenance or lifespan can be achieved by surrounding the muscle actuators with a specific bath of amphibian ringer's solution comprising antibiotic and antimycotic agents. To test these ideas, we construct two robotic build-ups, each comprising a freshly dissected pair of explanted semitendinosus muscles. For each build-up, pilot data are collected to characterize the robot's swimming mechanics and lifespan.
Methods
Muscle Removal and Maintenance
The surgical removal of muscle specimens designated for robotic actuation were performed according to procedures approved by the Committee on Animal Care, Northeastern University (Approval #0402-025-05). Briefly, adult frogs (Rana pipiens) were pithed, and both semitendinosus muscles were dissected free and removed with tendons intact. Before removal of the tissues from the animal, the length of each muscle belly was measured at an equilibrium or rest length. The resting length measurement was conducted on the intact muscle specimen with the limb positioned at an anatomically neutral position (see Table I for muscle lengths). After removal from the animal, the muscle, including its intact tendons, was weighed (see Table I for muscle mass). Each tendon was manipulated via tightly secured silk suture (size 5-0). Each muscle was then pinned at its rest length in a 100 mm Petri dish with a previously prepared SYLGARD (Dow Chemical) polydimethylsiloxane (PDMS) substrate.
Table I Muscle actuator parameters and swimming robot performance parameters (mean values, N = 4) at the maximum forward swimming speed for robotic build-ups, B1a and B1b.
Robot Muscle Mass (g) Muscle Len. (mm) Peak Muscle Strain Muscle Shortening Vel. (mm/s) Tail-beat Freq. (Hz) Tail Amp. (mm) Max. Robot Speed (mm/s) Wave Speed (mm/s) Wave Len. (mm) Slip
B1a 0.31 31 6.5% 25 3.1 16 31 121 39 0.26
B1b 0.34 31 6.5% 25 3.1 16 45 140 45 0.32
Shortly before harvesting the muscles, two fresh liters of amphibian ringer solution were prepared according to a protocol specifically designed for frog organ culture [14,15]. The amphibian ringer comprised: NaCl, 83.89 mM; NaHCO3, 28.11 mM; KCL, 1.5 mM; KH2PO4, 1.2 mM; MgSO4, 1.2 mM; CaCl2 Dihydrate, 1.3 mM; Glucose, 10 mM; MEM Amino Acid Mixture, 1:50 dilution (GIBCO #1130051); MEM Vitamin Mixture, 1:100 dilution (GIBCO # 1120052); Creatine, 1 mM; DL-Carnitine, 1 mM; Ferric Chloride, 0.9 μM; Human Serum Transferrin, 1.35 μM; Insulin, 1 mU/ml; L-Glutamine, 1:100 dilution; Sigma Chemical #A9909, 1:50 dilution (an antibiotic/antimycotic). A broad-spectrum, antibiotic/antimycotic was added out of necessity for long-term maintenance of the muscles, ex vivo. We observed, for periods greater than 24 hours, septic degradation of the muscle specimens in the absence of the antibiotic/antimycotic agents. After each muscle was placed within a Petri dish, a small volume of ringer solution was used to surround each muscle, the balance being used in the test tank for the swimming robot evaluations. The total amount of time between muscle removal from the animal to finalizing the muscle installation into the robotic swimmer was approximately 1 hour.
Test Tank Construction and Swimming Robot Design
The test tank was constructed from 6 mm (1/4") thick cast acrylic sheet, welded together with methylene chloride, with silicone fish tank adhesive being applied to form a water-tight seal at each joint. The test tank was 30 cm square and 6 cm deep.
The robotic platform (Figure 1) was specifically designed to accommodate the frog semitendinosus muscles. The actuators were a single pair of whole muscle explants from frog semitendinosus muscle, arranged as antagonists on either side of the robot in an open-frame architecture. This open-frame architecture exposed the explanted tissues to the amphibian ringer solution during robot operations. The robotic platform mass before installation of the muscle actuators was 12.15 g, and the overall length (L) was 12 cm. Of this total length, the fore or anterior 7 cm section comprised a rigid frame machined from acetyl (Delrin) with nylon threaded fasteners, while the aft or posterior 5 cm section comprised a compliant cast silicone tail. A closed-cell Styrofoam float was affixed to the rigid forward section to provide positive buoyancy. The compliant tail had a narrow rectangular section between the mounting flange and the insertion to the rigid Delrin backbone. This compliant segment (Figure 1) served as a hinge for single degree-of-freedom actuation, permitting mediolateral oscillations of the tail. This narrow compliant section also provided a restoring force to return the tail to its neutral position when no muscle force was applied.
Figure 1 The Biomechatronic Robotic Platform. The top image is a photograph (side view) of the device (robot B1a) shortly after initial testing. The bottom image is a schematic (to scale) with the float and embedded controller removed, showing the main components of the system: semitendinosus muscles (M), suture attachments (s), Styrofoam float (F), electrode wires (w), cast silicone tail assembly (T), rigid Delrin backbone (D), rigid Delrin head piece (H), lithium batteries (B), compliant hinge segment (k), cylindrical tail mounting boss (a), encapsulated microcontroller, infra-red sensor, and stimulator unit (C).
The single part silicone RTV (Dow Corning type 734 flowable silicone) tails were cast using a 5-part virgin Teflon mold machined to form a single solid tail assembly with all of the features shown in Figure 1. Casting of one-part silicones was accelerated by the addition of ~1 drop of water-based food coloring per 10 ml of silicone elastomer. This technique allowed tails of different mechanical properties to be readily color-coded during casting, and allowed the elastomer to be fully polymerized and set throughout the entire cross section within 15 minutes of initial mixing. Castings of this sort are not biocompatible for several days due to the emission of acetic acid. If placed in an aqueous environment too quickly with a living tissue, tissue damage would inevitably result. Thicker sections require longer waiting periods, but we found that storage on the shelf for at least one week prior to use was sufficient to achieve biocompatibility with no noticeable effects on the explanted tissues. The cylindrical mounting boss permitted different tail assemblies to be inserted or removed, simply by pressing the boss into a cylindrical receptacle in the Delrin spine. A 0.07 mm diametric interference fit was used. The tail mold allowed different tail lengths and base thicknesses to be cast by simply changing the two Teflon plates that formed the sides of the triangular mold cavity, allowing easy adjustment of the tail compliance. The final tail geometry resulted in sufficient compliance to allow the tail to assume a sigmoidal shape, with a wave traveling caudally when actuated in water at frequencies above ~2 Hz. After design iterations, the spring constant of the compliant tail was 0.42 Newton*cm/radian, and the stiffness remained the same throughout all subsequent experimental sessions.
The onboard electronics were based upon a previously published design for an implantable muscle stimulator [16], and thus the circuit architecture will not be reproduced here. Several minor modifications were made to the circuit hardware. The MAX630 DC-DC converter was not used. The system was powered by two 3 Volt, 48 mAh tabbed lithium batteries (Panasonic # BR1225-1VC) connected in series. The actual operating voltage of the batteries was ~2.8 V [16,17]. The embedded microprocessor (PIC16C54A, SSOP package), was operated from only the first battery in the series, at 2.8 VDC with a 40 kHz crystal oscillator to minimize the power consumption of the device [16,17]. The stimulator output buffer was powered by both lithium batteries in series and was constructed using logic level HEXFETs (International Rectifier # IRF7105) to provide capacitive discharge square pulse stimulation to each actuator at ~5.6 V. The pulse was sufficient to elicit a sub-maximal contraction of each semitendinosus muscle. To minimize the size of the on-board control electronics, a PC board was not used, rather each component was soldered by hand directly to the leads of each IC chip with jumper wires added as necessary.
Stimulation was controlled remotely via a unidirectional infra red (IR) link from a hand-held command module. The on-board fixed stimulation parameters were: amplitude = 5.8 V (alternating bipolar) [16], frequency = 80 Hz, pulse width = 100 μsec. The remote command module allowed for manual control of the onset of stimulation, the train duration (0 to 2550 ms, in 10 ms increments), the dwell time (time between stimulus trains (0 to 2550 ms, in 10 ms increments), and a setting to control either alternating stimulation between the antagonistic actuators for forward motion, or continuous one-sided muscle activation for steering control.
The electrodes were fashioned from medical grade TFE coated 40 AWG stainless steel multi-strand electrode wire (Cooner Wire). The distal ends were stripped to allow the electrode wire to be wrapped around each muscle, as described previously [16]. The finished on-board control modules were encapsulated using electronic grade epoxy, followed by 6 coats of Dow silicone elastomer #734 dispersed with toluene, according to the method described previously [17].
During robotic swimming operations, the fuel sources were a glucose-bearing ringer solution (~2 g/L glucose), and lithium batteries to power the embedded microcontroller and stimulator system. Due to the micro-power electronic design, the estimated battery life for the system was ~21 days (assuming a 10% stimulation duty cycle) [16,17].
The semitendinosus muscle was selected primarily due to its convenient size and tendon anatomy. It is easily dissected with both proximal and distal tendons attached. The proximal tendons of each semitendinosus muscle were sutured to the rigid Delrin head piece, and the distal tendons were sutured to the lateral mounting flange on each side of the tail base using 5-0 braided silk suture (Figure 1). The muscles were mounted symmetrically on opposite sides of the robotic platform to act as antagonists, providing a single degree-of-freedom reversible actuator for the base of the compliant tail. Muscle length was adjusted manually during installation by sliding the sutures through the tail flange to achieve the desired muscle length. Both muscle lengths were adjusted to set each muscle at rest length when the tail was in its neutral position. With no muscle force applied, the restoring torque of the silicone hinge-joint returned the tail to the neutral position, thus both muscles were at their rest length when neither was activated. This important feature is essential for muscle maintenance, as muscles maintained at stretched lengths are known to degenerate more rapidly than muscles held at lengths corresponding to the ascending limb of the length-tension curve [14].
Robotic Experiments and Performance Characterizations
Two robotic platforms were evaluated in terms of muscle actuator performance, swimming efficiency and locomotory maneuverability. Each robotic platform was designated "B1x", where "x" indicated the build-up, serialized as "a, b, c, ..." for each subsequent pair of explanted frog muscles. Two build-ups were constructed, B1a and B1b, each with a separate pair of freshly explanted frog semitendinosus muscles.
Prior to swimming evaluations, two liters of ringer solution (ringer composition in Methods: Muscle Removal and Maintenance) were poured into the test tank, providing a fluid depth of approximately 2.1 cm, enough for the robot to swim without touching the bottom of the tank. The tank temperature was measured but not controlled, and was allowed to stabilize at room temperature, approximately 22°C for the duration of each experiment. The ringer solution was aerated with unfiltered room air using 4 standard porous stone fish tank aerators, one placed at each corner of the tank, and connected to an aquarium aeration pump via silicone tubing. Aeration was discontinued briefly before each test run to minimize turbulence in the test tank. For each robotic build-up, or for each pair of explanted semitendinosus muscles, the test tank ringer solution was not replaced or replenished for the entirety of the robotic experimental session.
Muscle installation was carried out with the robotic platform partially immersed in ringer solution using #5 forceps (Fine Science Tools). After installation was complete, the muscles were allowed to acclimate for a period of approximately 5 minutes before stimulation. The robot was manually placed to allow forward motion through the bath, and muscle stimulus parameters, specifically stimulus train duration and dwell time, were varied manually until the maximum swimming velocity was achieved. To increase swimming speed, dwell period was decreased and train duration was increased until further decreases in dwell time or further increases in train duration did not result in additional increases in forward swimming speed. During experimentation, swimming speed was determined by measuring the amount of time required for the robot to swim across a known, fixed distance. Once the maximum swimming speed was achieved, the ventral view of the swimming robot was filmed (Sony Model #DCR-TRV820; 30 frames/sec), and the film was then digitized to determine tail-beat frequency, tail amplitude, and the wave speed and wave length of the propulsive body wave. In addition to forward straight-line swimming, muscle stimulus parameters were varied to investigate turning maneuvers. At a maximum forward swimming speed, the robot's open loop, alternating stimulation pattern between the antagonistic actuators was switched to a continuous one-sided muscle activation for steering control, causing the robot to turn in the direction of the single stimulated muscle (a medial turn resulting from one-sided medial muscle stimulation). Here again, the ventral view of the swimming robot was filmed, and the film was then digitized to determine the maximum turning radius.
For each tail-beat period, at least 10 video frames were captured, separated in time by 33 ms, depending on the swimming speed of the robot. A customized software program was used to digitize 10 points on each side of the outline of the ventral silhouette of the robot, for a total of 20 points for each image. A series of cubic spline functions were used to draw the best-fit line along these points [18,19], and a midline was constructed. Tail-beat frequency was measured by tracking a digitized point on the tail tip from the ventral view over the course of one tail-beat cycle and dividing by the elapsed time. Tail amplitude was determined by measuring the tip-to-tip linear distance at the two extremes of tail excursion and then dividing by two. As described by [20], mean propulsive wavelength was measured directly from the reconstructed midlines as the distance between two successive peaks present on the robot's body. Propulsive wave speed was calculated by dividing the distance between the anterior most point of the body exhibiting undulation and the tail tip by the time required for the crest of the wave to pass through these points.
To estimate the overall mechanical swimming efficiency of each robotic build-up, we calculated the robot's slip value, a dimensionless velocity [21]. A high slip value indicates a larger contribution to rearward, thrust-producing forces than lateral forces. Slip was calculated by dividing the robot's steady state swimming velocity by its propulsive wave speed.
To estimate muscle actuator performance at the maximum swimming speed, muscle strain and shortening velocity were estimated using the tail-beat frequency and amplitude measurements taken from the digitized films. After the swimming experiments were finalized, the change in linear distance between the robot's muscle attachment points was measured when the robot's tail was re-positioned from a neutral, straight position to the tail amplitude posture measured during straight-line swimming. As an estimate of peak muscle shortening strain, this linear-distance change was then divided by the muscle's resting length, or the muscle belly length when the tail was held straight (resting length measurement protocol defined in Methods: Muscle Removal and Maintenance). Still further, to estimate muscle-shortening velocity at the maximum swimming speed, the measured linear-distance change between muscle attachment points was divided by the time required for the tail to re-position from a neutral, straight position to the tail amplitude posture measured during straight-line swimming. This time period was measured from the digitized films and was equal to approximately one quarter of a tail-beat period.
For the turning maneuvers, the turning radius was estimated from the ventral video images by tracking the spatial trajectory of a point midway between the tail tip and the nose of the robot, a distance 6 cm from the tail tip along the midline of the robot when the tail assumed a neutral, straight orientation. The turning radius was the radius of a circle with an arc curvature equivalent to the midpoint trajectory curvature.
Semitendinosus Contractile Experiment: Maximum Shortening Velocity
To estimate the contractile efficiency of the robotic muscle actuators at the maximum swimming velocity, a separate experiment was conducted to determine the maximum shortening velocity of freshly dissected semitendinosus muscles of comparable size and rest length to that of the muscles employed in robotic build-ups, B1a and B1b. Six freshly dissected semitendinosus muscles were placed in a muscle characterization apparatus (Aurora Model 305B) and isotonic contraction experiments [22] were conducted to measure the muscles' maximum shortening velocity. The contractile experiment was conducted at the same temperature as the robotic experiments, or 22°C.
Results
Robotic Performance Characterizations
For the B1a and B1b robotic swimmers, the locomotory performance parameters at maximum swimming velocity are summarized in Table I. Table I also includes the muscle actuator mass and rest length for each robotic build-up. For both robot B1a and B1b, the total muscle mass did not exceed 6% of the total mass of the robot (B1a = 4.8%; B1b = 5.3%). Even with such a low relative actuator mass, swimming robots B1a and B1b achieved top speeds greater than 1/4 and 1/3 body lengths per second, respectively (here the robot's total length, 12 cm, was used as the normalization factor). For both robotic swimmers, forward swimming speed was readily controllable simply by decreasing the dwell period or by increasing the train duration. The maximum steady state, forward swimming speed was achieved with alternating actuator contractions of 110 ms train duration, with 40 ms dwell periods between each stimulus train, resulting in 3.1 tail-beats per second. Further increases in the stimulus train duration or further decreases in the stimulus dwell time did not result in additional increases in forward swimming speed.
Each robotic build-up was capable of the following controlled maneuvers: forward accelerations, decelerations, steady state gliding, and turning to the right or left. The robot was capable of surface swimming only, so all maneuvers were restricted to 2-dimensions. Turning was accomplished after forward momentum had been established by continuously activating only one actuator. The minimum gliding turn radius was 400 mm as estimated from the digitized video images of the robot's midpoint trajectory.
After swimming the full length of the test tank, the robot was manually repositioned to the opposite end of the tank where it began, once again, to swim across the tank width. Typically, a period of swimming activity (~3 min) was followed by a period of swimming inactivity (~30 min). Due to muscle fatigue, periods of inactivity were required to restore the robot's peak swimming velocity to at least 75% of its maximum value measured during the first session of robotic swimming (first 10 minutes of the robot's lifespan). Robot B1a swam for a sum total of 45 minutes over a 7.5 hour lifespan (10% duty cycle), after which its swimming velocity degraded below 75% of its maximum value even after a 30 minute period of swimming inactivity. In distinction, robot B1b swam for a much longer period – a sum total of 4 hours over a 42 hour lifespan (10% duty cycle) before its velocity degraded below 75% of its maximum value following a 30 minute period of swimming inactivity.
To compare the overall swimming efficiency of each robotic build-up, we calculated the propeller efficiency using the measure of slip (swimming velocity/ propulsive wave speed) (Table I). In a steady-state condition, at the maximum forward swimming speed, slip values for robotic build-ups, B1a and B1b, were 0.26 and 0.32, respectively. By comparison, slip values generally increase with swimming speed in fish, ranging from 0.2 to 0.7 in most fish [19,20]. The mechanical swimming efficiency of robots B1a and B1b, as determined by their respective slip values, were within the biological efficiency range.
Maximum Shortening Velocity and the V/Vmax Ratio at Maximum Swimming Speed
In a separate experiment from the robotic investigations, six freshly dissected semitendinosus muscles (mass = 0.34 ± 0.04 g; rest length = 30 ± 1 mm; Mean ± S.E., N = 6 muscles) produced a maximum shortening velocity, Vmax, of 78 ± 3 mm s-1 (Mean ± S.E., N = 6 muscles) in isotonic contractions. At the maximum swimming speed, the muscle actuators within robots B1a and B1b experienced a shortening velocity of 25 mm s-1 (Table I), giving a V/Vmax ratio of 0.32, an intermediate contraction velocity where muscle typically produces peak power and efficiency [7].
Discussion
Although a great deal of research has been conducted to advance a synthetic actuator technology with muscle-like properties, engineering science has not yet produced a motor system that can mimic the contractility, energetics, scalability and plasticity of living muscle tissue [1,2]. In this investigation, we examine the feasibility of using animal-derived muscle as an actuator for artificial devices. We construct a simple robotic platform powered by explanted living amphibian muscle and controlled by an embedded microcontroller via an infra red data link. Using an open loop control and a simple interface design, we present preliminary data that suggests that living muscle might one day be employed as a practical, controllable actuator. Hybrid robot B1b remained active for up to 42 hours, and during that time, performed basic swimming maneuvers such as starting, stopping, turning and straight-line swimming at speeds exceeding 1/3 body lengths per second. The muscle-actuated swimming robot also offered a reasonable swimming efficiency, as indicated by a slip value of 0.32 (see Table I).
Muscle Fiber Type and Control
The frog semitendinosus muscles employed in the robot were comprised predominantly of fast-twitch muscle fibers, and therefore provided higher mechanical power, at the expense of being considerably more fatigable, than would have been achievable using a slow-twitch muscle of comparable size. Ideally, a biomechatronic swimming robot would incorporate several muscle fiber types to permit both explosive as well as low-power locomotion and maneuvering. For the robotic platform of this investigation, it is important to note that the stimulation was non-physiologic in many ways. Each muscle was stimulated in bulk, with all fibers being subjected to approximately the same electric field. In living muscle in vivo, individual motor axons innervate one or more muscle fibers, establishing the fundamental neuromotor functional unit: a motor unit. In a sophisticated biomechatronic system, a motor-unit level of control would be desirable (fast vs. slow), both for controllability and for tissue phenotype maintenance.
Tissue Failure Modes
In this study, the performance of the muscle actuators eventually degraded to the point where they were no longer effective mechanical actuators. Several factors contributed to the observed tissue degradation. To begin with, explanted muscle generally has a very finite functional life expectancy [14,15], usually less than one day. Excluding such transient failure modes as metabolic muscle fatigue, the major failure modes of muscle in vitro generally fall into one of the following categories: (1) core necrosis due to lack of oxygenation/capillary perfusion and large diffusion distances, (2) sepsis, (3) exogenous toxicity, (4) electro-chemical damage resulting from excessive electrical stimulation, (5) accumulated contraction-induced injury, (6) sarcomeres heterogeneity leading to loss of thick and thin filament overlap in regions of muscle fibers (exacerbated by prolonged periods at or above the optimal length for force generation), and (7) direct mechanical damage to the muscle from external sources, such as the robot frame, attachment hardware, or electrodes.
For the tissue-actuated device of this investigation, several design considerations were made to minimize many of these failure modes. The bath was aerated to assist oxygen delivery to the tissues, although this strategy would only be helpful to the outer shell of muscle fibers no greater than ~200 μm from the surface. In addition, the level of muscle cell depolarization was kept to a minimum in order to limit electro-chemical damage [16]. Still further, the muscle actuators were attached to the robot frame at rest length in order to minimize the risk of excessive muscle strains and sarcomere heterogeneity. Clearly, when looking to the future, other failure modes must be considered when very long periods of ex vivo tissue maintenance are necessary. These include loss of muscle excitability and mass, phenotypic drift, and de-differentiation of the muscle from desired adult muscle phenotypes.
Muscle Actuator Source: Engineered Muscle versus Explanted Tissue
Even though organogenic mechanisms are poorly understood, it is nonetheless possible to engineer functional muscle organs from individual cells in culture [23-26], but currently these tissue constructs have several practical limitations that limit their usefulness as living actuators. Among these limitations are: (1) low contractility, similar to that during early stages of muscle development, (2) low excitability, thus requiring large amounts of electrical energy to adequately stimulate the tissue to contract, (3) the lack of perfusion, which limits the tissue cross section to a maximum radius of approximately 200 μm, and (4) the lack of suitable tissue interfaces, both neural and mechanical. Given such technological limitations, we chose in this study to employ explanted muscle tissues for robotic actuation. However, once these technical hurdles are overcome, engineered muscle actuators might offer important advantages to the construction of biomechatronic robots.
Future Work
The results of this investigation, although preliminary, suggest that some degree of ex vivo robustness and longevity is possible for natural muscle actuators if adequate chemical and electromechanical interventions are supplied from a host robotic environment. Clearly, an important area of future research will be to establish processes by which optimal intervention strategies are defined for a given hybrid-machine task objective. Another important area of research will be tissue control. It has been established that natural muscle changes in size and strength depending on environmental work-load, and when supplied with appropriate signals, changes frequency characteristic or fiber type [9-11]. Hence, an important area of future work will be to put forth strategies by which muscle tissue plasticity can be monitored and controlled. Finally, strategies must also be devised to control the force and power output of muscle, in the context of robotic systems, through the modulation of electrical pulses to the muscle cell. To achieve the long-term objective of functional, muscle-actuated robotic and prosthetic devices, we feel controlling machine movements through electrical stimulation, harnessing muscle tissue plasticity, and maintaining ex vivo contractility are critical areas for future research.
Conclusion
In this paper, we ask whether muscle tissue explants can be employed as mechanical actuators for robots in the millimeter to centimeter size scale. Using a very simple control and interface design, we present preliminary data that suggests that living muscle might one day be employed as a practical, controllable actuator. The robot of this investigation remained active for up to 42 hours, and during that time, performed basic swimming maneuvers such as starting, stopping, turning and straight-line swimming at speeds exceeding 1/3 body lengths per second. It is our hope that this work will lead to further studies of tissue actuated robots and prostheses that will result in an even wider range of biomechatronic machine capabilities.
Acknowledgment
The authors thank Dr. Richard Marsh for his invaluable assistance with the preparation of the amphibian ringer solution and the characterization of the semitendinosus frog muscle.
This work was supported by the Defense Advanced Research Projects Agency (DARPA #6890899, An Actin-Myosin Machine).
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Immun AgeingImmunity & ageing : I & A1742-4933BioMed Central London 1742-4933-1-11567992110.1186/1742-4933-1-1EditorialImmunity & Ageing: a new journal looking at ageing from an immunological point of view Vasto Sonya [email protected] Calogero [email protected] Managing Editor Gruppo di Studio sull'Immunosenescenza, Dipartimento di Biopatologia e Metodologie Biomediche, Università di Palermo, Corso Tukory 211, 90134 Palermo, Italy2 Editor-in-Chief Dipartimento di Biopatologia e Metodologie Biomediche, Università di Palermo, Corso Tukory 211, 90134 Palermo, Italy2004 29 10 2004 1 1 1 29 9 2004 29 10 2004 Copyright © 2004 Vasto and Caruso; licensee BioMed Central Ltd.2004Vasto and Caruso; 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 the elderly, many alterations of both innate and clonotypic immunity have been described. Alterations to the immune system in the elderly are generally viewed as a deterioration of immunity, leading to the use of the term immunosenescence. However, although many immunological parameters are often notably reduced in the elderly, retained function of both innate and clonotypic immunity in the elderly is tightly correlated to health status. Recognising the important role of the immune system in ageing, over the last few years, journals oriented towards gerontology and geriatric sciences have increasingly published articles dealing with the immunology of ageing, but a specialised journal in this area does not exist. Immunity & Ageing is a new Open Access, peer reviewed journal that aims to cover all the topics dealing with innate and clonotypic immunity which are relevant to ageing. The journal will provide an opportunity to focus on this topic, which is emerging as one of the critical mechanisms of ageing. Furthermore, as an online, Open Access journal, Immunity & Ageing will promote immediate accessibility to research, which is generally not possible for articles published in printed journals. We hope this forum, concentrating on the themes of ageing and immunology with a strong focus on human studies, will create a new perspective for viewing a world that is inevitably becoming older.
ageingimmunosenescenceinflammationopen access
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Immunity & Ageing is a new Open Access, peer reviewed journal that aims to provide a forum for articles examining ageing from an immunological point of view.
During the past century, humans have gained more years of average life expectancy than in the last 10,000 years; we are now living in a rapidly ageing world. The sharp rise in life expectancy, coupled to a steady decline in birth rates in all developed countries, has led to an unprecedented demographic revolution characterized by an explosive growth in the number and proportion of older people. The number of people aged 60 years or older exceeded 635 million in 2002, and is expected to grow to nearly 2 billion by 2050. The proportion of people aged 60 and over stands about 1 in 4 in many Western European countries as well as in Japan. Should the present trend continues, this ratio is expected to reach 1 in 3 by 2050 [1]. Among the aged, the oldest old (>85) make up the fastest growing category. As access to medical care improves worldwide, the rate of population ageing will accelerate. If global communications is making the world "young and fast", then global ageing is surely "maturing and slowing" it. In any case, these epidemiological facts underscore the importance of studies on successful and unsuccessful ageing and necessitate the prompt spread of knowledge about ageing in order to satisfactorily decrease the medical, economic and social problems associated with advancing years.
Ageing
Ageing is a post-maturational process that, due to a diminished homeostatic capacity and increased vulnerability, reduces responsiveness to environmental stimuli and is generally associated with an increased predisposition to illness and death. At the beginning of the 19th century, mortality was described as increasing exponentially with respect to progression through the lifespan [2]. This trend, also described in invertebrates, persists: in Western countries the mortality rate increases 25 times more rapidly in individuals over 60 years old compared to people aged 25–44. Causes of death in aged people are increased compared with individuals between 25 and 44 years old: cancer 43-fold, pneumonia and influenza 89-fold, heart disease 92-fold and stroke and chronic lung disease greater than 100-fold [3]. Thus far, to understand ageing mechanisms, much attention has been paid to gene mutations in invertebrates and caloric restriction in rodents. However, these data suggest a key role for immunity in the survival of the elderly because susceptibility to these diseases depends at least in part on optimal immune function [4,5]. So, a better understanding of the ageing immune system may provide the most important clues for slowing the inevitable decline associated with the passage of time.
Immunity in ageing
In the elderly, many alterations in innate and clonotypic immunity have been described and viewed as deleterious, hence the term immunosenescence. In 1969, Roy Walford published his landmark book, "The Immunologic Theory of Aging", and first coined the term immunosenescence [6]. Significantly, most of the areas that he pioneered during his illustrious research career remain the "hot" areas of current gerontological research. On the other hand, immunosenescence is a complex process involving multiple reorganizational and developmentally regulated changes, rather than simple unidirectional decline of the whole function [7,8]. However, some immunological parameters are commonly notably reduced in the elderly and, reciprocally good function is tightly correlated to health status [4,5].
Innate immunity in ageing
The process of maintaining life for the individual is a constant struggle to preserve his/her integrity. This can come at a price when immunity is involved, namely systemic inflammation [9]. Inflammation is not a negative phenomenon per se: it is the response of the immune system to the invasion of viruses or bacteria and other pathogens. The problem is that in the course of evolution the human organism was set to live 40 or 50 years. Today, however, the immune system must remain active for longer. This very long activity leads to a chronic inflammation that slowly but inexorably damages all the organs: this is the typical phenomenon linked to ageing and it is considered the major risk factor for age-related chronic diseases, such as osteoporosis, sarcopenia, type 2 diabetes, Alzheimer's disease and atherosclerosis, though progression seems also dependent on the genetic background of individuals [4,5,8,10,11]. Emerging evidence suggests that pro-inflammatory genotypes are related to unsuccessful ageing, and, reciprocally, controlling inflammatory status may allow a better chance of successful ageing [4,5,8,12]. In other words, age-related diseases are "the price we pay" for an active immune system that defends us thorough out life, but also has the capacity to harm us later, as its fine tuning becomes compromised [13]. In fact, our immune system has evolved to control pathogens, so pro-inflammatory responses are likely to be evolutionarily programmed to resist fatal infections with an increased resistance to pathogens. Thus, inflammatory genotypes are an important and necessary part of the normal host responses to pathogens in early life, but the overproduction of inflammatory molecules might also cause immune-related inflammatory diseases and eventually death later. Therefore, low responder genotypes might better control inflammatory responses and age-related disease development, resulting in an increased chance of long life survival in a facilitory environment with reduced pathogen load and medical care, such as might be present in Western societies [12,14-16].
Clonotypic immunity in ageing
Recently, longitudinal studies have shown that a cluster of immunological parameters can be used to evaluate the expectation and quality of life, i.e. the immunological risk phenotype [17,18]. Senescence of clonotypic immunity is most likely principally a result of alterations to T cells. Lifelong and chronic antigen load seems to be the major driving force of immunosenescence, which impacts on human lifespan by reducing the number of virgin antigen-non experienced T cells, and, simultaneously, fills the immunological space with expanded clones of memory and effector, antigen-experienced T cells [18-20]. Gradually, the T cell population shifts to a lower ratio of naïve cells to memory cells, the thymus pumps out fewer naïve T cells with age and those T cells remaining, especially the CD8+ subset, also show increased oligloclonality with age [4]. Thus, the repertoire of cells available to respond to antigenic challenge from previously unencountered pathogens is reduced. In addition, older organisms are often overrun by memory cells that carry a single type of T cell repertoire, i.e. clonal expansion. Thus, the memory cells from old individuals might recognize a limited set of antigens despite being plentiful in number. Many of the clonal expansions crowding an elderly person's immune system result from previous infections by persisting viruses [18,19].
In contrast to T cells, no evidence for a loss of B cell function has been found as neither the total number of B cells or immunoglobulin secreting cells have been shown to be profoundly decreased with age. However, the B-cell repertoire is influenced by ageing during an actual immune response, where the spectrum of expressed immunoglobulin genes, as well as the frequency of somatic mutations, affects the quality, though not necessarily the quantity, of the antibody response, which is highly relevant in clinical practice [7,21].
Immunity & Ageing. Why do we need an Open Access journal?
Considering the paramount function of the immune system during ageing, journals oriented towards gerontology and geriatric sciences are now publishing an increasing number of articles dealing with immunology and ageing, but a specialised journal in this area does not exist. Immunity & Ageing was conceived to cover all topics relevant to immunity and ageing in an interdisciplinary manner. It is clear that immunological mechanisms are involved in process and manifestation of ageing in many systems. An example is displayed by the increase in serum and tissue levels of circulation pro-inflammatory cytokines which is accompanied by typical cellular ageing phenomena such as telomeric loss, oxidative damage, DNA defects, accumulation of advanced glycosylation products, cellular loss and others. These defects may be stimulators of cytokine secretion, and subsequently, cytokines are released into the systemic circulation. This results in a slowly progressive endo-crinosenescence and neurosenescence. The different factors may influence each other in form of a vicious spiral [22].
The journal will provide an opportunity to focus the topic of immunology of ageing, which is emerging as one of the critical mechanisms of ageing which aspects should get to all scientists physicians and other professions who are involved in the different inter-related disciplines, and the bridge that is emerging between these fields.
Furthermore, as an online, Open Access journal, Immunity & Ageing will promote "immediate" accessibility to this fast moving area of research, which is generally not possible for articles published in printed journals. From a modern perspective, electronic publishing is obviously attractive for its speed, easy global access and low cost, which all present considerable advantages over print and are seen as attractive factors by authors, readers and publishers. Open Access appears to provide exactly what people would like: rapid publication for authors, free access to information for readers, and inexpensive, global availability to readers for publishers [23]. We believe knowledge should belong to those who want it and access should not be unjustly and unacceptably expensive or difficult. Immunity & Ageing will take advantage of the Open Access policy to make peer-reviewed information widely and almost immediately available. We hope this forum, focussing on the themes of ageing and immunology, will create a new perspective for looking at a world that is inevitably becoming older. Creating and pushing forward a research knowledge base in Immunity & Ageing provides a unique opportunity to dissect out some parameters which might make health span equate with increasing lifespan.
Acknowledgements
Sonya Vasto is a PhD student on the Pathobiology PhD course (directed by Calogero Caruso) of Palermo University. Calogero Caruso is indebted both to Claudio Franceschi and Graham Pawelec, mentors extraordinaire in the field of Immunology of Ageing and to his colleagues (Giuseppina Candore, Giuseppina Colonna-Romano and Domenico Lio) and PhD and Post-graduate students of Gruppo di Studio sull'immunosenescenza.
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Immun AgeingImmunity & ageing : I & A1742-4933BioMed Central London 1742-4933-1-21567992310.1186/1742-4933-1-2ReviewThe immunotherapy of Alzheimer's disease Weksler Marc E [email protected] Department of Medicine, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA2004 12 11 2004 1 2 2 18 10 2004 12 11 2004 Copyright © 2004 Weksler; licensee BioMed Central Ltd.2004Weksler; 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.
Only a small percentage of patients with Alzheimer's disease benefit from current drug therapy and for only a relatively short time. This is not surprising as the goal of these drugs is to enhance existing cerebral function in Alzheimer patients and not to block the progression of cognitive decline. In contrast, immunotherapy is directed at clearing the neurotoxic amyloid beta peptide from the brain that directly or indirectly leads to cognitive decline in patients with Alzheimer's disease. The single trial of active immunization with the amyloid beta peptide provided suggestive evidence of a reduction in cerebral amyloid plaques and of stabilization in cognitive function of half the patients who developed good antibody responses to the amyloid beta peptide. However, 6% of actively immunized Alzheimer patients developed sterile meningoencephalitis that forced the cessation of the clinical trial. Passive immunotherapy in animal models of Alzheimer's disease has provided similar benefits comparable to those seen with active immunotherapy and has the potential of being effective in the half of Alzheimer's disease patients who do not make a significant anti-amyloid beta peptide antibody response and without inducing T-cell-mediated encephalitis. Published studies of 5 patients with sporadic Alzheimer disease treated with intravenous immunoglobulin containing anti-amyloid beta peptide antibodies showed that amyloid beta peptide was mobilized from the brain and cognitive decline was interrupted. Further studies of passive immunotherapy are urgently required to confirm these observations.
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Introduction
The headline "Minimal benefit is seen in drugs for Alzheimer's disease" summarized an article in the New York Times concerning drug therapy of Alzheimer's disease [1]. The inescapable conclusion was that present drug therapy benefits only a very small percentage of the 4.5 million Americans with Alzheimer's disease patients. This is not surprising as the goal of today's drugs is to enhance existing cerebral function in Alzheimer patients and not to attack the basic causes of their progressive cognitive decline.
In contrast to today's drug therapy for Alzheimer's disease, immunotherapy is directed at the neurotoxic amyloid beta peptide that directly or indirectly leads to cognitive decline in patients with Alzheimer's disease. Most investigators believe that the accumulation of amyloid beta peptide in the brain of elderly adults is not only a hallmark of Alzheimer's disease but is the primary cause of cognitive decline [2]. There is secure evidence from animal studies and preliminary evidence from patients with sporadic Alzheimer disease that immunotherapy can block the accumulation of the neurotoxic amyloid beta peptide in the brain and cognitive decline in patients with Alzheimer's disease. Reducing the level of amyloid beta peptide in the brain can be achieved by decreasing the production of the amyloid beta peptide and/or by increasing its clearance from the brain. Decreased production of amyloid beta peptide would follow the inhibition of the beta and gamma secretases, enzymes that cleave the 40/42 amino acid amyloid beta peptides from the 770 amino acid transmembrane amyloid precursor protein (APP) or the augmentation of the activity of the alpha secretase that lowers amyloid beta peptide production [3,4]. Although inhibitors of beta or gamma secretases are known to suppress amyloid beta peptide generation from APP in cultured cells, the available inhibitors are too toxic for clinical use. Furthermore, it is likely that removal of cerebral amyloid beta peptide will be necessary to reverse the accumulated amyloid beta peptide that is already present at the time when Alzheimer's disease is recognized.
Induction or infusion of anti-amyloid beta peptide antibodies prevents the accumulation of cerebral amyloid beta peptide from the brain of mouse models of familial Alzheimer's disease and may work in humans with sporadic Alzheimer's disease [6,7]. Even more encouraging are the reports that immunotherapy reverses cognitive decline in the mouse models of familial Alzheimer's disease and may act similarly in elderly humans with sporadic Alzheimer's disease [8-10]. This article will review and place in historical perspective the development of immunotherapy, in general, and its application to experimental murine models of familial Alzheimer's disease and to a small number of elderly patients with sporadic Alzheimer's disease so far studied.
Immunotherapy
Immunity was first recognized by the fact that people who recovered from an infection were often protected from re-infection. Thus, Thucydides wrote of the Athens epidemic of plaque that occurred in 430 BC "the same man was never attacked twice – at least fatally" [11]. Twelve hundred years later, Rhazes wrote that smallpox struck children, rarely late in life, and generally only once [12]. The fact that clinical disease conveyed immunity raised the question whether the induction of a mild smallpox infection might protect individuals from this often fatal disease. Variolization, the inoculation with pustular material from a smallpox patient, was first reported in the 17th century in China to protect from virulent smallpox infection [13] and brought to wider attention in 1714 by Timoni's letter to the Royal Society of London reporting on variolization in Turkey [14]. Despite the clinical efficacy of variolation, the dose of a living, virulent, pathogen was difficult to control, leading, at times, to serious morbidity or mortality.
The next advance in immunotherapy followed the realization that exposure to a related, less virulent, infectious organism such as cowpox, protected individuals from the virulent pathogen, smallpox. In England during the second half of the 18th century the link between the fine complexion (no scars of smallpox) of milkmaids and their exposure to cowpox (vaccinia) was recognized [15]. But it was Edward Jenner's publication in 1798 that proved that inoculation with the coxpox, "vaccination", protected susceptible individuals from small pox [16]. In the years since Jenner's publication, many vaccines have been developed to protect humans and animals against infectious diseases [17]. Most vaccine stimulate an immune response against the infectious microorganisms that cause disease although some vaccines stimulate an immune response to toxins they produce [18]. The primary use of vaccines had been to prevent human disease.
At the end of the 19th century, von Behring and Kitasato discovered that immunity to diphtheria and tetanus was conveyed by serum antibodies against the exotoxins of these bacteria [19]. These investigators, knowing that serum antibodies conveyed immunity, showed that serum anti-diphtheria antibodies that had been induced in one animal could be transferred and, thereby, could cure another animal showing symptoms of the disease. Thus, was born the concept of immunotherapy. The clinical impact of this finding would be great as 50,000 German children died each year of diphtheria at the end of the 19th century. The first successful application of immunotherapy to humans occurred in 1891 when a child suffering from diphtheria was cured by an infusion of horse serum containing anti-diphtheria antibodies. The horse serum containing anti-toxin antibodies could be lifesaving although many patients, receiving large doses of xenogeneic proteins, developed serum sickness resulting from the patients' immune response to the foreign serum. During the first part of the 20th century passive immunotherapy of other infectious diseases including pneumococal pneumonia entered clinical practice but was replaced when antibiotics became available during the second third of the 20th century.
Today, passive immunotherapy is attracting renewed interest as a new means to treat neurodegenerative, infectious, autoimmune, and neoplastic diseases [7,20,21]. Passive immunotherapy has, thanks largely to remarkable progress in monoclonal antibody and recombinant DNA technologies, become one of the hottest fields of therapeutics. Humanized or human monoclonal antibodies have increased the efficacy of passive immunotherapy and eliminated serum sickness following injection of xenogeneic serum. Now passive immunotherapy has been developed for patients with chronic diseases including atherosclerotic, neoplastic, and neurodegenerative diseases.
Active Immunotherapy of Alzheimer's Disease
Immunotherapy of Alzheimer's disease followed the development of a mouse model of familial Alzheimer's disease. In 1995, an APP-transgenic mouse expressing a mutant, human APP gene isolated from a Swedish family with inherited Alzheimer's disease was developed [22]. These mice develop cerebral diffuse deposits of amyloid peptide and amyloid plaques by middle age and were, therefore, a useful model of familial Alzheimer's disease. Four years later, Schenk and his colleagues reported that repeated immunization of such mice with amyloid beta peptide prevented or reversed accumulation of amyloid deposits in the brain of these mice [6]. Despite the dramatic effects of active immunotherapy on cerebral histopathology in APP-transgenic mice, the key question – did immunotherapy prevent cognitive decline – remained to be answered. Eighteen months later it was reported that the cognitive decline seen in the APP-transgenic mice was blocked by active immunization [23-25]. No toxicity was observed following active immunization of APP-transgenic mice, despite some concern that the administration of the neurotoxic amyloid beta peptide might cause untoward effects [26].
These observations offered great promise for the treatment of patients with familial Alzheimer's disease and, perhaps, elderly patients with sporadic Alzheimer's disease. A clinical trial of active immunization of elderly patients with sporadic Alzheimer's disease was organized and initiated in 2001. However, there were reasons to question whether the benefits seen in a middle-aged mice model of familial Alzheimer's disease could be directly extrapolated to elderly patients with sporadic Alzheimer's disease.
First of all, aging is associated with a decreasing immune response [27]. Thus, active immunization with influenza or tetanus vaccines induces less protective immunity in old than young persons or experimental animals. It was reported that a large percentage of old mice and elderly humans following active immunization with amyloid beta peptide did not generate a robust anti-amyloid beta peptide antibody response [9,28]. Secondly, active immunization of elderly humans and old mice stimulates an increase in autoimmune responses despite the lower immune response to the foreign antigen [29].
The clinical trial of active immunization of patients with Alzheimer's disease with amyloid beta peptide, started in 2001, was stopped a year later after 4 patients in the actively immunized group developed sterile meningoencephalitis [30]. Approximately 6% of the 298 actively immunized Alzheimer's disease patients eventually developed sterile encephalitis. One patient with sporadic Alzheimer's disease died 12 months after the last immunization with amyloid beta peptide from a pulmonary embolus. Post-mortem examination of the brain in this patient revealed CD4+ T cells in a perivascular distribution [31]. While neither the function nor the specificity of the T cells infiltrating the brain was determined, it is possible that the age-associated tendency to generate autoimmune reactions led these patients to generate autoreactive CD4+T cells that entered the brain and contributed to encephalitis.
No comprehensive report of the clinical study of active immunization has yet been published but oral presentations and published results from a subset of actively immunized Alzheimer's disease patients have provided some information [9]. It appears that: (i) all patients with sterile encephalitis had been actively immunized with amyloid beta peptide; (ii) there was no correlation between the level of serum anti-amyloid beta peptide antibodies and risk of sterile encephalitis; (iii) certain patients with sterile encephalitis had no detectable anti-amyloid beta peptide antibodies in serum; (iv) one-half of the elderly patients with sporadic Alzheimer's disease who were immunized with amyloid beta peptide did not generate significant titers of anti-amyloid beta peptide antibodies; and finally, (v) in a subset of patients with sporadic Alzheimer's disease, those patients who generated significant levels of serum anti-amyloid beta peptide antibodies had little or no cognitive decline during the year of observation following active immunotherapy.
In summary, active immunization with amyloid beta peptide in elderly patients with Alzheimer's disease appears to be less effective and more toxic than in the middle-aged APP-transgenic mouse model of familial Alzheimer's disease. These differences may reflect the greater age of the patients with sporadic Alzheimer's disease and the decreased antibody responses to vaccines and the paradoxical increase in autoimmune responses in the elderly.
Passive Immunotherapy of Alzheimer's Disease
Administration of anti-amyloid beta peptide antibodies would bypass immune senescence and would not be expected to lead to T cell-mediated encephalitis. Furthermore, anti-amyloid beta peptide antibodies not only dissolved aggregates of amyloid beta peptide in vitro but also inhibited aggregated amyloid beta peptide-mediated cytotoxicity in vitro [32]. In vivo, passive immunotherapy of APP-transgenic mice with anti-amyloid beta peptide antibodies prevented or reversed cerebral amyloid deposition depending on whether treatment was begun before or after cerebral amyloid deposition had occurred [7]. It has been reported that passive immunotherapy of APP-transgenic mice prevented age-associated cognitive decline on in the APP-transgenic mice after a 6 week course of treatment with anti-amyloid beta peptide antibodies even before there was any detectable decrease in cerebral amyloid plaque number [33]. Preliminary clinical studies showed that infusing a preparation of human intravenous immunoglobulin (IVIg) containing anti-amyloid beta peptide antibodies into 6 elderly patients with sporadic Alzheimer's disease showed significant cognitive improvement during the 6 months of therapy [10]. Elan announced that a phase 1 clinical study of passive immunotherapy with humanized monoclonal anti-amyloid beta peptide antibody in patients with mild to moderate Alzheimer's disease had been started at the end of 2003 .
The potential benefit of anti-amyloid beta peptide antibodies in humans with sporadic Alzheimer's disease was also inferred from studies of cognitive function in a subset of actively immunized patients with Alzheimer's disease [9]. In these patients, there was a direct correlation between the level of serum anti-amyloid beta peptide antibodies and cognitive function one year after active immunization. Thus, the patients with the highest serum levels of anti-amyloid beta peptide had little or no cognitive decline while cognitive function declined markedly in the nearly 50% of patients who generated little or no detectable serum anti-amyloid beta peptide antibodies after active immunization.
Passive immunotherapy of Alzheimer's disease would require repeated administration of anti-amyloid beta peptide antibodies. For this reason, human anti-amyloid beta peptide antibodies should be used to prevent an immune response to the currently available murine monoclonal immunoglobulins. Several methods are known to obtain human anti-amyloid beta peptide antibodies: (i) purification of specific antibodies from human IVIg; (ii) humanization of murine anti-amyloid beta peptide antibodies by replacing framework portions of the murine anti-amyloid beta peptide antibodies with human framework sequences using recombinant DNA technology [34]; (iii) generation of human monoclonal anti-amyloid beta peptide antibodies in vitro by human immunoglobulin phage library display techniques or in vivo by immunization of mice whose immunoglobulin loci have been replaced by human Ig genes [34].
Human IVIg, purified from human plasma, was initially developed as replacement therapy for immunodeficient patients but IVIg has also been shown to be effective therapy in patients with a variety of autoimmune diseases [35]. Recently, we and others have demonstrated that human serum and IVIg have a significant quantity of human anti-amyloid beta peptide antibodies [36,37]. Such polyclonal human anti-amyloid beta peptide antibody preparations inhibit amyloid beta peptide-induced neurotoxicity in vitro.
There is no evidence that anti-amyloid beta peptide antibodies induce sterile encephalitis, observed following passive immunization of several strains of APP-transgenic mice or in the small number of elderly patients with Alzheimer's disease. However, it may be premature to conclude that passive immunotherapy with anti-amyloid beta peptide antibodies does not induce cerebral pathology. It should be remembered that most of the APP-transgenic mice strains tested do not develop amyloid vascular deposits (congophilic angiopathy) that occurs in elderly patients with Alzheimer's disease. It was reported that infusion of murine anti-amyloid beta peptide monoclonal antibodies specific for the N-terminal epitope of the amyloid beta peptide into a strain of APP-transgenic mice, which develop congophilic angiopathy, cerebral hemorrhage was observed [38]. This untoward effect appears to depend upon the epitope specificity of the anti-amyloid beta peptide antibodies.
The Choice of Anti-amyloid Beta Peptide Antibodies for Alzheimer's Disease
Pre-clinical data and inferences drawn from immunotherapy in patients with sporadic Alzheimer's disease suggest that passive immunotherapy with anti-amyloid beta peptide antibodies is preferable to active immunotherapy for the treatment of elderly patients with sporadic Alzheimer's disease. However, which anti-amyloid beta peptide antibodies would have greatest therapeutic efficacy and least risk of untoward effects for patients with Alzheimer's disease remains to be determined. However, there is general agreement that anti-amyloid beta peptide antibodies to be administered repeatedly to patients should not stimulate an antibody response to the infused immunoglobulin. There is evidence that humans not only can make immune response to therapeutic antibodies but that such immune responses compromise the action of the therapeutic antibodies [39]. To date, the only human anti-amyloid beta peptide antibodies reported to improve cognitive function in elderly patients with sporadic Alzheimer's disease are those contained in human IVIg [11]. In contrast to preparations containing polyclonal human anti-amyloid beta antibodies, several laboratories have produced humanized anti-amyloid beta peptide antibodies including the Elan preparation now in phase I trial. It has not been reported whether this humanized anti-amyloid beta peptide antibody is active in APP-transgenic mice. The therapeutic efficacy of candidate human anti-amyloid beta peptide antibodies can be compared by measuring their capacity to decrease or reverse cerebral amyloid beta peptide accumulation and cognitive decline in RAG-2-deficient, APP-transgenic mice. We have bred these mice that lack lymphocytes and are incapable of generating an immune response to the human anti-amyloid beta peptide antibodies to test antibodies considered for immunotherapy of Alzheimer's disease.
If the therapeutic benefit of polyclonal human anti-amyloid beta peptide antibodies is confirmed, it remains to be determined which antibody or antibodies are responsible for the therapeutic effect. Whether a single monoclonal antibody will be effective in humans as it has been in APP-transgenic mice is not certain. In some infectious diseases, a single monoclonal antibody has not been less protective than a mixture of several monoclonal antibodies [40]. Thus, while each antibody specificity in polyclonal human anti-amyloid beta peptide antibodies may be at a lower concentration than that of a monoclonal antibody, the synergistic effect of multiple antibody specificities may have advantage.
Monoclonal anti-amyloid beta peptide antibodies are known to differ in their fine specificity: isotype, affinity, as well as epitope, aggregate, and Fc specificity [41]. Whether it will be possible to choose an anti-amyloid beta peptide monoclonal antibody based on these characteristics is far from certain. It is likely that testing in immune deficient APP-transgenic mouse would be performed prior to the treatment of patients with Alzheimer's disease.
Monoclonal anti-amyloid beta peptide have different specificities. There are three major epitopes on the amyloid beta peptide: (i) antibodies to the N-terminal epitope (amino acids 1–6) of the amyloid peptide bind to aggregated amyloid beta peptide in vitro as well as cerebral and vascular deposits in vivo and APP (ii) antibodies specific for the central region (amino acids 15–25) of the amyloid peptide bind to APP but not to aggregated amyloid beta peptide in vitro, amyloid plaques or vascular amyloid deposits (iii) antibodies specific for the C-terminal region have been less well studied but reported to lack a therapeutic effect in APP-transgenic mice. This may be the reason why the N-terminal-specific anti-amyloid beta peptide antibodies but not central region-specific antibodies cause cerebral hemorrhage presumably from vessels with amyloid beta deposits [42].
Anti-amyloid beta peptide antibodies that differ in epitope and Fc specificities, dissolve cerebral amyloid plaques and block cognitive decline in APP-transgenic mice [41]. Anti-amyloid beta peptide antibodies, specific for the N-terminal region of the amyloid peptide, are reported to enter the brain, bind to cerebral amyloid plaques, dissolve the plaque, and mediate Fc-mediated endocytosis followed by catabolism of the amyloid beta peptide within glial cells [7]. However, the Fc-mediated pathway is not the only route to the dissolution of amyloid plaques. Direct application of anti-amyloid beta peptide antibodies that bind to amyloid plaques but do not express the Fc region of the molecule dissolve cerebral amyloid plaques [43]. Furthermore, anti-amyloid peptide antibodies dissolve amyloid plaques in APP-transgenic mice that do not express Fc receptors.
Surprisingly, treatment of APP-transgenic mice with anti-amyloid beta peptide antibodies specific for the central region of the amyloid peptide that do not stain cerebral amyloid plaques ex vivo and were not detectable within the brain also cleared cerebral amyloid peptide and plaques [33]. A novel explanation for the mechanism of action of anti-amyloid beta peptide antibodies that do not enter the brain. The "peripheral sink" hypothesis suggests that cerebral amyloid beta peptide in all its forms (monomer, oligomer, fibrils) are in equilibrium with amyloid beta peptide in the blood and, that anti-amyloid peptide antibodies, which cannot cross the blood-brain barrier, deplete cerebral amyloid beta peptide by its mobilization into the blood. It was shown that within a few hours of administering central region-specific anti-amyloid beta peptide antibodies, which do not enter the brain, the level of amyloid beta peptide in the blood increases as much as 1000 fold. Furthermore, the magnitude of the increase in total amyloid beta peptide levels in the blood following a single injection of anti-amyloid beta peptide antibody is a surrogate marker of cerebral amyloid beta peptide load [44]. Thus, it appears that similar effects – decreased cerebral amyloid load and cognitive loss can occur following treatment of APP-transgenic mice with different anti-amyloid beta peptide monoclonal antibodies by central (entry into the brain) or peripheral (entry into the blood) mechanisms.
Both N-terminal-specific and central region-specific anti-amyloid beta peptide antibodies but not C-terminal-specific anti-amyloid beta peptide antibodies also bind to APP the ubiquitous transmembrane cellular protein. C-terminal-specific anti-amyloid beta peptide antibodies can distinguish between amyloid beta 1–40 and 1–42 peptide. As amyloid beta 1–42 peptide is more neurotoxic peptide and forms the nidus of cerebral amyloid plaques antibodies to this amyloid beta peptide might be the most effective antibody for passive immunotherapy as they target the most pathogenic form of the amyloid beta peptide without binding to APP or the less pathogenic amyloid beta 1–40 peptide. However, the C-terminal specific anti-amyloid beta peptide antibodies have been reported not to clear cerebral amyloid beta peptide [41]. Recently, it has been reported that a C-terminal specific anti-amyloid beta peptide antibody does clear amyloid plaques [45].
Finally, there is considerable interest in the greater neurotoxicity of soluble amyloid beta peptide oligomers than either the amyloid beta monomers or fibrils [46]. If this proves to be case, it would be important to test the therapeutic efficacy of monoclonal antibodies to amyloid beta peptide oligomers in APP-transgenic mice [47].
Conclusion
Passive immunotherapy of sporadic Alzheimer's disease offers the potential of reversing the pathologic accumulation of cerebral amyloid beta peptide. To date the only preparation of human anti-amyloid beta peptide antibodies that have been reported to reverse cognitive defects in patients with sporadic Alzheimer's disease are polyclonal anti-amyloid beta peptide antibodies contained in human IVIg. Selection of human anti-amyloid beta peptide antibodies for clinical trial can be tested for therapeutic effect in vivo by their treatment of immunodeficient APP-transgenic mice.
Competing Interests
The authors declare that they have no competing interests.
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| 15679923 | PMC544955 | CC BY | 2021-01-04 16:36:28 | no | Immun Ageing. 2004 Nov 12; 1:2 | utf-8 | Immun Ageing | 2,004 | 10.1186/1742-4933-1-2 | oa_comm |
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Immun AgeingImmunity & ageing : I & A1742-4933BioMed Central London 1742-4933-1-31567992410.1186/1742-4933-1-3CommentaryIs immunotherapy an effective treatment for Alzheimer's disease? Licastro Federico [email protected] Calogero [email protected] Dipartimento di Patologia Sperimentale, Università di Bologna, Via S. Giacomo 14, 40126 Bologna, Italy2 Gruppo di Studio sull'Immunosenescenza, Dipartimento di Biopatologia e Metodologie Biomediche, Università di Palermo, Corso Tukory 211, 90134 Palermo, Italy2004 12 11 2004 1 3 3 19 10 2004 12 11 2004 Copyright © 2004 Licastro and Caruso; licensee BioMed Central Ltd.2004Licastro and Caruso; 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.
Immunotherapy in patients with Alzheimer's disease (AD) is rapidly becoming a hot topic of modern geriatric and clinical gerontology. Current views see immunization with Aβ peptide, the amyloidogenic protein found in senile plaque of AD patient's brains, or the infusion of preformed antibody specific for human Aβ, as possible therapeutic approaches to improve the cognitive status in the disease. Animal models of the disease have provided positive results from both approaches. Thus, an initial clinical trial using immunization with human Aβ in AD patients was started, but then shortly halted because of an unusually high incidence (6%) of meningoencephalitis. A long and currently ongoing debate in the scientific community about the pro or contra of vaccination or passive immunization with Aβ in AD is thereafter started. Here, the authors would like to stress few points of concern regarding these approaches in clinical practice.
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Immunotherapy in patients with Alzheimer's disease (AD) is rapidly becoming a hot topic of modern geriatric and clinical gerontology. M.E. Weksler [1] in the article entitled "The immunotherapy of Alzheimer's disease" published in Immunity and Ageing discussed this theme.
Current views see immunization with Aβ peptide, the amyloidogenic protein found in senile plaque of AD patient's brains, or the infusion of preformed antibody specific for human Aβ, as possible therapeutic approaches to improve the cognitive status in the disease.
Animal models of the disease have provided positive results from both approaches, since either vaccination with human Aβ or infusion of preformed antibodies specific for Aβ, have resulted in a decrease of amyloid plaques density in the brain of amyloid precursor protein (APP) transgenic mice [2,3]. Improved memory performances after Aβ vaccine on APP transgenic mice have also been claimed [4]. Several other studies from transgenic mice have there after reinforced the suggestion that vaccination or passive immunotherapy might result in a relevant clinical effect in human patients (see the Weksler article [1]).
An initial clinical trial using immunization with human Aβ in AD patients was started and then shortly halted because of an unusually high incidence (6%) of meningoencephalitis (see the Weksler article [1]). A long and currently ongoing debate in the scientific community about the pro or contra of vaccination or passive immunization with Aβ in AD is thereafter started.
Here, we would like to stress few points of concern regarding these approaches in clinical practice.
1) The claimed animal model for AD is unfortunately incomplete even if useful model for the human disease.
2) Vaccination with human Aβ in mice induces an immune response against a foreign protein, i.e. human Aβ, and the mouse Aβ homolog does not appear to be involved. On the contrary, Aβ vaccination in man may potentially induce an autoimmune like disease in the brain and other peripheral tissues of susceptible patients. At the moment we do not have the ability to predict which patients will suffer destructive immune responses.
3) The effects of both vaccination or passive immune therapy in AD brains might be non-specific, as already suggested by a recent report [5] and by the original histopathological investigation from AD patients deceased after meningoencephalitis [6]. This notion was then reinforced by the report of these authors [7].
4) Vaccine therapy is not always effective in the elderly, since immune defects of variable degree are often present in old persons [8]. In old clinically ill AD patients this immune activation might fail or activate noxious auto-aggressive immune responses. Once again we cannot predict which patient will experience one or the other condition after the vaccination.
5) Historically vaccination has been successful in the prevention of the diseases much and much less in the therapy of ongoing diseases. A significant gain would be achieved by Aβ vaccination or passive immune therapy, if these manipulations will work in the very early stages of the disease. At the moment, no clinical data on this topic are available and those from animal models have not extensively addressed this topic.
6) A general consideration on AD is also mandatory. In fact, clinical signs of dementia show up after extensive synapse and neuron loss have already occurred in the brain. How can an immune response restore an already compromised nervous circuit or revive dead neurons? The very modest decrement of cognitive deterioration rate claimed in a proportion of AD patients receiving the vaccine is a marginal therapeutic goal. In fact, the disease has not been cured, and the modest clinical slow down in the AD progression takes big prices, i.e. patients and care givers will continue to suffer the catastrophic effects of the disease for a longer time.
We feel that Aβ vaccination and in a less extent passive immune therapy are now exciting experimental protocols for animal research and experimental neurology investigations, probably premature for clinical applications.
==== Refs
Weksler ME The immunotherapy of Alzheimer's disease Immun Ageing 2004
Schenk D Barbour R Dunn W Gordon G Grajeda H Guido T Hu K Huang J Johnson-Wood K Khan K Kholodenko D Lee M Liao Z Lieberburg I Motter R Mutter L Soriano F Shopp G Vasquez N Vandevert C Walker S Wogulis M Yednock T Games D Seubert P Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse Nature 1999 400 173 177 10408445 10.1038/22124
Bard F Cannon C Barbour R Burke RL Games D Grajeda H Guido T Hu K Huang J Johnson-Wood K Khan K Kholodenko D Lee M Lieberburg I Motter R Nguyen M Soriano F Vasquez N Weiss K Welch B Seubert P Schenk D Yednock Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease T Nat Med 2000 6 916 919 10.1038/78682
Morgan D Diamond DM Gottschall PE Ugen KE Dickey C Hardy J Duff K Jantzen P DiCarlo G Wilcock D Connor K Hatcher J Hope C Gordon M Arendash GW A beta peptide vaccination prevents memory loss in an animal model of Alzheimer's disease Nature 2000 408 982 985 11140686 10.1038/35050116
Akiyama H McGeer PL Specificity of mechanisms for plaque removal after A beta immunotherapy for Alzheimer disease Nat Med 2004 10 117 118 14760408 10.1038/nm0204-117
Nicoll JA Wilkinson D Holmes C Steart P Markham H Weller RO Neuropathology of human Alzheimer disease after immunization with amyloid-beta peptide: a case report Nat Med 2003 9 448 425 12640446 10.1038/nm840
Nicoll JAR Wilkinson D Holmes C Stear P Markham H Weller R reply Nat Med 2004 10 118 119 10.1038/nm0204-118
Vasto S Caruso C IMMUNITY&AGEING: a new journal looking at ageing from an immunological point of view Immun Ageing 2004 1 1 15679921 10.1186/1742-4933-1-1
| 15679924 | PMC544956 | CC BY | 2021-01-04 16:36:28 | no | Immun Ageing. 2004 Nov 12; 1:3 | utf-8 | Immun Ageing | 2,004 | 10.1186/1742-4933-1-3 | oa_comm |
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Immun AgeingImmunity & ageing : I & A1742-4933BioMed Central London 1742-4933-1-41567993310.1186/1742-4933-1-4ResearchAssociation between monocyte Fcγ subclass expression and acute coronary syndrome Calverley David C [email protected] Taya [email protected] Elizabeth [email protected] Denice D [email protected] Susan [email protected] Wendy J [email protected] Thomas A [email protected] Howard N [email protected] Alan D [email protected] Department of Medicine, University of Colorado, Denver, CO; USA2 Department of Medicine, University of Southern California Keck School of Medicine, Los Angeles, CA; USA3 Department of Preventive Medicine, University of Southern California Keck School of Medicine, Los Angeles, CA; USA4 Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA; USA2004 12 11 2004 1 4 4 8 10 2004 12 11 2004 Copyright © 2004 Calverley et al; licensee BioMed Central Ltd.2004Calverley 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
Atherosclerosis lesions contain abundant immunoglobulins complexed with oxidized LDL (OxLDL) that are endocytosed by macrophages to form foam cells. While recent evidence supports a role for the macrophage scavenger receptor pathway in 75–90% of OxLDL uptake, in vitro evidence suggests another potential uptake pathway could involve autoantibody binding to IgG subclass-specific Fc receptors.
Objective and Methods
To address this mechanism from an in vivo standpoint, the objective of this study was to utilize flow cytometry to prospectively determine monocyte Fcγ (FcR) I, II, and III receptor expression levels in patients with acute coronary syndrome (ACS, n = 48), diabetes mellitus (DM, n = 59), or neither (C, n = 88).
Results
Increased FcR I expression was found in the ACS versus DM groups [geometric mean, (95% CI) = 2.26 (2.07, 2.47) versus 1.83 (1.69, 1.98) (p < 0.001)] and versus C [1.90 (1.78, 2.03) (p = 0.005)]. Similar relationships were found with both the FcR II receptor [ACS mean = 4.57 (4.02, 5.19) versus DM 3.61 (3.22, 4.05) (p = 0.021) and versus C 3.86 (3.51, 4.24) (p = 0.09)] and FcR III receptor [ACS mean = 1.55 (1.44, 1.68) versus DM 1.36 (1.27, 1.46) (p = 0.038) and versus C 1.37 (1.30, 1.45) (p = 0.032)]. There was no difference between DM and C groups in FcR I, II or III expression.
Conclusions
This in vivo data supports a possible second OxLDL-autoantibody macrophage uptake mechanism through an Fc receptor-mediated pathway and a potential relationship between atherosclerotic plaque macrophage FcR levels and ACS.
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Introduction
Atherosclerosis is a chronic inflammatory process that results from hyperlipidemia and complex interactions involving other genetic and environmental factors. OxLDL plays a central role in the atherogenic process through generation of highly immunogenic neodeterminants for the immune system [1]. Natural autoantibody titer to a number of these epitopes and extent of immune complex formation may correlate with plaque size and rate of progression, and plaques have been shown to contain OxLDL/autoantibody immune complexes [2-5]. It is clear that both innate and adaptive immunity can modulate lesion progression and composition, and most studies to date have indicated a proatherogenic influence of the immune system on this process [1,4].
Recent evidence supports the macrophage scavenger receptors SR-A and CD36 as a mechanism responsible for up to 90% of uptake of OxLDL that leads to foam cell formation with no other scavenger receptors compensating for their absence in a knockout mouse model [6]. Earlier evidence involving in vitro incubation of both human monocyte-derived macrophages and the monocytic cell line THP-1 with human LDL-rabbit anti-apo B immune complexes demonstrated a potential role for the FcγRI receptor in its uptake [7]. A second in vitro study also suggested a potential Fc receptor role through inhibition of immune complex uptake when Fab or F(ab')2 fragments were substituted for an intact anti-apo B antibody [8]. Though findings from the latter two studies may have been partly explained by contributions from the scavenger pathway, it is reasonable to speculate the Fc receptor pathway maybe playing a small but important role as well [9-11].
Immune complexes with modified lipoproteins have recently emerged as an important coronary artery and macrovascular disease risk factor in DM [12,13]. Evidence supports an increased content of macrophages in the atherosclerotic lesions of persons with DM that is thought to be due to altered levels of cytokines [12]. Furthermore, while DM itself does not increase levels of LDL, the small dense LDL particles found in type 2 DM are more atherogenic because they are more easily glycated and are thought to be more susceptible to oxidation [14,15].
In recent work our group has shown FcγRII expression to be increased in the platelets of patients experiencing an acute atherothrombotic event, or who are healthy with two or more atherosclerosis risk factors [16]. Non-acutely ill diabetes patients have significantly elevated expression levels and this may play a role in the increased sensitivity of their platelets to activation by subendothelial collagen [16-19]. We speculate that Fc expression levels and activity on macrophages and platelets may represent another link between the immune system and atherosclerosis progression and plaque disruption.
In view of the controversy regarding the mechanism of cholesterol uptake by monocyte-macrophages in atherosclerosis and diabetes [20,21] and the previous lack of in vivo data to help elucidate any role of the Fc receptors in this process, we have prospectively determined IgG-binding receptor expression levels for each Fcγ receptor subclass on the monocytes of three groups: (1) patients admitted to the hospital with ACS, (2) well patients with no history of heart disease but one or more atherosclerosis risk factors (ARF's) that included DM, and (3) control patients (with no history of ACS or DM).
Materials and Methods
All 195 patients were randomly chosen for study participation from a larger group who fit study inclusion criteria and gave written informed consent. Forty-eight patients in the study had heart disease (HD) and were within 2 hours of onset of an ACS (myocardial infarction or unstable angina), 59 were DM outpatients (both type 1 and type 2 were included) with no known history of HD, and an additional 88 outpatients without HD or DM were randomly chosen as controls (C). The number and nature of ARF's was documented for each group (Table 1).
Table 1 Demographic characteristics at enrollment
Characteristic Group 1 (ACS) Group 2 (DM) Group 3 (C) p-value1
Total patients (% with MI Group 1) 48 (52) 59 88
Mean age (years) 56 55 56 0.862
Male (%) 35 (73) 24 (41) 55 (63) 0.002
Positive family history (%)3 6 (13) 21 (36) 21 (24) 0.018
Current cigarette smoker (%) 16 (33) 10 (17) 5 (6) <0.001
Hypertension (%) 29 (60) 29 (49) 20 (23) <0.001
Abnormal lipid profile present (%) 24 (50) 12 (21) 17 (19) <0.001
Diabetes mellitus present (%) 15 (31) 59 (100) 0 (0) <0.001
1. Based on chi-square test.
2. Based on ANOVA F-test
3. One patient in group 2 had missing data with positive family history
(Abbreviations: ACS: acute coronary syndrome; DM: diabetes mellitus; C: control; MI: myocardial infarction)
Blood was collected in 3.8% trisodium citrate and divided into 50 μl aliquots to which 5 μl of a saturating concentration of anti-FcR I (32.2), anti-FcR II (IV.3), anti-FcR III (3G8), or a negative class-specific control antibody (MOPC-141, Sigma) was added. Following a 15 minute incubation, 5 μl of FITC-sheep anti-mouse antibody (Sigma) was added and a second 15 minute incubation done. The pellet was washed twice before 5 μl of phycoerythrin-conjugated anti-CD14 (Becton Dickinson) was added and incubated for 15 minutes. The solution was diluted with 1 ml ammonium chloride lysing solution and incubated for 10 minutes or until the solution was clear, and the pellet washed twice before flow cytometry to determine relative receptor expression levels was carried out according to the manufacturer's specifications (Becton Dickinson). Monocytes were readily identifiable from other blood cells by their forward and side scatter properties along with CD14 expression.
Following establishment of saturating concentrations for each antibody, mean inter and intra-individual coefficients of variation for each of the three Fc receptors were calculated in the antibody labeling assay employing blood samples from five healthy laboratory volunteers who met control patient criteria and FcR I values found to be 3.2 and 9.7%, FcR II 11.7 and 16.1% and FcR III 4.1 and 26.9% respectively.
The ratios of FcR I, FcR II, FcR III and MOPC-141 antibody expression were calculated for each patient and the logarithm of the ratios were used to analyze the results. An analysis of variance (ANOVA) was performed to compare the groups of (1) these 3 ratios with the 3 groups of patients, (2) the 3 ratios with the total number of major ARFs, (3) the 3 ratios with each ARF, such as hypertension, diabetes, and smoking, and (4) the 3 ratios with MI, and unstable angina in group 1. The overall p-values were based on the ANOVA F-test. If the overall F-test p-value < 0.05, the LSD method (least significant difference) [22] was used for multiple comparison. The geometric means and the associated 95% confidence intervals were calculated to summarize the data. Patient baseline data between groups was analyzed using the chi-square test. To perform statistical analysis, SAS software, version 8.2, was used (SAS Institute, Inc., Cary, NC).
Results
Significantly increased FcR I expression was found in ACS patients compared with DM patients [geometric mean FcR I expression, (95% CI) = 2.26 (2.07, 2.47) versus 1.83 (1.69, 1.98) (p < 0.001)] and compared with C [1.90 (1.78, 2.03) (p = 0.005)] (Table 2, Figure 1). Similar relationships between the three groups were found to exist employing antibodies specific to the FcR II receptor: ACS geometric mean (CI) = 4.57 (4.02, 5.19) versus DM 3.61 (3.22, 4.05) (p = 0.021) and versus C 3.86 (3.51, 4.24) (p = 0.09) and the FcR III receptor: ACS geometric mean (CI) = 1.55 (1.44, 1.68) versus DM 1.36 (1.27, 1.46) (p = 0.038) and versus C 1.37 (1.30, 1.45) (p = 0.032). There was no difference between DM and C groups in FcR I, II or III expression (p = 0.73, 0.66, and 0.99 respectively).
Table 2 Mean monocyte FcR expression in 195 study patients
FcR I FcR II FcR III
N Geometric mean (95% CI) p-value Geometric mean (95% CI) p-value Geometric mean (95% CI) p-value
Unadjusted analysis
Groups <0.0012 0.0242 0.0212
Group 1 (ACS) 48 2.26 (2.07, 2.47) group 1 vs. 2: 0.0013 4.57 (4.02, 5.19) group 1 vs. 2: 0.0213 1.55 (1.44,1.68) group 1 vs. 2: 0.0383
Group 2 (DM) 59 1.83 (1.69, 1.98) group 1 vs. 3: 0.0053 3.61 (3.22, 4.05) group 1 vs. 3: 0.093 1.36 (1.27, 1.46) group 1 vs. 3: 0.0323
Group 3 (C) 88 1.90 (1.78, 2.03) group 2 vs. 3: 0.733 3.86 (3.51, 4.24) group 2 vs. 3: 0.663 1.37 (1.30, 1.45) group 2 vs. 3: 0.99
Adjusted analysis1
Groups 0.0102 0.0612 0.102
Group 1 (ACS) 48 2.33 (2.14, 2.55) group 1 vs. 2: 0.0073 4.57 (4.00, 5.22) group 1 vs. 2: 0.0503 1.58 (1.45,1.71) group 1 vs. 2: 0.103
Group 2 (DM) 59 1.95 (1.78, 2.13) group 1 vs. 3: 0.093 3.71 (3.24, 4.24) group 1 vs. 3: 0.473 1.41 (1.30, 1.53) group 1 vs. 3: 0.223
Group 3 (C) 88 2.05 (1.87, 2.25) group 2 vs. 3: 0.563 4.11 (3.59, 4.71) group 2 vs. 3: 0.383 1.44 (1.32, 1.56) group 2 vs. 3: 0.433
1. Adjusted by smoking and hypertension status
2. p-value based on F-test
3. p-value based on Tukey-Kramer test
Figure 1 Monocyte Fcγ receptor subclass expression levels of 48 patients with heart disease (HD) and 59 patients with DM compared with 88 control patients with neither HD nor DM. HD patients display significantly increased expression levels of all 3 subclasses versus controls. * p = 0.002, 0.037, and 0.014 for FcR I, FcR II, and FcR III respectively when HD group is compared to control group.
There were no statistically significant associations with increased expression of any Fc receptor and gender, family history of premature coronary disease, diabetes, or abnormal lipid profiles. Current cigarette smoking significantly increased expression of FcR I, 2.24 (2.01, 2.50), compared to absence of current cigarette smoking, 1.91 (1.82, 2.01) (p = 0.010) (Table 2 adjusted analysis). FcR II was significantly increased among the patients with hypertension, 4.29 (3.88, 4.74) compared to those without hypertension 3.73 (3.44, 4.05) (p = 0.034). There was a slight association between age and FcR I, II or III expression (p = 0.042, 0.050, 0.022 respectively). Expression levels in younger (age < 45) and older (age > 55) groups were higher than the middle age group. No difference was found in FcR expression with respect to ARF number (with the lone exception of increased FcR III in patients with 2 or more ARFs compared with less than two), or between the ACS subgroups of acute MI and unstable angina. When FcR expression is compared between all diabetes and non-diabetes patients in the 3 groups, there is no difference in monocyte FcR I, II, or III expression.
Discussion
It can be speculated from this in vivo data that phagocytosis of OxLDL-autoantibody immune complexes by plaque-associated macrophage through an Fc-mediated pathway could be a second uptake mechanism in addition to that involving the scavenger receptors. The potential clinical implication behind these findings is that while marrow and blood monocyte scavenger receptors SR-A and CD36 have not demonstrated inter-individual variability in their basal expression levels (prior to initial uptake of OxLDL or differentiation to macrophages) [23,24], the variable expression of Fcγ receptors found in this series of patients maybe playing a role in the extent of OxLDL immune complex uptake by atherosclerosis plaques. The fact we were able to document relatively increased surface expression of all three receptor classes in patients with ACS, along with increased FcR I in those who smoked and FcR II in those with hypertension, supports this hypothesis. Any precise pathophysiological implication behind these findings, though, or any cause and effect relationship between monocyte Fc expression and ACS is presently uncertain.
No difference was noted in expression of any Fc receptor between the diabetes and control groups. Given that the control group was the largest of the three and that there was a difference noted in the expression levels of all three receptors between the two smaller groups, it can be concluded that increasing the sample size of either of the two groups with similar expression levels could possibly lead to an increase in the difference between them, but this difference is still unlikely to be of any significance compared with that between the ACS group and the other two groups.
There was an interesting trend in both the unadjusted and adjusted analyses (Table 2) in which the control groups had consistently higher monocyte Fc expression levels compared with the diabetes groups. One potential explanation for this would be the hypothesis that those with diabetes may have reduced FcR expression levels (with a possible consequent decreased uptake in oxidized LDL) compared with non-diabetes subjects in response to relatively higher levels of molecular mediators that support atherosclerosis progression and a pro-inflammatory, pro-thrombotic environment. This may reflect a biological ying-yang type of response that leads to an attempt at dampening the effects of molecular players capable of contributing to atherothrombotic events.
An issue pertinent to this study would be the possible effects of inflammation on monocyte FcR expression levels. In diabetes patients it would be reasonable to speculate a significant number of activated cells in the circulation would be unlikely since in most patients with atherosclerosis the inflammatory reaction is circumscribed to the vessel wall. Overexpression of monocyte Fc receptors may have been a possibility in ACS however. Figure 1 shows the ratio of ACS/control mean FcR expression levels to be very similar between Fc receptor subtypes. The extent of increased expression associated with inflammation-associated monocyte activation has been shown to be variable between FcR subtypes [25]. In this respect FcR II and III represent the receptors primarily involved in the inflammatory response in vivo. Since all three receptors had uniform increases in expression levels in ACS compared with controls (with the FcR I ratio being the highest of the three), it may be reasonable to attribute a relatively minimal effect of acute inflammation to the ACS data. Circulating monocyte activation may also have returned relatively close to baseline as a consequence of blood being drawn around 2 hours after symptom onset in most cases. The average circulation time of blood monocytes in response to an inflammatory stimulus may fall to as little as 30 minutes [26].
Observational studies of this nature have certain limitations related to their design and patient selection. As an example, selection bias needs to be considered in any study involving a population of volunteers associated with an atherosclerosis prevention trial (Groups 2 and 3). Even though the ethnic composition of the groups was the same, the implication of this selection bias is that the results are not generalizable to the population at large and this is attested to by the demographic characteristics in Table 1.
Through bridging innate and adaptive immune processes, macrophages play an important role in the progression of atherosclerosis and mediating plaque disruption that is considered to be the inciting event in the majority of coronary thrombi [27,28]. In this respect there is continual migration of monocytes between neighboring endothelial cells as well as two-way migration of monocytes between blood and OxLDL-containing foam cells when there is separation of endothelial cells associated with the fatty streak [29]. This observation and the inherent difficulty in isolating monocytes from tissues compared with serum both served as justification for utilizing blood monocyte Fc expression as a surrogate for plaque macrophage expression [30].
The overall effect of the humoral immune response on atherogenesis is likely to be complex. Of note, for example, is that the FcγRII receptor has an inhibitory role on B cells that are rarely seen in plaques, while it mediates phagocytosis and release of inflammatory mediators from cells of the myeloid lineage when cross-linked by immune complexes [31,32]. Thus FcR binding by opsonized OxLDL could induce either negative or positive regulation of immune cell responses. Elucidation of the immune mechanisms involved in atherogenesis will continue to evolve and lead to new insights into the molecular pathways associated with disease progression. Ultimately these insights will contribute towards the full explanation behind the clinical diversity of atherosclerosis expression in patients who appear to have equal risk.
Competing Interests
The authors declare that they have no competing interests.
Acknowledgments
This work was supported in part by grants from the NIH (HL03740), the Wright Foundation, and NCI Cancer Center Core Grant # P30 CA 14089. The authors are grateful to staff of the USC Atherosclerosis Research Unit for assistance with data collection.
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| 15679933 | PMC544957 | CC BY | 2021-01-04 16:36:28 | no | Immun Ageing. 2004 Nov 12; 1:4 | utf-8 | Immun Ageing | 2,004 | 10.1186/1742-4933-1-4 | oa_comm |
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Immun AgeingImmunity & ageing : I & A1742-4933BioMed Central London 1742-4933-1-51567992910.1186/1742-4933-1-5ResearchAre zinc-bound metallothionein isoforms (I+II and III) involved in impaired thymulin production and thymic involution during ageing? Mocchegiani Eugenio [email protected] Robertina [email protected] Catia [email protected] Elisa [email protected] Nazzarena [email protected] Marco [email protected] Immunology Ctr. (Section Nutrition, Immunity and Ageing) Res. Dept. INRCA, Ancona, Italy2 Immunosenescence Unit, Department of Pathobiology and Biomedical Methodologies, University of Palermo, Palermo, Italy2004 12 11 2004 1 5 5 18 10 2004 12 11 2004 Copyright © 2004 Mocchegiani et al; licensee BioMed Central Ltd.2004Mocchegiani 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
With advancing age, thymic efficiency shows progressive decline due to thymic involution allowing impaired cell-mediated immunity and the appearance of age-related diseases. The intrinsic cause of thymic involution is still undefined. Chronic inflammation and high glucocorticoids (GCs) may be involved. However, transgenic mice, with increased GC sensitivity and over expression of GC receptors, display delayed age-associated thymic involution. This fact suggests that other substances may affect thymic involution. Among them, both isoforms of metallothioneins (MTs) I+II and III are the major candidates because their increments leads to organ atrophy in constant stress and are induced by IL-6, which increases in ageing. Enhanced MTs in ageing allows constant sequester of zinc ions and no subsequent zinc release leading to low zinc ion bioavailability for thymic efficiency. This sequester is very limited in very old age. Thus, we have investigated the MTmRNA (I+II and III) in the thymus from young, old and very old mice.
Methods
MTmRNA and IL-6mRNA (RT-PCR) in the thymus from different donors were tested. Concomitantly, TECs proliferation, zinc ion bioavailability (ratio total thymulin/active thymulin), thymulin activity and corticosterone were tested from different donors.
Results
Both isoforms of MTmRNA and IL-6mRNA increase in old thymus coupled with low zinc ion bioavailability, reduced TECs proliferation, impaired thymulin activity and enhanced plasma corticosterone in comparison with young. Conversely, although the thymus is involuted in very old mice because of no changes in thymus weight in comparison to old mice, reduced MTmRNA, especially MT-I+II isoforms, and low IL6mRNA occur. Concomitantly, good zinc ion bioavailability, maintained TECs proliferation, satisfactory thymulin activity and reduced corticosterone are observed in very old mice.
Conclusions
The concomitant increments by high IL-6 of both MT isoforms in the thymus from old mice may be involved in thymic involution because provoking low zinc ion bioavailability, which is relevant for thymic efficiency. By contrast, the limited increments of MTs by low IL-6 induce good zinc ion bioavailability and satisfactory thymic efficiency in very old mice. Therefore, abnormal increased MTs may provoke complete thymic involution during ageing and the possible appearance of age-related diseases. If their increments are instead limited by low inflammation, healthy ageing and longevity may be reached.
Thymic involutionMetallothioneinsIL-6glucocorticoidszincTECsinflammationageinglongevity
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Introduction
The thymus gland is a central lymphoid organ in which bone marrow-derived T cell precursors undergo a complex process of maturation and differentiation leading to migration of positively selected thymocytes to the T cell-dependent peripheral areas [1]. Although thymocytes proliferation and differentiation persist throughout life, they diminish with ageing. Older thymuses are significantly atrophied and have fewer thymocytes than younger ones. Therefore, the thymus undergoes an age-dependent degenerative process, which allows a progressive loss of thymocytes as well as thymic lymphoid tissue becoming involuted, atrophic and full of fat [2]. Thymic involution is particularly important in relation to immunosenescence because leading to an impaired T cell-mediated immunity with the subsequent appearance of some age-related diseases [3]. The loss of thymocytes in ageing is also due, other than to diminished size of thymic cortex, to decreased production of thymic hormonal factors, which are important for thymocytes maturation, differentiation and proliferation [4]. Thymic hormonal factors, such as thymulin, thymopentin and thymosines, are produced by the Thymic Epithelial Cells (TECs), which number and proliferation decrease in ageing together with thymocytes [5]. The following one another of thymic negative events during ageing have been attributed to concomitant increments of glucocorticoids (GCs). Specific GCs receptors are present both on thymocytes and TECs leading the thymic cells to undergo apoptosis via Fas [6]. However, it has been recently reported in transgenic mice with increased GC sensitivity and over expression of GC receptors, a delayed age-associated thymic involution when compared with wild-type mice. These mice display a higher number of thymocytes and, surprisingly, thymic apoptosis is unaffected [7]. These data suggest that endogenous GCs may not be directly involved in thymic atrophy in ageing or, at least, they may act concomitantly or synergistically with other substances. In this context, some proteins, such as zinc-bound metallothioneins (MT) (isoforms I+II and III), may be involved in age-related thymic involution for the following reasons. Firstly, MT induction is controlled by GCs and pro-inflammatory cytokines (IL-6) [8], which increase in ageing and inflammation [9]; and also high IL-6 is involved in thymic dysregulation [10]. Second, MT increases in ageing and strictly related to high IL-6 and GCs [11]. Third, high MT are harmful in immunosenescence because they sequester zinc and are unable, within old lymphocytes, in the zinc release [11], which is in turn pivotal for immune efficiency and in conferring biological activity to thymulin [12], and thymulin activity, immune efficiency and free zinc ion bioavailability decrease in ageing [13]. Fourth, concomitant increments of MT-I+II and III during persistent stress like-conditions, as it occurs in ageing [14], lead to pancreas atrophy in stressed mice [15]. Although, MT-III isoform may be only present in the brain [16], its existence also in peripheral organs has been reported [17]. Following these considerations, we have investigated the presence of MT I+II and III gene expression and zinc content in the thymus from young, old and very old mice. Concomitantly, the IL-6 gene expression and TECs number and proliferation in the thymus from different donors have been evaluated as well as thymulin activity, corticosterone and zinc plasma levels. We have chosen very old mice because the MT (I+II) gene expression is low, like in younger, allowing satisfactory peripheral immune response [11].
Materials and Methods
Mice
Balb/c male inbred mice were used at the age of 2–3 months (young = n.10 mice), at the age of 20 months (old = n.10 mice) and at the age of 28–30 months (very old = n.10 mice). Although the maximum thymus sizes as well as peaks in thymocytes maturation and differentiation occur at 2–4 weeks of age in mice [18], no differences in thymulin activity and TECs number exist among 1 and 2–3 months of age [19]. Therefore, the choice of young mice at 2–3 months of age for the present study is appropriate. Mice were housed in plastic non-galvanized cages (5–6 mice for cage) and fed with standard pellet food (Nossan, Italy) and tap water ad libitum. Under our housing condition, the life span of Balb/c mice was of 30 months [13]. Since about 50% of survival occurred at 20 months of age, mice at this age were considered old [13]. Mice were maintained on a 12-h light/12-h dark cycle from 7:00 a.m. to 7:00 p.m. at constant temperature (20 ± 1°C) and humidity (50 ± 5%). Mice were sacrificed under ether anaesthesia. Heparinized blood samples were collected by cardiac puncture for plasma determinations of corticosterone, thymulin and zinc. Freshly thymuses were frozen in liquid nitrogen for MT-I+II, MT-III and IL-6 mRNA expressions and for testing zinc content.
RNA isolation and RT-PCR analysis
Total RNA was extracted from frozen thymus using Tri-Reagent according manufacture s protocol (Sigma, USA). 3 μg of RNA sample were reverse transcribed adding Olio d(T) and kept at 70°C for 10 min. dent, Raise inhibitor and MMLV reverse transcriptase were subsequently added and incubated at 37°C for 1 h. Samples were heated at 95°C to inactivate enzymes and stored at 20°C. PCRs were performed using sense and antisense primers as follows: MT-I: 5'-ATGGACCCCAACTGCTCCTGCTCCACC-3', 5'-GGGTGGAACTGTATAGGAAGACGCTGG-3' (259 bp) MT-III:5'-ATGGACCCTGAGACCTGCCCCTGTCCT-3', 5'-GGCCTCTGCCTTGGCCCCCTCTTCACC-3',(183 bp); β-actin: 5'-GGACTCCTATGTGGGTGACGAGG-3', 5'-GGGAGAGCATAGCCCTCGTAGAT-3' (366 bp); IL-6: 5'-ATGAAGTTCCTCTCTGCAAGAGACT-3', 5'-CACTAGGTTTGCCGAGTAGATCTC-3' (615 bp). Conditions for amplification were as follows: for MT-I each cycle consisted of 94°C 0.30 min, 50°C 0.30 min, 72°C 0.30 min with 30 cycles; for MT-III each cycle consisted 94°C 45 sec., 55°C 30 sec., 72°C 1.5 min with 30 cycles; for β-actin each cycle consisted 94°C 1 min, 61°C 1 min, 72°C 1 min with 24 cycles; for IL-6 each cycle consisted 94°C 1 min, 65°C 2 min, 72°C 3 min with 40 cycles. The products of amplification were size-fractionated by 2% agarose gel electrophoresis and visualized by staining with ethidium bromide. Semi-quantitative analysis of the amplified products was performed with an image analyser (Gel-doc 2000 instrument, Bio-Rad, USA). The results were evaluated as a relative unit determined by normalisation of the density of each band to that of the β-actin one. This method reflects MT protein production tested with Ag+ saturation method [11].
Plasma Active Thymulin (AT) and total thymulin (TT)
Plasma active zinc-bound thymulin (AT), as extensively described elsewhere [19], was measured using a bioassay based on the ability to restore the inhibitory effect of azathioprine on rosette formation in spleen cells from young Tx mice. Results were expressed as log-2 of the maximal dilution of tested plasma able to induce this phenomenon [19]. In order to avoid interference due to zinc, zinc sulphate at final concentration of 200 nM was added up to plasma samples. This fact shows the total amount of thymulin produced (active thymulin+ inactive thymulin) (TT) [19]. The ratio TT/AT is an index of zinc ion bioavailability because of strict inverse correlation between ratio TT/AT and plasma zinc levels. In particular, ratio >2 = low zinc ion bioavailability; ratio <2 = mild zinc ion bioavailability; ratio = 1 normal zinc ion bioavailability [19].
Plasma zinc and thymus zinc content
Plasma and tissue zinc content were determined in Atomic Absorption Spectrophotometer (AAS) against zinc reference standards (Sigma USA). Plasma zinc was determined after plasma dilution 1:5. Thymus tissue (1 gr) was put in muffle furnace at 550°C overnight. The ash obtained was diluted with 3 ml of 3 N HCl and transferred to a 25 ml volumetric flask and further diluted with 3 ml of 0.36 N HCl. The determination of zinc was then performed at AAS.
Plasma Corticosterone
Plasma corticosterone level (ng/ml) was determined by RIA rat-corticosterone-3H kit (ICN Biomedicals, CA, USA) and referred against a standard curve. The percentage of cross-reaction with other steroid was <0.01. The sensitivity was of 0.05 ng/ml of corticosterone.
Immunocytochemistry studies
a) TECs characterization
Anti pan-cytokeratin IgG1/FITC MoAb (Sigma, USA) diluted 1/25 and anti-keratin MoAb (Sigma, USA) diluted 1/20 were used. For this latter, guinea pig IgG/FITC (Sigma, USA) diluted 1/60 was used as second antibody. These MoAbs are specific to detect TECs (cortical and medullary) [20].
b) TEC separation and percentage
TECs were separated with method described by Kurz et al. [20]. Briefly, the thymus from young, old and very old mice after 6 h of culture was minced into small fragments and incubated with collagenase (1 mg/ml, Sigma, USA) in PBS for 1 hr at 37°C (1 ml of collagenase solution/thymus). The choice of 6 h of culture is because the maximum thymulin production and TECs number and proliferation occurred at this time of culture in experiments of thymulin kinetic (from 1 h to 12 hrs) from young thymic cultures [21,22].
The suspension was then centrifuged (2 min, 400 g) and the pellet suspended in 1 ml of Dulbecco s modified Eagle medium/Ham s F12 medium (1:1) (DMEM/F12, Gibco, Germany). The cells were subjected to two-steps trypsin (0.1 and 0.25%, respectively) and 0.001% DNase treatment in order to avoid fibroblasts [19]. After three washes in PBS, the cells were dissociated by cautious triturating through Eppendorf tips and incubated in 3 ml of DMEM/F12 medium for 2–3 h at 37°C in humidified 5% CO2-atmosphere in order to make to adhere the cells. The supernatant containing unattached TEC was seeded into another plastic flask containing DMEM/F12 medium supplemented with 10% horse serum and put in culture in humidified 5% CO2-atmosphere. The cultures were inspected for morphologically visible fibroblasts (spindle shaped cells). In cases of significant contamination, the cells were washed with PBS and underwent again to trypsinization [20]. Separated TECs were washed three times in PBS. An aliquota (103) was resuspended in 1 ml of medium and underwent to TEC percentage analysis. Percentages of separated TECs were counted in 1.000 cells at fluorescence microscope [22]. Tests were performed after pre-fixation with cold methanol in the slides. Controls were performed without the primary antibodies.
c) TECs proliferation
After TECs separation, another aliquota (40 × 103) was resuspended in 4 ml of DMEM/F12 medium for TEC proliferation analysis, which was approached using [3H] thymidine incorporation using 96 microtiter plates (Nunc, Denmark). 40 × 103 TECs were put in 40 wells (100 μl/well = 103 TECs/well). 10 wells were used as young; 10 wells as old; 10 wells as very old. Concomitantly, 1 μCi [3H]-thymidine/well (Amersham, UK) was added. The plates were incubated in humidified 5%-CO2 atmosphere for 6 hrs. Automatic harvester collected the samples and the amount of incorporated radioactivity was determined in a liquid scintillation beta-counter (Perkin-Elmer, USA).
Statistical analysis
Two-tailed Student s t test, and ANOVA test (one-way) evaluated differences between means. Correlations were determined by linear regression analysis by the least square method. Differences were evaluated by analysis of covariance. Differences were significant when p < 0.05.
Results
MT-(I+II and III) and IL-6 mRNAs and zinc content in the thymus from young, old and very old mice
Table 1 shows that MT-I+II and MT-III increase in old mice in comparison with young (p < 0.001). The same increment is also observed in very old mice as compared to young ones (p < 0.01), but at lower levels than old especially for MT-I+II. The increments of both isoforms of MT in old mice are correlated with high gene expression of IL-6 when compared to young mice (p < 0.01). The increments of IL-6 from the thymus of very old mice are lower, but still significant when compared to young (p < 0.05). Conversely, the zinc content within the thymus is very high in old mice as compared to young and very old mice (p < 0.01). Since AAS tests zinc-bound and zinc unbound [12], this last finding is not so surprising because it suggests that a large amount of zinc ions are bound to MT in the thymus from old mice. As a consequence, free zinc ions are not available for thymic efficiency in old age. Significant positive correlation exists between zinc content and MT-I+IImRNA from the thymus of young, old and very old mice (r = 0.83, p < 0.01). The thymus weight from old and very old mice is strongly reduced in comparison to young mice (p < 0.001), but with no changes between old and very old mice (Table 1).
Table 1 MT-I+II, MT-III, IL-6 mRNAs and zinc content in the thymus from young, old and very old mice.
Mice MT-I+II (MT-I/βactin) IL-6 (IL-6/βactin) MT-III (MT-III/βactin) Zinc content (μg/gr.) Absolute thymus weight (mg)
Young 0.18 ± 0.02 0.14 ± 0.03 0.48 ± 0.02 62.3 ± 11.2 30.6 ± 5.0
Old 3.52 ± 0.3* 0.23 ± 0.02§ 1.65 ± 0.03* 107.4 ± 27.5** 13.6 ± 2.0§
Very old 1.29 ± 0.6+ 0.18 ± 0.04+ 1.63 ± 0.02* 77.4 ± 8.7 15.4 ± 2.3§
*p < 0.001 when compared to young mice; +p < 0.01 when compared to old mice;
**p < 0.01 when compared to young and very old mice; §p < 0.01 when compared to young mice;
++p < 0.05 when compared to old mice
Thymic efficiency, plasma zinc and Corticosterone in young, old and very old mice
Table 2 shows that thymulin activity is strongly reduced in old mice in comparison with young (p < 0.001). Thymulin activity is instead satisfactory in very old mice when compared to old ones (p < 0.05), even if its plasma value does not reach to that observed in young mice (Table 2). These data reflect the number (in percent) and the proliferation of TECs. Both TECs number and proliferation are reduced in old mice when compared to young and very old mice (p < 0.01), even if the TECs proliferation is lower in very old mice than in young ones, but still significant in comparison with old mice (p < 0.05) (Table 2). The proliferation data in young mice agree with testing TECs proliferation in pure murine TECs cell line (IT-45RI), as previously shown [21].
Table 2 Zinc ion bioavailability, corticosterone, thymulin and TECs number and proliferation in young, old and very old mice
Mice Thymulin activity (log-2) AT/TT (log-2 (zinc ion bioavailability) Corticosterone (ng/ml) Plasma zinc (μg/dl) % TECs TECs proliferation (cpm)
Young 5.5 ± 0.5 1.1 ± 0.3 153 ± 18.3 110 ± 11 53 ± 11 750 ± 25
Old 1.07 ± 0.3* 3.0 ± 0.3** 265 ± 16.6** 80 ± 5.7++ 21 ± 7** 227 ± 34**
Very old 2.5 ± 0.3+ 1.0 ± 0.3 180 ± 12.4+ 87 ± 43++ 40 ± 12+ 450 ± 39+
*p < 0.001 when compared to young mice; +p < 0.05 when compared to old mice;
**p < 0.01 when compared to young and very old mice; ++p < 0.01 when compared to young mice.
The ratio total thymulin (TT)/active thymulin (AT) represents the zinc ion bioavailability. More high is the ratio (≥ 2) less zinc ion bioavailability is present, whereas ratio < 2 or equal to 1 means satisfactory or good zinc ion bioavailability, respectively [19]. The ratio TT/AT is higher in old mice in comparison with young and very old mice (p < 0.01) (Table 2). This fact means that a good zinc ion bioavailability exists in very old mice, as in younger ones, despite plasma zinc levels are lower in very old mice than in young ones (p < 0.01) and, at the same time, not different to those observed in old mice (Table 2). With regard to plasma Corticosterone, higher values are observed in old mice when compared to young and very old mice (p < 0.01) (Table 2).
Significant inverse correlation exists between zinc and Corticosterone (r = -0.71, p < 0.01), whereas significant positive correlation exists between thymulin activity and TECs number and proliferation (r = 0.81, p < 0.01; r = 0.79, p < 0.01, respectively) from young, old and very old mice.
Discussion
Although MT-III isoform may be present exclusively within the brain [16], some peripheral organs (testis, prostate, epididymis, tongue, ovary, uterus, stomach, heart, pancreas and seminal vesicles) may also express MT-III isoform together with MT-I+II [17]. We herein present for the first time the concomitant gene expression of MT-I+II and MT-III also in the thymus. Both isoforms of zinc-bound MT (I+II and III)mRNA increase within the thymus of old mice, but with a minor extent of MT-I+II isoform in the thymus from very old mice. Concomitantly, the gene expression of IL-6 is higher in old mice than in young and very old ones. The zinc content within the thymus is enhanced in old mice respect to young and very old mice, whereas some thymic functions (thymulin activity and TECs number and proliferation) are impaired in old mice and preserved in very old mice. These last findings regarding to zinc content and thymic efficiency seems contradictory between old and very old mice. Really, they are not contradictory. Since AAS tests zinc-bound and zinc-unbound [13], the higher zinc content in the old thymus is largely due to high zinc-bound MTs, which recall zinc from the periphery, via zinc transporters ZnT1–4 [23], and sequester a lot amount of zinc ions [22]. The inflammation provokes zinc loss with subsequent impairment of immune response [24]. Thus, such a recall and sequester by MT are due to the great inflammation by high IL-6 and GCs because zinc ions have not to be lost. But, free zinc ions are subsequently not available for thymic efficiency due to inability of MT in zinc release in constant inflammation [14]. Conversely, limited recall of zinc ions occurs in very old mice because the inflammation is less deep. As a consequence, the zinc content in the thymus from very old mice is lower and, at the same time, more free zinc ions are available for thymic efficiency by low zinc-bound MT. Anyway, the present data show that abnormal increments of both isoforms of MT are present in the atrophic thymus from old and very old mice, but with less extent of MT-I+II in very old mice.
MT-I+II and III are expressed in the brain with a balance between the two isoforms (25). When one isoform increases, the other decreases due to a possible genetic control of MRE region on the chromosome 8, which brings the two isoforms [26]. Concomitant increments of the two isoforms within the hippocampus from old rats leads to impaired number and functions of synapses coupled with low zinc ion bioavailability [27]; and synaptic function is zinc-dependent [28]. The same phenomena occur in age-related neurodegenerative diseases [29], suggesting a possible role of increased MT-I+II and III in neurodegeneration [27]. Moreover, the concomitant presence of MT isoforms provokes the atrophy of the pancreas in stressed mice [15]. Therefore, enhanced MT I+II and III in the old thymus may lead to the thymic involution and atrophy because of an unbalance between the two MT isoforms. The mechanism may be largely due to the constant sequester of zinc ions by MT (I+II and III) with no subsequent zinc release leading to low zinc ion bioavailability for thymic endocrine activity and TECs proliferation [13]. In this context, IL-6 and glucocorticoids (GCs) may play key roles because IL-6 and GCs affect MTmRNA [8] and, in turn, abnormal high GC levels are involved in thymic atrophy through the activation of GC receptors on TECs and thymocytes [6]. A lack of free zinc ions also provokes thymic atrophy [30]. We have found enhanced IL-6mRNA within the thymus from old mice. Concomitantly, strong increments of plasma corticosterone are observed. Such increments are strictly related to high MTmRNA and low zinc ion bioavailability. These findings suggest that the chronic inflammation, via IL-6 and GCs, allows high MTs induction, low zinc ion bioavailability and subsequent thymic atrophy in old mice. The thymus is obviously atrophic in very old mice. But, the MT-I+IImRNA and IL-6mRNA are lower than old mice as well as reduced corticosterone. High corticosterone provokes zinc loss by urine and faeces [31]. Corticosterone is low in very old mice (Table 2). Thus, very old mice display more free zinc ion bioavailability with subsequent more thymic efficiency and preserved TECs number and proliferation. TECs produce thymulin, a zinc-dependent thymic hormone [12]. Zinc-bound MTs transfer zinc to thymulin in TECs [32]. The less MT-I+IImRNA in very old mice may thus allow less sequester of zinc ions. Alternatively, an easier release of zinc by MT for thymic efficiency might occur due to reduced inflammation by low IL-6mRNA and corticosterone. Anyway, the thymus from very old mice is still efficient despite it is involuted because the thymus weight between old and very old mice does not change (Table 1). This means that the thymic reconstitution in old age might not be necessary because the age-related loss of thymic efficiency appears to be only quantitative and not qualitative [33]. Thus, it might be sufficient to maintain inflammatory status and MTs homeostasis below a critical threshold in order to preserve thymic efficiency. Further experiments in thymic output from very old mice and in genes involved in thymocytes maturation and differentiation (Rag 1 and Rag 2) [2] are in progress in our lab.
On the other hand, MT-ImRNA is lower in lymphocytes from human nonagenarians coupled with satisfactory thymic and peripheral immune efficiency and good zinc ion bioavailability [11]. Moreover, very old mice display still efficient thymic functions during liver regeneration after partial hepatectomy (model of acute and constant inflammation) [34]. In addition, the presence of involuted thymus in stressed MT transgenic mice [22] further supports the involvement of high MT in thymic involution during ageing.
However, the thymic involution is not irreversible phenomena because zinc treatment in old mice restores thymic efficiency with a re-growth of thymic cortex [19]. This finding suggests that in ageing the thymus is in quiescent phase, which is less deep in very old age due probably to more zinc ion bioavailability, via MTs homeostasis, and less inflammation. Indeed, zinc also affects cell cycle and, therefore, cellular proliferation [35]. Thus, the thymus from very old mice is still active and not quiescent. The satisfactory zinc ion bioavailability coupled with the maintenance of TECs proliferation in the thymus from very old mice is in line with this interpretation. This means that in vivo condition TECs from very old mice are still capable to proliferate at the occurrence, for example in presence of external noxae in order to have a sufficient thymulin production for a prompt immune response, as occurring in human centenarians, who are high responder individuals showing a great capacity in remodelling thymulin activity [36].
In conclusion, concomitant increments of zinc-bound MT-I+II and III within the thymus may lead to thymic involution in ageing because they sequester zinc and are unable in the subsequent zinc release, which is indispensable for thymic efficiency. The cause may be related to chronic inflammation by high IL-6 and GCs. Without excluding a direct role of GCs in thymic involution [6], GCs may synergistically act with MT isoforms because GCs also affect MTmRNA [8]. Less inflammation by low IL-6 and GCs in very old mice allows reduced MTmRNA with subsequent satisfactory zinc ion bioavailability and, therefore, preserved thymic efficiency.
However, if the thymic involution may be a necessary event in order to avoid autoimmune phenomena in ageing is an intriguing point to be investigated. Indeed, high IL-6 provokes an enlargement of the thymus with the appearance of autoimmune phenomena [10]. Thus, if on one hand high MTs may be of protection inducing thymic involution in order to escape autoimmune phenomena by high IL-6, on the other hand high MT are harmful because leading to low zinc ion bioavailability for thymic efficiency. However, in this context, the involvement of zinc and MT in the efficiency of extrathymic T-cell pathway [13] has to be also considered, because this pathway is prominent in ageing and autoimmunity in order to compensate thymic failure [37]. Very old mice display satisfactory thymic efficiency (present study) and good extrathymic T-cell functions [38]. Moreover, thyroid autoantibodies are rare in healthy centenarians [39]. Therefore, the thymic involution, via MTs homeostasis, has to be limited or controlled concomitantly with the appearance of efficient extrathymic T-cell functions in order to reach healthy ageing and longevity. In other words, a correct balance between thymic involution and extrathymic T-cell functions has to exist in ageing. Otherwise, a complete thymic atrophy by abnormal high MT-I+II and III, via high IL-6 may provoke continuous immune dysfunctions (thymic and extrathymic). Altered genetic controls between the two MT isoforms on MRE region of chromosome 8 may be involved, representing an interesting field of investigation in immunosenescence. Works are in progress in our lab.
Acknowledgements
Supported by INRCA, Italian Health Ministry (R.F. 216/02 to EM) and EU Commission (Project ZINCAGE, n. FOOD-CT-2003-506850, Coordinator. E. Mocchegiani). Robertina Giacconi and Catia Cipriano are PhD students in the Pathobiology PhD curriculum (directed by Prof. C. Caruso) of Palermo University and this work is in partial fulfillment of the requirement for the PhD.
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| 15679929 | PMC544958 | CC BY | 2021-01-04 16:36:28 | no | Immun Ageing. 2004 Nov 12; 1:5 | utf-8 | Immun Ageing | 2,004 | 10.1186/1742-4933-1-5 | oa_comm |
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Immun AgeingImmunity & ageing : I & A1742-4933BioMed Central London 1742-4933-1-61567992510.1186/1742-4933-1-6Short Report+874(T→A) single nucleotide gene polymorphism does not represent a risk factor for Alzheimer's disease Galimberti Lorenza [email protected] Beatrice [email protected] Carmen [email protected] Silvia [email protected] Susanna [email protected] Simona Delli [email protected] Carlo [email protected] Giorgio [email protected] Department of Geriatrics, Ospedale Maggiore IRCCS, University of Milano, Milan, Italy2 Department of Clinical Medicine, Prevention and Medical Biotechnology, University of Milano-Bicocca, Mila, Italy2004 12 11 2004 1 6 6 30 9 2004 12 11 2004 Copyright © 2004 Galimberti et al; licensee BioMed Central Ltd.2004Galimberti 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 the recent years, several cytokines have been associated with Alzheimer's disease (AD) development and progression and many studies have correlated this risk with polymorphisms in the genes encoding these molecules. Also the type 1 cytokine interferon (IFN)-γ belongs to a cytokine class that affects the immune function; in fact it plays a major role in defence against viruses and intracellular pathogens but also in the induction of the immune-mediated inflammatory response. The aim of this study was to evaluate the role of IFN-γ in AD by studying the association of +874T→A IFN-γ gene polymorphism with AD. We included in this study 115 AD patients (70 women, 45 men, mean age 80) and 90 sex and age-matched healthy controls (HC, 51 women, 39 men, mean age 82) from northern Italy. Genomic DNA was extracted with the salting-out method from whole blood of all subjects; the genotyping at IFN-γ loci was assessed with ARMS-PCR. The data obtained from the +874T→A IFN-γ gene polymorphism analysis of AD patients and HC lack of any statistically significant differences also when stratified according to gender. In conclusion these results confirm the previous shown lack of association between +874T→A IFN-γ gene polymorphism and the risk of AD. However, other polymorphisms have been demonstrated to influence IFN-γ transcription and since natural killer cells of AD patients show higher production of the cytokine, further analysis will be necessary to clarify the role of this gene in the pathogenesis of the disease.
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In the human brain several cell types are responsible for initiating and amplifying a specific inflammatory response. In Alzheimer disease (AD) signs of an inflammatory activation of microglia and astroglia are present both inside and outside amyloid deposits. Cell cultures and animal models suggest an interactive relationship between inflammatory response activation, reduced neuronal functioning and amyloid deposition. Furthermore cells associated with extracellular plaques within AD brains can produce a variety of cytokines, chemokines and other related proteins that influence plaque and tangle formation [1]. For these reasons cytokines could play a critical role in the pathogenesis of AD. In the recent years, several cytokines have been associated with AD development and progression and many studies have correlated this risk with polymorphisms in the genes encoding these molecules [2-4]. Inside this research area we described that single nucleotide polymorphisms (SNP)s of the intereleukin(IL)-10 and IL-6 genes were associated with highest risk of AD with apparent interaction between these two genes [5]; these results supported the theory that the overall risk of developing AD may be governed by a 'susceptibility profile' and reflected the combined influence of inheriting multiple high-risk alleles. Also the type 1 cytokine interferon (IFN)-γ belongs to a cytokine class that affects the immune function; in fact it plays a major role in defence against viruses and intracellular pathogens but also in the induction of the immune-mediated inflammatory response [6]. It has been reported that the polymorphism +874(T→A) of the gene encoding IFN-γ is associated with a different production of this molecule, in particular the T allele correlates with increased levels of the cytokine [7]. In regards to the hypothesis that IFN-γ SNP may represent a genetic risk factor for AD, a recent study did not support this possibility [8]. Therefore with reference to this paper we try to confirm their hypothesis in a sample of patients, that were already genotyped for apolipoprotein E (ApoE), IL-10 and IL-6 [5]. We included in this study 115 AD patients (70 women, 45 men, mean age 80 ± 2) and 90 sex and age-matched healthy controls (HC, 51 women, 39 men, mean age 82 ± 2) from northern Italy. The clinical diagnosis of AD fulfilled the international criteria of the DMS IV and NINCDS-ADRDA; every patient had a recent brain magnetic resonance imaging (MRI)/computed tomography (CT) scan available. Cognitive performances were assessed according to the Mini-Mental State Evaluation (MMSE). Only AD and HC without clinical signs of inflammation (e.g. normal body temperature, no concomitant inflammatory condition) were eligible in order to minimize the risk of clinical or sub-clinical inflammatory processes. Blood chemistry tests were done and subjects with an abnormal red blood cell sedimentation rate or altered albumin and transferrin plasma levels were excluded. AD patients were further selected according to their C reactive protein (CRP) plasma levels and anyone with CRP above 5 mg/L (mean + 2 standard deviations of control values) were not eligible. Informed consent was obtained from all the subjects or their relatives. Genomic DNA was extracted with the salting-out method from whole blood of all subjects; the genotyping at IFN-γ loci was assessed with the same amplification method (ARMS-PCR) described by Scola et al. [8]. The genotype frequencies in the study groups were compared by the chi-square (χ) test in order to calculate significant different SNP distribution between AD patients and controls (Table 1). The data obtained from the +874T→A IFN-γ gene polymorphism analysis of AD patients and HC lack of any statistically significant differences also when stratified according to gender (data not shown). The percentage of the different genotypes was similar in AD compared with HC (T/T: 17.4% vs. 16.8% ; T/A: 50.4% vs. 55.1% ; A/A 32.2% vs. 28.1%) and consequently also the allele distribution shows no differences (T: 42.6% vs. 44.4% ; A: 57.4% vs. 55.6%). Our genotype distribution looks very similar to that found by Scola et al. [8]. In conclusion these results confirm the lake of association between +874T→A IFN-γ gene polymorphism and the risk of AD. Nevertheless other polymorphisms have been demonstrated to influence IFN-γ transcription and since natural killer cells of AD patients show higher production of the cytokine, further analysis will be necessary to clarify the role of this gene in the pathogenesis of the disease [9,10].
Table 1 IFN-γ genotype and allele distribution
Genotype Allele
T/T(H) T/A(I) A/A(L) T A
AD 20(17,4%) 58(50,4%) 37(32,2%) 98(42,6%) 132(57,4%)
HC 15(16,8%) 49(55,1%) 25(28,1%) 79(44,4%) 99(55,6%)
Genotype χ2 = 0.488, df = 2, p = 0.783
Allele χ2 = 0.232, df = 1, p = 0.63
In brackets there are the corresponding phenotype high (H), intermediate (I) and low (L).
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Meda L Baron P Prat E Scarpini E Scarlato G Cassatella MA Rossi F Proinflammatory profile of cytokine production by human monocytes and murine microglia stimulated with beta-amyloid J Neuro immunol 1999 93 45 52
Mrak RE Griffin WST Interleukin-1, neuroinflammation, and Alzheimer's disease Neurobiol Aging 2001 22 903 908 11754997 10.1016/S0197-4580(01)00287-1
Pola R Flex A Gaetani E Dal Lago A Gerardini L Pola P The -174 G/C polymorphism of the interleukin-6 gene promoter is associated with Alzheimer's disease in an italian population Neuroreport 2002 13 1645 1647 12352619 10.1097/00001756-200209160-00015
Arosio B Trabattoni D Galimberti L Bucciarelli P Fasano F Calabresi C Cazzullo CL Vergani C Annoni G Clerici M Interleukin-10 and interleukin-6 gene polymorphisms as risk factors for Alzheimer's disease Neurobiol Aging 2004 25 1009 1015 15212825 10.1016/j.neurobiolaging.2003.10.009
Billiau A Heremans H Vermeire K Matthys P Immunomodulatory properties of interferon-gamma. An update Ann NY Acad Sci 1998 856 22 32 9917861
Pravica V Perrey C Stevens A Lee JH Hutchinson IV A single nucleotide polymorphism in the first intron of the human IFN-gamma gene: absolute correlation with a polymorphic CA microsatellite marker of high IFN-gamma gene Hum Immunol 2000 61 863 866 11053629 10.1016/S0198-8859(00)00167-1
Scola L Licastro F Chiappelli M Franceschi C Grimaldi LM Crivello A Colonna-Romano G Candore G Lio D Caruso C Allele frequencies of +874T -> A single nucleotide polymorphism at the first intron of IFN-gamma gene in Alzheimer's disease patients Aging Clin Exp Res 2003 15 292 295 14661818
Miyake K Nakashima H Akahoshi M Inoue Y Nagano S Tanaka Y Masutani K Hirakata H Gondo H Otsuka T Harada M Genetically determined interferon-gamma production influences the histological phenotype of lupus nephritis Rheumatology 2002 41 518 524 12011374 10.1093/rheumatology/41.5.518
Solerte SB Cravello L Ferrari E Fioravanti M Overproduction of IFN-gamma and TNF-alpha from natural killer (NK) cells is associated with abnormal NK reactivity and cognitive derangement in Alzheimer's disease Ann NY Acad Sci 2000 917 331 340 11268360
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Aust New Zealand Health PolicyAustralia and New Zealand Health Policy1743-8462BioMed Central London 1743-8462-1-11567992210.1186/1743-8462-1-1EditorialAustralia and New Zealand Health Policy: a new journal Duckett Stephen J [email protected] Professor of Health Policy La Trobe University, Victoria, 3086, Australia2004 17 11 2004 1 1 1 19 8 2004 17 11 2004 Copyright © 2004 Duckett; licensee BioMed Central Ltd.2004Duckett; 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.
Australia and New Zealand Health Policy is a new journal which aims to promote debate and understanding about contemporary health policy developments in Australia and New Zealand. Although there are other international journals focussing on health policy, there are no Australian or New Zealand journals with this focus.
One of the aims of Australia and New Zealand Health Policy is to focus on contemporary critiques and contemporary developments. Accordingly an e-journal format is particularly appropriate. Australian and New Zealand Health Policy is an open access journal which means that all articles will be freely and universally accessible online which, amongst other things, means that all articles will be freely and universally accessible online without any barriers to access, which increases their visibility.
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Editorial
Welcome to Australia and New Zealand Health Policy a new journal which aims to promote debate and understanding about contemporary health policy developments in Australia and New Zealand. Health policy is regularly in the media and is a high profile issue at election times. In Australia the health system has been characterised by conflicts over values and policy choices over the decades. So pervasive is this conflict that Sax entitled his 1984 book about health services, "A Strife of Interests" [1]. Health policy in New Zealand has also had a turbulent time over the past decade [2,3]. Health policy changes in Australia and New Zealand are thus ripe for analysis. Australia and New Zealand Health Policy aims to provide a prestigious venue for analysis and critique of health policy in the two countries.
Why a new journal?
Although there are other international journals focussing on health policy, there are no Australian or New Zealand journals with this focus. Other related-area local journals are medical, public health or hospital-related. Although the local journals publish occasional policy articles, this area is not their principal interest, nor are they necessarily the journals which policy-oriented academics or policy practitioners scan to keep abreast of developments.
The absence of a health policy journal serving Australia and New Zealand has long-term consequences for the development of systematic analysis of and research into health policy. One consequence is that there is no forum where health policy developments are documented and tracked, a lacuna which precludes cumulative analyses of trends. Australia and New Zealand Health Policy will address this by publishing annual reviews of policy developments.
As one of the aims of Australia and New Zealand Health Policy is to focus on contemporary critiques and contemporary developments, an e-journal format is particularly appropriate. Debate will also be stimulated by providing for 'Comment' on published articles, in a way analogous to a letters column.
Australia and New Zealand Health Policy is a peer reviewed journal. In keeping with its policy-applied focus, articles will be refereed by two referees, preferably one with a strong practitioner background, such as currently or recently employed in a policy role in a health authority, and one from an academic background. At least one of the referees will have substantive content knowledge relating to the article. Articles published in Australia and New Zealand Health Policy will be listed in PubMed and permanently archived in PubMed Central as well as certain other national archives.
Journal scope
Australia and New Zealand Health Policy contributes to understanding of health policy development and practice with a particular focus on Australia and New Zealand. It welcomes submissions which:
• Review and critique contemporary health policy issues;
• Identify major trends in health policy and emerging policy issues, including new evidence about the effect of policy changes;
• Identify impacts of health services research on new policies;
• Identify major public policy and governance trends and their application to health policy;
• Analyse contemporary health policy themes which cut across a range of policy areas;
• Report on international policy developments and new international comparisons of health policy involving Australia and/or New Zealand; or
• Critique contemporary health policy developments.
Open Access
Australia and New Zealand Health Policy is an Open Access journal, which means:
• All articles will be freely and universally accessible online without any barriers to access, which increases their visibility.
• You and your peers will be free to print out copies of your article, email it to colleagues, and post it on the web because of the BioMed Central copyright and license agreement.
Open access journals are funded by article processing charges rather than journal subscriptions. The costs are therefore borne by the authors, their institutions of from their grants. That is, all access to journals is free to readers via the web (BMC online-only journals). Authors from institutional supporters are exempt from authorship charges. The institutional supporters pay a sliding scale based on the number of staff and postgraduate students in biomedical sciences.
Institutional subscription has a number of benefits. In addition to the direct benefits in terms of waived author fees, there are public policy benefits in supporting an open access journal regime such as Biomed Central. Open access journals are one mechanism for putting pressure on regular journal publishers to moderate their price increases.
Unfortunately there are no New Zealand institutional subscribers to BioMed Central at present, which means that New Zealand authors will face article processing charges, although no article processing charges will be payable on manuscripts submitted in the first six months following the launch of the journal. Article processing charges are also usually regarded as a legitimate charge against research grants. In the medium term, alternative arrangements, such as institutional support, should be encouraged, although after this time the editor-in-chief will be able to grant a limited number of discretionary processing charge waivers.
The first articles
Australia and New Zealand Health Policy commences its publication program with a series of articles which describe and evaluate health policy developments in Australia in 2003. Responses or commentaries on these articles would be welcome. It is the aim of the Editorial Board to encourage a cluster of articles at the start of each year about health policy developments in Australia and New Zealand in the previous calendar year. Authors who have an interest in reviewing contemporary developments are encouraged to submit manuscripts on these themes early in the New Year so that the articles can contribute in a timely way to health policy debate in the two countries.
S.J.Duckett
Editor-in-Chief
==== Refs
Sax S A strife of interests: Politics and policies in Australian health services 1984 Allen & Unwin, North Sydney
Davis P Ashton T Eds Health and public policy in New Zealand 2001 Oxford University Press, Auckland
Gauld R Revolving doors: New Zealand's health reforms 2001 Institute of Policy Studies and the Health Services Research Centre, Victoria University, Wellington, NZ
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Aust New Zealand Health PolicyAustralia and New Zealand Health Policy1743-8462BioMed Central London 1743-8462-1-21567993010.1186/1743-8462-1-2CommentaryAustralian primary care policy in 2004: two tiers or one for Medicare? Swerissen Hal [email protected] Director, Australian Institute for Primary Care La Trobe University Australia2004 17 11 2004 1 2 2 13 8 2004 17 11 2004 Copyright © 2004 Swerissen; licensee BioMed Central Ltd.2004Swerissen; 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 recent primary care policy debate in Australia has centred on access to primary medical (general practice) services. In Australia, access is heavily influenced by Commonwealth Government patient rebates that provide incentives for general practitioners not to charge copayments to patients (bulk billing). A steady decline in key access indicators (bulk billing) has led the Howard Government to introduce a set of changes that move Medicare from a universal scheme, to one increasingly targeted at providing services to more disadvantaged Australians. In doing so, another scene in the story of the contest between universal health care and selective provision in Australia has been written. This paper explores the immediate antecedents and consequences of the changes and sets them in the broader context of policy development for primary care in Australia.
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Introduction
Primary health care and community care can be thought of as a set of health programs and services. Most discussions of the primary health and community care services sector suggest that it has the following characteristics: (1) It is the first point of contact with the health system. This may occur through general practice, community health services, and pharmacies. There is also some overlap between primary care and hospital emergency departments, particularly for less complex and intensive presentations. (2) Services are provided in community and ambulatory settings and at home. (3) There is an emphasis on continuing relationships between service providers and consumers over extended periods of time. (4) Services have a more comprehensive and holistic approach. (5) There is an emphasis on early detection and illness prevention services such as maternal and child health programs and population health programs including health promotion.
Primary health and community care is the most visible and commonly used part of the health system. In 1999–00 the Commonwealth provided approximately $6 billion through the Commonwealth Medical Benefits and Pharmaceutical Benefits Schemes. States and Local Government provided approximately $1.8 billion for 'community and public health' which includes allied health, counselling, nursing and a range of primary and secondary prevention and health promotion programs. The Commonwealth, through direct outlays ($6 million) and private health insurance premium rebates ($97 million), also provided $103 million for dental services, with the States and Territories contributing $305 million. This does not include the substantial funds committed to the various forms of community support for people with disabilities, chronic illness and mental illness [1].
Primary health and community care services face unique challenges. Over the past three decades primary health services have come under significant pressure to address a more complex and diverse range of community needs.
Deinstitutionalisation, the introduction of new health and information technologies, the increasing prevalence of chronic disease and more general social and economic trends have had a significant impact on primary health and community support services. This has resulted in concerns about the equity, quality and efficiency of services and programs. Arguably, there is a need for a national primary health care policy in Australia. One that would address system integration, care pathways and team practice, work force development, payment arrangements, governance, performance management and accountability. However, current Commonwealth reforms are focused on important, but relatively, narrowly focused solutions to the decreasing affordability and access for general medical services. This article focuses primarily on the recent debate that surrounds this issue.
Recent Policy
Medicare is a Commonwealth Government, tax funded, social insurance scheme that provides rebates for general (primary) and specialist medical services and optometry. In Australia, it is the principal national program for ensuring equitable access to primary medical services. Over the last two years there has been a fiercely contested debate about the future of Medicare.
Medicare was introduced to provide universal access to affordable medical care. Up until recently Medicare simply provided a rebate of 85% of the Commonwealth Government determined schedule fee for medical and diagnostic services. Practitioners were free to charge patients a copayment as well. Where they did not apply a copayment, they could bill the Commonwealth for the rebate and receive bulk payments direct from the Commonwealth for these services, thereby avoiding administrative costs and delay. This payment method, which became known as bulk billing, ensured that services were effectively free to the patient at the point of service.
Medicare has been very successful, particularly for general practice services. It is strongly supported by the Australia community because it provides affordable access to medical services. Despite historical resistance by the Australian Medical Association, it has been widely supported by practitioners because it provides them with a universal, simple and predictable revenue stream.
Bulk billing increased steadily from the introduction of Medicare in 1984/85 to approximately 70% in the mid-1990s. Bulk-billing rates for GP services have generally been about 10% higher than the overall bulk-billing rates over the last decade, reaching a plateau of about 80% in the mid-1990s. They declined significantly after 2000. Average GP bulk billing fell to 68% by September 2003. GP bulk billing rates are now similar to the overall CMBS bulk billing rates for all services [2].
As bulk billing rates declined, disquiet and concern about access to medical services rose amongst stakeholder interests. More generally, the overall decline in bulk billing came on top of considerable disparity in equity of access between rural and urban settings. Bulk billing rates in inner city areas with high per capita GP ratios were 30% higher than those in rural settings with low per capita ratios. A number of remote rural areas had difficulty attracting any GPs at all.
Analysis of the reasons for the decline in bulk billing and the disparities between rural and metropolitan settings suggest a strong relationship between the supply of GPs and the capacity to charge copayments and the importance of the steady decline in the relative value of Commonwealth rebates for GP services over time.
There is considerable evidence that GPs manage demand for their services to maintain their income [3]. As the number of GPs increased with introduction of Medicare, particularly in inner city areas, per capita utilisation of GP services increased sharply. Average out of pocket costs for patients fell as bulk billing increased. The Commonwealth effectively provided the 'floor price' for services in areas of high supply and high competition leaving patient throughput rates as the primary means for increasing revenue. Urban areas with greater levels of disadvantage had higher bulk billing rates and shorter consultation times. In higher socio-economic status areas, where patients have a greater capacity to pay, there were lower bulk billing rates and longer consultation times.
In the decade to 2003, changes to GP training, migration and demographic ageing lead to a stabilization and decline in the supply of GP services. Over the same period, the relative value of Medicare rebate income for GP services fell by about 10 percent compared with average weekly ordinary time earnings. A decline that was probably even greater when compared to specialist incomes. In response, GPs began to experiment with price increases (co-payments) to improve their relative incomes [4].
Interestingly, bulk billing rates in relatively under supplied rural settings remained relatively stable at about 50% of consultations. This is about the level of consultations one would expect people on low incomes, who are eligible for concessional welfare benefits, to use. A finding that suggests that patient capacity to pay sets a 'floor' bulk billing rate at about this level [4].
It is worth noting that many of these effects were predictable from the reforms to General Practice introduced in the early 1990s. In particular, tight management of GP supply through changes to training programs and restrictions on overseas trained medical practitioners were introduced in order to reduce growth in aggregate Medicare expenditure. However, the reforms recognised that a move away from fee for service payment would also be required in the longer term. To this end a Better Practice Program to pay GPs on a per capita basis was introduced.
Over time, it was intended that a significant proportion of Medicare payments would be made by practice based, per capita payments. Progressively this would have allowed a shift toward more comprehensive, integrated practice and a greater focus on quality and preventive services. However, while the supply of GPs was successfully constrained, per capita payments remained a marginal component of the payment system.
In response to concerns about the fall in the bulk billing rate, the Commonwealth Government proposed a "Fairer Medicare" package in April 2003. The package introduced a participating practice scheme. GP practices that agreed to charge a no gap fee to concessional patients were to be eligible for increased Medicare rebates for these patients. The level of the proposed increase for the rebate was $1 in metropolitan city practices, $2.95 in non-metropolitan city practices, $5.30 in rural centre practices, and $6.30 in outer rural and remote areas.
Participating practices were to continue to have the capacity to determine fees for non-concession cardholders, including the option of bulk billing. However, if they chose not to bulk bill these patients, they were to be able to charge the patient the co-payment and claim the Medicare rebate direct from the Health Insurance Commission through HIC online billing facilities. Effectively, non-concession cardholders were to be charged a gap payment thereby avoiding the transaction costs involved in claiming a rebate through the Medicare scheme themselves.
A new MBS safety net was to be available for those covered by concession cards with out-of-pocket costs greater than $500 in a calendar year. Charges in excess of the scheduled fee were to be included, as were the costs of specialist and diagnostic services. Eighty per cent of out-of-pocket costs above the $500 threshold were to be met through this safety net.
Private health insurers were to be able to offer insurance coverage for the cumulative cost of out-of-hospital medical services over $1,000 for a family in a calendar year. This included costs above the scheduled fee across a range of out-of-hospital services, including GP and specialist consultations and diagnostic tests. The Commonwealth estimated that insurance products for this coverage were likely to cost around $50 per year for families, and the 30% private health insurance rebate was to apply to these products.
The Government's package also included proposals to introduce additional medical school places, additional GP training places, additional nurses and allied health professionals in general practice, and measures for veterans.
The Fairer Medicare package resulted in considerable debate and criticism, much of which was considered by the Senate Select Committee on Medicare [5]. In part the Committee concluded that:
• Equitable access to general practice services regardless of income or geography is fundamental to good health care.
• GP income from bulk billing had not kept pace with increases in average weekly ordinary time earnings and this had contributed to declining bulk billing rates and increased out of pocket charges.
• Shortages in the supply of GPs are emerging as result of compositional changes in the workforce, changes in practice patterns and population ageing.
• The Commonwealth's 'Fairer Medicare' proposals were inconsistent with the principles of Medicare.
• The differential rebate payments for concessional patients were unnecessary because these patients were already largely receiving bulk billed services
• The introduction of the new safety net arrangements creates a two tier system of access to GP services
It became apparent that the Senate would not pass the legislation required to enact the Commonwealth's package. Consequently, the Commonwealth presented its 'MedicarePlus' extensions and revisions to the original proposal in November 2003 [6]. This package was passed by the Australian Senate with the support of four independent Senators.
The MedicarePlus proposals dropped the participating practice scheme. Instead the Commonwealth proposed to increase the rebate for all concessional patients by $5 in metropolitan areas and $7.50 in remote, rural and regional areas (including the State of Tasmania). The increased rebate was also extended to children under 16. The safety net provisions were modified to provide an 80% rebate for out of hospital medical costs for concessional patients and those whose income fell below specified tax thresholds after $300 of out of pocket expenses and after $700 for the remainder of the population.
Other aspects of the original proposal were largely retained and extended. These included training places for GPs, medical graduates and nurses. Additionally, it was proposed to introduce a Medicare Benefits Schedule item for nursing support in general practice and improved internet access and online billing for GPs. MedicarePlus also provides rebates for up to five allied health consultations delivered to patients with a chronic condition or complex care needs, for and on behalf of a GP. Similarly, dental treatment care plans will be funded for these patients where they have significant dental problems that exacerbate their condition. The total estimated cost for MedicarePlus to 2006/07 was estimated at $2.85 billion.
Policy Analysis
Initial reactions to the Commonwealth's proposals were mixed. A number of patient and provider groups have criticized the new arrangements as undermining the principle of universality that underpins Medicare. Criticisms have also focused on the narrow focus of MedicarePlus on fees for general practitioners.
More specifically, MedicarePlus is likely to have differential effects on affordability and access to GP services in rural and metropolitan settings. In metropolitan settings, the introduction of a $5 differential rebate for bulk billing concessional payments is sufficient to increase net GP incomes to about the AWOTE relativities that applied prior to the decline in bulk billing.
However, with current levels of bulk billing still at over 65% in metropolitan areas, virtually all concessional patients are already bulk billed. The proposal is therefore subject to substantial dead weight loss. No incentives to bulk bill non concessional patients (other than non concessional children aged less than 16) are included.
In metropolitan areas, the gap between the average Medicare rebate and the average patient billed service is around $13 for patients not covered by the differential rebate, compared to $8 for concessional patients and children under 16. Within system constraints, GP incomes are optimized by bulk billing concessional patients and children under 16 and charging copayments for other patients. Doing so also largely addresses patient capacity to pay issues. In the absence of major changes to supply or GP costs, it is therefore likely that bulk billing rates in metropolitan areas will continue to decline over time, until they stabilise at around the level of services for concessional patients plus non concessional children under 16 (around 60% in metropolitan areas, assuming children under 16 have average population consultation rates).
The proportion of children is highest in outer metropolitan regions and lowest in the inner city. Bulk billing rates have declined most in outer metropolitan areas. It is therefore plausible that this measure will have the greatest differential impact in outer metropolitan regions.
In rural settings, where there are GP shortages, the differential rebate (which is higher than in metropolitan settings) could substantially increase GP incomes. However, GP supply factors ensure GPs have considerable capacity to increase copayments within the limits of patient capacity to pay. In general, bulk billing rates in rural settings are now at or below the consultation rate for concessional patients. The new arrangements are therefore likely to protect bulk billing rate for concessional patients and are likely to see the rate increase to the level of concessional consultations (around 55 – 60%).
The effect of differential rebates for non concessional children under 16 on overall bulk billing rates is less clear in rural settings. With the increased rebate, there remains a gap of approximately $5.50 between the new rebate and the average patient billed service. As for metropolitan settings, there are no additional incentives to bulk bill other non concessional patients. Given the greater capacity to charge copayments, this measure may be less successful in encouraging bulk billing than in metropolitan areas.
The safety net provisions in MedicarePlus have significant inflationary potential for out of hospital medical service fees. Concessional patients and those who qualify for Family Tax Benefit A are eligible for an 80% rebate on out of hospital costs once they incur $300 of out of pocket costs. There is no cap on the rebate under the safety net. Average out of pocket costs for patient billed GP services are currently about $13. The safety net is therefore reached in 20–25 consultations. The safety net provisions will be invoked more quickly when specialist medical practitioners, diagnostic imaging services and pathology are required. Average copayments are two or three times higher for specialist medical practitioners than for GPs.
Effectively, the introduction of the safety net removes constraints on medical practitioners associated with concerns about patient capacity to pay. This introduces moral hazard for practitioners and consumers. Practitioners have incentives to increase their fees and provide more services than necessary knowing the safety net will protect patients. Patients have incentives to consume more services than are necessary because they are effectively insured by the safety net.
However, the initial threshold and value of copayments act as balancing disincentives for utilization. Clearly if the initial threshold and the copayments were less, the hazard would be greater and vice versa. This trade off is likely to impact differently depending on need, capacity to pay and supply factors.
For example, there may be paradoxical adverse effects for patients with significant ongoing health costs who are currently bulk billed because GPs and specialists have concerns about their capacity to meet aggregate out of pocket costs over time. This is particularly true for aged pension recipients with chronic illness. With the introduction of the safety net, the potential for incurring unmanageable costs is significantly reduced and therefore bulk billing rates for this group may decline. Whether effects like these are experienced in practice will depend on factors such as the real value of GP rebates, patient need, capacity to pay, GP supply and regulatory constraints.
Overall, the design features of the Howard Government recent changes to Medicare are intended to, and will produce a two tier system. Access to primary medical services for people on low incomes will be relatively well protected, but those above the income threshold will see a steady decline in bulk billing and an increase in out of pocket costs for these services. Additionally, the poorly designed safety net will have inflationary consequences.
Future Directions
The policy and political contest around Medicare has an extended pedigree. The conservative Liberal/National Party Coalition has long held the position that government should primarily provide health services for those who are unable to provide for themselves and that those who are able to make their own way should do so, particularly by taking out private health insurance. From this perspective the role of government is to provide an appropriate regulatory environment, incentives and sanctions to take up private insurance and a targeted safety net for the disadvantaged. Their preferred model was developed and refined in the 1950s and 1960s during the period of the Menzies Government and reintroduced in stages during the late 1970s and early 1980s by the Fraser Government [7].
On the other hand, the Australian Labor Party has advocated tax funded, universal access to publicly funded health care provided on the basis of need, rather than capacity to pay. The Whitlam Government settled the basic architecture of Labor's approach in the early 1970s. Setting aside the brief flirtation with a national community health program for primary care, it established a universal system of public hospital access through the States and a tax funded, social insurance scheme to underwrite equitable access to medical and related services.
Notwithstanding Howard Government claims of strong support for Medicare, the pendulum has now swung a considerable distance back toward the traditional Liberal/National Party preferred model. If history is a guide, now that the incentives to take out private health insurance and the safety net is in place, the next steps are regulatory mechanisms to exclude higher income earners from accessing publicly funded health services.
While debates about access and equity are critical, they are only part of the overall picture. Recontesting the basic access and equity principles of the health system every decade or so misses a number of important emerging issues.
There is now emerging evidence that closer integration of clinical decision-making and purchasing for enrolled populations in primary care settings through funds pooling and local agreements and contracts has the potential to increase innovation, reduce costs and improve outcomes. These principles are being explored or actively implemented in a number of countries comparable to Australia, including the United Kingdom and New Zealand [8].
There is clearly a need to reconsider the development of a national policy for primary health and community support services. Such a policy might include the following elements to address the issues which have been discussed above:
• National primary health and community care goals and objectives. For example, these might broadly set out equity, efficiency and quality criteria for the Australian primary health and community support system.
• National performance indicators. For example, these indicators could be used to report on and benchmark the quality, access, efficiency and utilisation of the primary health and community support system and its impact on acute, sub acute and residential care.
• Population based planning, allocation and monitoring. For example, funding allocation models and system governance arrangements based on the health care needs of geographically defined residential populations (e.g. Divisions of General Practice, Area Health Authorities, Districts, Primary Care Partnerships) that promote continuity of care and service integration could be considered.
• Coordinated service pathways for health issues and conditions. For example, consistent best practice models linking prevention, early intervention, primary care, acute care, rehabilitation and community support should be developed for all major chronic diseases, mental illness and alcohol and drug problems.
• Payment systems. For example a program to develop integrated payment models and systems for primary and community support services could be established and linked to Commonwealth/State agreements (e.g. AHCAS, HACC) and own purpose funding streams. This might include consideration of capitated, case based, and contract funding to replace or compliment existing arrangements for primary care services.
• National workforce planning and analysis for primary health and community support services.
• A national evaluation, research and development program in primary health and community support services.
• National planning and priority setting processes for primary health and community care to ensure greater alignment of Commonwealth and State priorities.
==== Refs
Australian Institute of Health and Welfare Health Expenditure Bulletin No.17, Australia's Health Services 1999–00 2001 Canberra Australian Institute of Health and Welfare
Australian Government Department of Health & Ageing (2004) Medicare Statistics 2004 Canberra Australian Government Department of Health & Ageing
Richardson J Peacock S Supplier induced demand reconsidered Working Paper 81 1999 Melbourne: Centre for Health Program Evaluation
Swerissen H Duckett SJ Livingstone C An analysis of potential inflationary effects on health care costs for consumers associated with the Government's 'A fairer Medicare', and the Opposition proposal 2003 A Report for the Department of the Senate, Canberra
Sax S A Strife of interests: politics and policies in Australian Health Services 1984 Sydney: Allen and Unwin
Mays N Syke S Malbon G Goodwin N The purchasing of health care by primary care organizations 2001 Buckingham Open University
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Aust New Zealand Health PolicyAustralia and New Zealand Health Policy1743-8462BioMed Central London 1743-8462-1-31567993210.1186/1743-8462-1-3CommentaryRecent developments in national Aboriginal and Torres Strait Islander health strategy Anderson Ian PS [email protected] Centre for the Study of Health and Society & VicHealth Koori Health Research and Community Development Unit, Department of Public Health, University of Melbourne, Melbourne, Australia2004 18 11 2004 1 3 3 13 8 2004 18 11 2004 Copyright © 2004 Anderson; licensee BioMed Central Ltd.2004Anderson; 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 this paper I will describe some of the sentinel events in Aboriginal and Torres Strait Islander health policy and strategy during 2003 and the early part of 2004. This will involve discussion on the:
• National Strategic Framework in Aboriginal and Torres Strait Islander Health
• National Strategic Framework for Aboriginal and Torres Strait Islander Peoples Mental Health and Social and Emotional Well Being 2004–2009
• National Aboriginal and Torres Strait Islander Health Performance Framework
• The roll-out of the Primary Health Care Access Program
• The National Aboriginal and Torres Strait Islander Social Survey and the National Indigenous Health Survey
These developments are consistent with a policy agenda that has evolved, in general terms, since the release of the National Aboriginal Health Strategy in 1989. However, I will also consider significant developments in the broader context for Aboriginal and Torres Strait Islander affairs, particularly the decision made in early 2004 by the Howard government to abolish the Aboriginal and Torres Strait Islander Commission (ATSIC). While the key events and developments that are reported in this paper elaborate on an agenda that has been developing for more than a decade, the decision to abolish ATSIC is likely to have a revolutionary impact on the future development of Aboriginal health strategy.
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Introduction
Following the lead of the National Aboriginal Health Strategy (NAHS) [1], national strategy in this field has focussed on health sector reform and the development of inter-sectoral strategies to improve Indigenous health outcomes. In 1995, the health portfolio assumed responsibility for the management of the Australian government's Aboriginal health program from the Aboriginal and Torres Strait Islander Commission (ATSIC). Since that time, mechanisms have been established that provide a platform for collaborative, inter-governmental planning, engaging with both the Aboriginal community sector and the non-health sectors of government [2-4]. The key elements of this national planning framework include:
• Framework Agreements in Aboriginal and Torres Strait Islander Health (multi-party agreements between the Australian government; State and Territory governments; the Aboriginal and Torres Strait Islander Commission and the Aboriginal community controlled health sector);
• Joint Planning Forums (established at a jurisdictional level with responsibility for the developing State and regional Aboriginal and Torres Strait Islander health plans).
The NAHS has been the guiding framework for action in this field since it was endorsed in 1989. Consequently, it was significant that the Australian Health Ministers Conference endorsed its successor on the 31st of July 2003, the "National Strategic Framework for Aboriginal and Torres Strait Islander Health" (hereafter, the "National Framework"). Agreement has also been recently brokered that details strategies for Indigenous social and emotional well-being, which is one of the Key Result Areas for the "National Framework". Significant progress has also been made in the development of a national performance management framework for Aboriginal and Torres Strait Islander health that aligns with the "National Framework".
The agenda in Aboriginal and Torres Strait Islander health strategy that was adopted by the health portfolio post 1995 had focussed on reform priorities focussed on the development of [3]:
• The capacity of primary health services to respond to Aboriginal and Torres Strait Islander health need (with a particular focus on financing and workforce);
• disease and risk strategies that aimed to improve Aboriginal and Torres Strait Islander health outcomes;
• the evidence base for policy and practice in this sector (through strategic research and improvements in the quality of health and related data).
With respect to primary care capacity, the roll-out of the Primary Health Care Access Program (PHCAP) continues to be one of the central planks of this agenda and I will provide an overview of recent progress. Significant progress has also been made over the last couple of years in the development of the Australian Bureau of Statistics Indigenous Survey program, which promises to enhance the information available to assist decision-making within the sector. I will provide a report on the recent developments in the roll-out of this program.
While the recent developments in Aboriginal and Torres Strait Islander health policy and strategy represent an evolution of a health reform agenda that has been developing for more than a decade, the abolition of ATSIC points to a much more revolutionary change in the broader institutional and programmatic context for Aboriginal affairs. ATSIC had play a critical role in integrating Australian government programs in Indigenous affairs and providing an institutional structure that facilitated Aboriginal and Torres Strait Islander input into policy and program development. ATSIC, for instance, continued to play a role in health strategy following the transfer of specific health program responsibilities in 1995. It retained, for instance, responsibility for the delivery of environmental health service. A memorandum of understanding was developed between the Department of Health and Human Services and ATSIC to support collaboration between the sectors [6]. Consequently, the decision to abolish the ATSIC and radically overhaul of the administration of Commonwealth programs in Aboriginal Affairs has potential implications for national health strategy. These are discussed later in this paper.
Discussion
National Strategic Framework in Aboriginal and Torres Strait Islander Health
The National Aboriginal and Torres Strait Islander Health Council (NATSIHC) oversaw the development of the "National Framework". However, this process was stalled by political conflict between the key stakeholders. In December 2000, Council members representing the National Aboriginal Community Controlled Health Organisation (NACCHO) resigned in protest over a consultation draft of the 'National Framework'. NACCHO, which is the peak body representing the Aboriginal community controlled health sector, was concerned with [7]:
The way the Draft is written distances Aboriginal and Torres Strait Islander people, undermines the concept of Aboriginal community control of primary health care service delivery and diminished structures which NACCHO believe are still useful. The document's tone and language is wrong in a number of ways...
Following further negotiation, NACCHO withdrew its resignation, and the NATISHC was reconstituted with revised membership and Terms of Reference [8]. Despite this successful outcome, this ruction in the relationship with NACCHO illustrates the tenuous nature of partnership structures and processes in this sector – an issue that I will return when discussing the issues that may potentially flow on following the abolition of ATSIC.
The agreed "National Framework" consists of two documents:
• The "National Strategic Framework for Aboriginal and Torres Strait Islander Health – Framework for action by Governments", which sets out a five- to ten-year reform agenda in 9 key result areas [5].
• The "National Strategic Framework for Aboriginal and Torres Strait Islander Health – Context", which outlines the rationale for the Framework and its context [9].
There are nine Key Result Areas set out in the Framework including [5]:
• Community controlled primary health care: building community capacity so that individuals and communities can better address and manage their own health needs.
• Health system delivery framework: improving the responsiveness of the mainstream health system to Indigenous Australians and developing stronger partnerships between mainstream and Indigenous-specific services.
• A competent health workforce: improving the training, supply, recruitment and retention of appropriately skilled health professionals, health service managers and policy officers in both mainstream and Indigenous-specific health services.
• Emotional and social well-being: improving outcomes with respect to mental health, suicide, family violence, substance misuse and male health (through non-health sectors strategies).
• Environmental health: improving the delivery of safe housing, water, sewerage and waste disposal.
• Wider strategies that impact on health: undertaking action in portfolios outside the health sector and implementing health gain strategies in the areas of education, employment transport, food and nutrition, custodial health, aged and disability services, recreation and exercise.
• Data, research and evidence: aiming to improve the quality of information about how well the health sector is meeting the needs of Indigenous Australians.
• Resources and finance: aiming to provide an optimal level of resources for Indigenous health commensurate with levels of need, costs of delivering services and community capacity to deliver health outcomes.
• Accountability: both to communities and to governments for the delivery and effectiveness of health services.
The "National Framework" was endorsed as a plan to guide all Australian governments in a coordinated, collaborative and multi-sectoral approach to achieving Aboriginal and Torres Strait Islander health gain over the next decade. It does not have a specific funding program attached to its implementation, although arguably, the roll-out of the Primary Health Care Access Program (described later) will provide additional capacity to the implementation of the "National Framework". It is also possible that the National Framework will guide the allocation of any new resources made available through the joint planning processes established under the Framework Agreements.
To further these ends, it is significant that the "National Framework" was endorsed through each government's cabinet process, providing a whole-of-government commitment to its implementation in each State and Territory and at the Commonwealth level. Each jurisdiction is developing its implementation plan against which it will report annually on progress and outcomes in health portfolios and biennially on whole of government progress. The plans will identify the specific strategies and timeframes for each action area. The National Aboriginal and Torres Strait Islander Health Council will develop a plan for an independent mid-term and final evaluation.
The National Strategic Framework for Aboriginal and Torres Strait Islander Peoples Mental Health and Social and Emotional Well Being 2004–2009
The "Social and Emotional Well Being Framework (SEWB Framework)" [10] is based on Aboriginal health values that emphasise the need for a holistic and 'whole of life' approach to achieving the conditions for well-being. Although this framework encompasses the traditional field of mental health, these issues are situated in an approach that also addresses the emotional and social well-being of Indigenous Australians and their communities.
The nine guiding principles for the "SEWB Framework" were been extracted from "Ways Forward" [11], an earlier strategy that established the importance of this holistic approach in this area of health. In supporting this holistic approach the "SEWB Framework" articulates strategies that support self-determination and culturally valid understandings of health. It further recognises the impact of trauma, grief, loss, discrimination and human rights issues on the social and emotional well being of Aboriginal and Torres Strait Islander communities.
In 2003 the Social Health Reference Group (SHRG) (established to oversee its development) conducted extensive consultations on a draft framework document. Since then the 'SEWB Framework' has endorsed by the NATSIHC and the National Mental Health Working Group in November 2003, and the Standing Committee on Aboriginal and Torres Strait Islander Health in December 2003. It was anticipated that the final 'SEWB Framework' document would be endorsed out of session by the Australian Health Ministers Advisory Council by the middle of 2004.
The Aboriginal and Torres Strait Islander Health Performance Framework
The development of the Aboriginal and Torres Strait Islander Health Performance Framework has built on the foundations of earlier work which has established the key elements of this framework, including the:
• national performance indicators in Aboriginal and Torres Strait Islander health for the Australian Health Ministers Advisory Council [3];
• service activity reporting for Aboriginal community controlled health services [12];
• Australian government health portfolio indicators [13]; and
• the reporting against key indicators of Aboriginal and Torres Strait Islander disadvantage for the Council of Australian Governments [14].
It is intended that the Aboriginal and Torres Strait Islander Health Performance Framework will both integrate those government performance reporting processes that have already been developed; streamline reporting processes in Indigenous health and, ensure the strategic management of policy relevant and quality information in published reports (such as the National Health Performance Committee, the Productivity Commission's Report of Government Services and the Indigenous Disadvantage Report) [15]. As starting point, the National Health Performance Committee Framework, which has already been endorsed by the Australian Health Ministers Conference will be used as a guide to the relevant measurement domains for Aboriginal and Torres Strait Islander specific framework [15]. It is also intended that the Aboriginal and Torres Strait Islander Health Performance Framework will support the implementation of the 'National Framework' by:
• mapping the relationship between the Key Result areas of the 'National Framework' and key domains of health performance (effectiveness, safety, responsiveness etc); and
• identifying priorities for data development and improvement based on priorities of the 'National Framework'.
Roll-out of the Primary Health Care Access Program
The Primary Health Care Access Program (PHCAP) was introduced in the 1999–2000 Federal Budget to improve access to primary health care for Aboriginal and Torres Strait Islander people. PHCAP achieves this by funding increased primary health care provision, such as additional general practitioners, nurses, Aboriginal Health Workers, and through preventive and health promotional activities, such as diabetes education and management. Funds are also used for supports to service provision such as capital works and equipment. The program also aims to work with existing health services to ensure they are responsive to the needs of Aboriginal and Torres Strait Islander people.
On average across Australia, PHCAP aims to bring the level of Commonwealth funding for Indigenous primary health care to three times the average MBS usage for all Australians. The key objectives of PHCAP are [3]:
• Increased availability of appropriate primary health care services where they are currently inadequate;
• Local health systems that better meet the needs of Aboriginal and Torres Strait Islander people; and
• Individuals and communities that are empowered to take greater responsibility for their own health.
Services can be provided through a mix of arrangements, including Indigenous specific, mainstream or a combination of these. Funding can also be used to support mechanisms to assist service providers to deliver better services and enable individuals and communities to become more involved in improving their health.
Up until March 2004, new and additional services have been funded in Central Australia (5 regions), Queensland (3 regions) and South Australia (4 regions) through PHCAP, as well as continued funding of services provided through the former Aboriginal Coordinated Care Trials (Yael Cass, Office for Aboriginal and Torres Strait Islander Health, Australian Department of Health and Ageing, personal communication).
During 2003 a more streamlined approach to the management of PHCAP rollout was developed [4,16], resulting in more than 200 proposals to improve access to primary care being developed in each state and territory. This was in discussions with members of the state or territory forum or partnership, and drawing on regional plans and other work that has been undertaken over the last several years.
From these proposals, $11.8 million in funding was approved on 14 March 2004 for:
• additional health professional and support staff, for example, over 20 more health professional positions in the Kimberley region of WA;
• capital works for health clinic upgrades and the construction of staff housing in remote communities;
• minor capital purchases such as medical equipment; and
• one-off health promotional activities and health board support and training.
Longer term strategies around enhancement of local service systems, to ensure they are more accessible for Aboriginal and Torres Strait Islander people, and ensuring the commitment by state/territory governments to at least maintain their funding commitments, will continue to be pursued. While the PHCAP program has provided a significant injection of resources into what is generally considered an inadequately funded primary heath care system, the amount made available through this program still does not meet its programmatic benchmarks and targets [4].
Roll-out of the Australian Bureau of Statistics Indigenous Survey Program
One of the development priorities established by the heath portfolio when it took responsibility for the administration of the Aboriginal health program was to develop the evidence base to support policy reform and the development of health service capacity [3].
The National Health Survey, undertaken by the Australian Bureau of Statistics with funding support from the Australian Department of Health and Ageing, collects information about the health status, use of health services and facilities, socio-economic status and health-related aspects of the lifestyle of Australians. The Indigenous component of this survey aims to benchmark information on a range of health issues and enable comparisons between the health characteristics of Indigenous and non-Indigenous Australians and to allow trends in the health of Indigenous Australians to be monitored over time.
The Indigenous Health Survey that was run in 2001, collected data from approximately 3,500 individuals which was reportable at the national level [17]. In 2004, the Indigenous Health Survey will collect information from 11,000 Indigenous participants in order to be able to provide statistics at the national and State/Territory levels, and some geographical areas. It will also, for the first time, provide information on mental health. It is anticipated that the data collected will be reported in 2005.
In parallel with the health survey program the Australian Bureau of Statistics collected data for the 2002 National Aboriginal and Torres Strait Islander Social Survey from August 2002 to April 2003 [18]. It is planned to repeat this survey a six yearly intervals. A summary of findings has been published that covers topics such as family and culture, health, education work, income and housing law and just and transport.
A revolution in program administration in Aboriginal Affairs
On 20 April 2004, the Prime Minister, John Howard and the Minister for Aboriginal Affairs, Senator Amanda Vanstone, announced the intention of the Australian government to abolish the ATSIC.
ATSIC had been established in 1989 when the program responsibilities of the Commonwealth Department of Aboriginal Affairs and the Aboriginal Development Corporation were merged into a structure that enable the regional allocation of resources through elected regional councils. The board of Commissioners, elected by ATSIC regional councils, was responsible for national policy development and the oversight of national programs. At the Commonwealth level, ATSIC had the lead agency responsible for the administration of a range of programs such as: community development and employment (CDEP); housing and infrastructure; cultural heritage, broadcasting services; legal services; native title, land rights and the Indigenous land fund, etc.
The agency also played a critical role in co-ordinating and integrating the Aboriginal strategy across the different government program areas. ATSIC and the health portfolio had collaborated in the implementation of health infrastructure priority projects [19]. The Memorandum of Understanding developed between the two portfolios enabled collaborative planning across a range of programs that impacted on Indigenous health outcomes. The advantage of this institutional arrangement for cross sectoral strategy is that these programs might otherwise have been dispersed across a number of different government departments or instrumentalities. Further, ATSIC provide a structure for engaging Indigenous participation that broader than the sector specific mechanisms.
ATSIC played a pivotal institutional role in the development of 'whole of government' strategies across the Australian government. It was in effect the only institutional mechanism (with the exception of time limited inter-departmental committees) that enabled this. This was until the Council of Australian Governments (COAG) resolved (in 2000 and 2002) to trial, in up to 10 regions across the country, innovative administrative arrangements, developed in partnership with Indigenous communities, which aimed to provide "more flexible programs and services based on priorities agreed with communities" [20].
From its first term in 1996, the Howard Coalition government had a conflictual relationship with the Commission. However, government confidence in the ATSIC Board deteriorated significantly under the chairmanship of Geoff Clark (first elected chairperson in 1999) to the extent that the Minister for Indigenous Affairs suspended him on the ground of misbehaviour (under section 40 of the ATSIC Act 1989) [21]. A review of ATSIC was undertaken during the period December 2002–October 2003. It recommended that ATSIC should be retained as the primary vehicle for representing the aspirations of Aboriginal people to all levels of government and that its existing program responsibilities should also be retained pending a determination of its role in the context of [a] broader examination of service delivery [22]. The review also recommended a comprehensive program of reform primarily focussed at strengthening the capacity of regional councils and improving the relationships between ATSIC and the Australian government and between ATSIC's elected and administrative arms. Prior to the completion of the review the Coalition government moved to structurally separate ATSIC into an elected arm (ATSIC) and an executive agency, Aboriginal and Torres Strait Islander Services (ATSIS). ATSIS retained, under Ministerial delegation, program administrative responsibilities.
The Federal cabinet, nevertheless, resolved to a more radical agenda than outlined in the review findings, and announced its intention to abolish ATSIC, its regional councils and the mainstreaming of the administration of all the programs for which ATSIC had been responsible. It is proposed that the elected advisory structures will be replaced by a government appointed national council. It is also proposed that Indigenous specific program dollars will be quarantined and a whole of government approach is to be developed for the delivery of Indigenous specific funding. The key elements of this reform package have been positioned within the broader context of a government commitment to reforms aimed at producing "'joined up' government and the 'seamless' delivery of programmes" [23]. This new framework for Indigenous policy and program administration also include the establishment of a:
• Ministerial Taskforce: which would operate as a cabinet committee, provide collaborative leadership at a government level and set strategic directions.
• Secretaries group: which would support Ministerial decision-making, coordinate across government agencies, and oversight annual reporting.
• National Indigenous Council: in which the Minister would appointment Indigenous leaders in health, education, employment, law and justice to provide advice and monitor performance.
The proposed mechanisms and structures that would be established to deliver this 'joined-up' framework including regional partnership agreements and community shared responsibility agreements (detailing mutual obligations). It is also proposed to establish Indigenous co-ordination centres which will provide a single shopfront in regional and remote Australia for indigenous specific programs, lead the negotiation of regional partnerships an shared responsibility agreements but maintain line responsibility to mainstream departments.
The impact of this radical reform agenda to national Aboriginal health strategy is difficult to predict. One the one hand the actual changes to the administration of the health program is insignificant (leaving aside some potentially critical issues in budget development). A mainstream department has administered this program since 1995. One the other hand, the implementation of this reform agenda could have potentially very significant consequences for the development of inter-sectoral strategies in Indigenous health. This depends on the success in implementing the new mechanisms, and on their effectiveness. Furthermore, the political consequences of this radical agenda on the relationship between the Australian government and Aboriginal and Torres Strait Islander peoples have yet to really become clear. Specific partnership arrangements that have been developed within the health sector are tenuous – as is evident by the politics in the development of the "National Framework". These partnerships are critical to successful implementation of strategy in Indigenous health. Consequently, deterioration in the broader relationship between Indigenous Australians and the Australian government may have significant negative consequences for the partnership processes specific to the health sector. Even though 2003 was a year in which policy and strategy in Indigenous health made no or new radical departures, it was a year of considerable tumult in relations between the Australian government and Indigenous peoples. The ramifications of this are only now beginning to unfold.
Abbreviations
ATSIC Aboriginal and Torres Strait Islander Commission
NAHS National Aboriginal Health Strategy
NATSICH National Aboriginal and Torres Strait Islander Health Council
NACCHO National Aboriginal Community Controlled Health Organisation
PHCAP Primary Health Care Access Program
Competing interests
The authors declare that they have no competing interests.
Acknowledgments
Core funding for the VicHealth Koori Health Research and Community Development Unit is provided by the Victorian Health Promotion Foundation and the Commonwealth Department of Health and Ageing. The author would like to express his thanks particularly to Elizabeth Harding from the Office for Aboriginal and Torres Strait Islander Health who provided an initial briefing on key events during the last 12 months and assisted the author in accessing key documents. The author would also like to express his thanks to those key informants who reviewed a draft of this paper. The opinions expressed in this paper are the responsibility of the author alone.
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National Aboriginal Health Strategy Working Party A national Aboriginal health strategy 1989 Canberra: National Aboriginal Health Strategy Working Party
Australian National Audit Office The Aboriginal and Torres Strait Islander health program, Commonwealth Department of Health and Aged Care Audit Report No 13a 1998 Canberra: Australian National Audit Office
Anderson I National strategy in Aboriginal and Torres Strait Islander health: a framework for health gain? Discussion paper no 6 2002 Melbourne: VicHealth Koori Health Research and Community Development Unit, University of Melbourne
Dwyer J Silburn K Wilson G National strategies for improving Indigenous health and health care, consultant report no 1 for the review of the Australian Government's Aboriginal and Torres Strait Islander primary health care program 2004 Canberra: Commonwealth of Australia
National Aboriginal and Torres Strait Islander Health Council National strategic framework for Aboriginal and Torres Strait Islander health – framework for action by governments 2003 Canberra: Commonwealth of Australia
Aboriginal and Torres Strait Islander Commission & Commonwealth Department of Health and Human Services Memorandum of understanding between the Department of Health and Human Services and the Aboriginal and Torres Strait Islander Commission (ATSIC) Canberra 1995
National Aboriginal Community Controlled Health Organisation (NACCHO) Peak Aboriginal health body pulls out of government advisory group in protest [press release] NACCHO AGM 8 December 2000
Australian Department of Health and Aged Care Annual report 2000–2001, part 2 outcome reports (Outcome 7: Aboriginal and Torres Strait Islander health) publication content last modified 26 November 2001, page last modified 1 February 2002, access date: 10 May 2004
National Aboriginal and Torres Strait Islander Health Council National strategic framework for Aboriginal and Torres Strait Islander health – context 2003 Canberra: Commonwealth of Australia
Department of Health and Ageing Social and emotional well being framework, a national strategic framework for Aboriginal and Torres Strait Islander mental health and social and emotional well being (2004–2009) [draft] 2004 Canberra: Australian Government
Swan P Raphael B Ways forward: national consultancy report on Aboriginal and Torres Strait Islander mental health 1995 Canberra: Australian Government Publishing Service
Commonwealth Department of Health and Ageing and National Aboriginal Community Controlled Health Organisation Service activity reporting 1998–1999 key results 2001 Canberra: Commonwealth Department of Health and Ageing
Commonwealth Department of Health and Ageing Department of Health and Ageing annual report 2001–2002 2002 Canberra: Commonwealth Department of Health and Ageing
Steering Committee for the Review of Government Service Provision (SCRGSP) Overcoming Indigenous disadvantage: key indicators 2003 2003 Canberra: Productivity Commission
Office for Aboriginal and Torres Strait Islander Health Policy context developing an Aboriginal and Torres Strait Islander health performance framework 2003 Canberra: Australian Department of Health and Ageing [unpub. paper]
Australian Department of Health and Ageing 2004–05 portfolio budget statement Budget related paper no 111 2004 Canberra:Commonwealth of Australia
Australian Bureau of Statistics and the Australian Institute of Health and Welfare The health and welfare of Australia's Aboriginal and Torres Strait Islander peoples 2003 2003 Canberra Commonwealth of Australia
Australian Bureau of Statistics National Aboriginal and Torres Strait Islander social survey 2002 2004 Canberra: Commonwealth of Australia
Commonwealth Department of Health and Family Services 'Submission from the Commonwealth Department of Health and Family Services to the House of Representatives Standing Committee on Family and Community Affairs Inquiry into Indigenous Health' in Inquiry into Indigenous health, submissions authorised for publication, national organisations 1997 1 Canberra: House of Representatives Standing Committee on Family and Community Affairs 215 316
Management Advisory Committee Connecting government, whole of government responses to Australia's priority challenge 2004 Canberra: Commonwealth of Australia
Minister for Immigration, Multicultural Affairs and Indigenous Affairs Suspension of Mr Geoff Clark as ATSIC Commissioner [press release] IPS 060/2003 2003 Canberra: Australian Government
Hannaford J Huggins J Collins B In the hands of the regions – a new ATSIC, report of the review of the Aboriginal and Torres Strait Islander Commission 2003 Canberra: Commonwealth of Australia
Shergold P Connecting government Canberra: Department of Prime Minister and Cabinet 20 April 2004
| 15679932 | PMC544962 | CC BY | 2021-01-04 16:38:26 | no | Aust New Zealand Health Policy. 2004 Nov 18; 1:3 | utf-8 | Aust New Zealand Health Policy | 2,004 | 10.1186/1743-8462-1-3 | oa_comm |
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Aust New Zealand Health PolicyAustralia and New Zealand Health Policy1743-8462BioMed Central London 1743-8462-1-41567992610.1186/1743-8462-1-4CommentaryHealthy children, healthy country: the use of governing instruments in shifting the policy paradigm Leggat Sandra G [email protected] School of Public Health, La Trobe University, Bundoora, Australia2004 18 11 2004 1 4 4 18 8 2004 18 11 2004 Copyright © 2004 Leggat; licensee BioMed Central Ltd.2004Leggat; 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 evidence on early childhood strongly suggests the need to shift child health policy from the current focus on social welfare to a socio-ecologically based approach. This paper reviews three governing instruments, exhortation, expenditure and regulation, that have been used by governments in Australia and discusses the relative effectiveness of these approaches in shifting the child health policy paradigm.
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The evidence for healthy public policy for children
There can be no keener revelation of a society's soul than the way it treats its children [1].
Research evidence has demonstrated that the experiences of early childhood can have a profound lifelong impact on a child's health, wellbeing and competence [2]. The importance of the early years of life in influencing future outcomes, such as crime, obesity, heart disease, mental health problems and poor school outcomes has been identified and highlighted [3]. While there are many factors found to influence rising crime rates, various researchers have identified children with manifested behaviour disorders in early childhood [4], academic difficulties and non-engagement in schooling [5], and the quality of neighbourhood supervision and support as contributing factors to criminal behaviour [6].
Education, literacy and other social determinants of health can influence the coping skills of children, which provide the basis of learning, behaviour and health throughout life [7]. Poverty, whether measured in absolute or relative terms, has a negative effect on children's health [8,9]. In particular, poverty is associated with developmental delay, poor school achievement and employment futures, behaviour problems, increased incidence of chronic illness, visual and hearing defects and dental problems [10]. Parental poverty and exposure to unhealthy environments (eg smoking; low levels of literacy; nutrition; emotional support) reduce a child's life chances. Studies in neurobiology, neurodevelopment and early intervention show that the time period from conception to school age is a critically important time for brain development, setting the scene for prevention of some of the identified adverse outcomes through early identification and intervention [11].
Consistent with the increasing evidence, many governments have identified support in early childhood as a lifelong determinant of health, wellbeing and competence, as a matter for policy development, initiating actions to ensure comprehensive child development strategies for their societies. This approach requires a whole of government response, integrating health, welfare, education and other relevant parts of government. The evidence suggests that healthy public policy for infants, children and their parents is dependent on understanding of the socio-ecological factors supported by integrated, multidisciplinary and intersectoral policy and programs.
In Canada, Britain and the United States, targeted interventions in the antenatal period, infancy and childhood, including parenting skills programs, are recognized for their potential to support healthier families. A socio-ecological model of health is increasingly perceived to be the most appropriate approach for the early years of life agenda. Consistent with this approach, the United Kingdom program 'Sure Start', has been positively reviewed by the UK Audit Office and is considered by many to be the standard for the whole of government approach [12]. In addition, a recently released ten year plan is attempting to significantly change the way in which children are treated throughout UK systems [13].
The Canadian experience is widely quoted as best practice [2], with both federal and provincial investment in early childhood (See, for example [14-16]). In the US, during the Presidents' Summit for America's Future held in April 1997, Presidents Bill Clinton, George Bush, Jimmy Carter and Gerald Ford and First Lady Nancy Reagan stressed the importance of early childhood, calling the nation to action. American policy in this area has built upon influential reports that have led to investment in early childhood in most states [12].
The adoption of healthy public policy for children based on this socio-ecological framework has been inconsistent throughout Australia. In an attempt to explore these inconsistencies, this paper reviews the use of three governing instruments, that is exhortation, expenditure and regulation, by national and state governments in Australia. Governing instruments are the major mechanisms governments use to seek compliance, support and implementation of public policy. Governing instruments range from minimum coercion by exhortation, through expenditure, taxation, regulation, to maximum coercion through public ownership [17]. The following sections describe the impact of the use of exhortation, expenditure and regulation on the implementation of healthy child policy.
Consensus building – exhortation as the national instrument of choice
During the 1990s the Australian Government identified the health of children and young people as a key policy area, with a series of policy documents:
• The National Health Goals and Targets for Australian Children and Youth (1992)
• The National Health Policy for Children and Young People (1995) and associated Implementation Plan (1996)
• The National Health Policy for Young Australians (1997).
These documents provided broad national goals for children and young people:
• Reducing preventable premature mortality
• Reducing the impact of disability
• Reducing the incidence of vaccine preventable disease
• Reducing the impact of conditions occurring in adulthood with their origins or early manifestation in childhood or adolescence
• Enhancing family and social functioning
Although the evidence supporting a broader definition of child health was strong, the focus of these National Health Goals and Targets remained heavily focused towards surveillance and the reduction of injury and illness, perhaps reflecting a comfort with current and past approaches.
To date there has been little evidence of an integrated, multidisciplinary approach to child health at the national level. The 2003/04 federal budget did not provide the broad whole of government approach recommended for child health, with only a few targeted interventions, such as the National Meningococcal C Campaign, and a much greater focus on the health needs of the aging population. In 2003, the Australian of the year, Fiona Stanley suggested that the social and economic policies of the Government were not effective in tackling the issues associated with ensuring healthy children and young people [18].
The platform for a paradigm shift was established in 2001 with the appointment of the Minister for Children and Youth Affairs and the subsequent statement in 2002 of the intent to develop a National Agenda for Early Childhood. The consultation paper Towards the Development of a National Agenda for Early Childhood signaled a changing paradigm, with a whole of child and life course approach addressing promotion, prevention and early intervention for all children.
The last years have seen the creation of ever more advisory groups, partnerships and inquiries with a mandate to influence child health policy. The Child and Youth Health Intergovernmental Partnership (under the auspice of the Australian Health Ministers' Advisory Council) was convened in December 2001 to develop a national child public health strategy and advise on the National Agenda for Early Childhood. Their draft strategy framework Better Child Public Health: A Strategic Approach to Building Capacity – A National Action Plan 2004–2007 has been developed and is being used in consultation and capacity building initiatives. In October 2002 the Minister for Children and Youth Affairs referred an inquiry into improving children's health and well being to the Standing Committee on Family and Community Affairs. The Australian Council for Children and Parenting (ACCAP), an advisory body to the Minister for Children and Youth Affairs, was granted a two year term from July 2003, with a focus on strategic advice in the areas of early childhood intervention and prevention, parenting and child protection, foster care and emerging early childhood initiatives, including advising about the continuing development of the National Agenda for Early Childhood.
As described above, the policy approach at the national level has focused almost entirely on exhortation, the least coercive instrument, where support and compliance are sought voluntarily through persuasion and discussion. In comparison with other countries, such as Britain and Canada, the lack of a common and shared understanding of the socio-ecologic approach and its implications has made it difficult to show any significant advances in this area. In fact, the recent demise of the Child Health Unit within the Australian Government Department of Health suggests less focus on child health.
Nationally, child health has not been heavily addressed through other policy instruments, such as expenditure, taxation, or regulation, although more recently the Commonwealth Government Department of Family and Community Services (DFaCS) established the 'Communities for Children' initiative as part of the Stronger Families and Communities Strategy. Communities for Children will directly fund 35 Australian communities between $1 and 4 million over four years to support parents, neighbourhoods and the wider community to give children the healthy start they need [19]. Importantly, there was little evidence of a community development or even a consultative approach in the implementation of this program, with the perception that Communities for Children has not been set up to respond to the greatest need.
It has been suggested that system change can be accomplished by motivating institutions, systems and actors to move in common directions and develop structures that sustain these efforts over time [20]. This requires a high level of trust among the participants, such that they eventually share common goals and voluntarily seek to achieve common ends. Success in using exhortation as a policy instrument requires that information not only flow from government, but also to it [21]. The strong use of exhortation at the national level may be seen as the only way to encourage change, given the shared responsibility for child welfare among the various levels of government in Australia. Yet it is precisely this divided accountability and responsibility that has been identified 'as the greatest barrier to the reform of children's services' [[22] pg. 980].
The use of exhortation may be successful at motivating common approaches but will be much less effective at ensuring the sustaining structures are developed. This is apparent in the existing committee structures, which still operate from within the government structures and are thus unable to cross the 'silos' to promote the needed whole-of-government approach. To be effective in changing the paradigm in this area, the exhortation process will require back up by more coercive governing instruments.
Conflicting expenditures – potential for uncertain outcomes in Victoria
In comparison with other Australian states, Victoria has been slow to provide visible translation of the socio-ecological model of health for children and young people in a coordinated and systematic way to state policy. A recent review of Victorian paediatric services suggested that Victoria needed to establish a child and young people focus ensuring appropriate mechanisms to plan, coordinate and monitor across government departments and service providers [23]. It was suggested that a structure was required to coordinate child health among of the various portfolios in the Victorian Department of Human Services – Health, Housing, Welfare, and Disability – as well as among the broader Government departments, contributing to the whole of government approach required for early childhood intervention programs.
The lack of coordinated focus on child health in Victoria is perhaps the result of a lingering policy focus on health surveillance. Despite the increasing evidence that surveillance and screening programs have limited effectiveness in child health [24,25], it is only recently that Victoria has increased the focus on the social determinants of health [26,27]. Most recently, Victoria has committed to the 'Best Start' program and will pilot it as demonstration projects in 10 communities across the State with an investment of $7.6 million. Best Start is auspiced by the Departments of Human Services and Education and Training and is focused on reducing the impact of disadvantage (from any cause) and enhancing the life chances of all children by strengthening the universal preventative system [28]. The aims of Best Start are multi-level including the social, emotional and physical well-being of children, capacity building of parents and carers and communities to assist them to become more child friendly, while focusing on specific interventions for socially disadvantaged families [29]. The demonstration projects are required to follow a prescribed implementation and evaluation process attempting to measure what works, under what circumstances and for whom, to ultimately improve services elsewhere in the State. Five approved demonstration sites with a total of $7.6 million are ensuring a 'brighter future' for the children of Frankston, Hume, Shepparton, Whittlesea and Yarra Ranges, while the rest of the State's children wait in the dark.
Despite the intentions of Best Start, existing government funding and reporting in the area of maternal and child health is still largely focused on surveillance [26]. The Victorian approach to policy implementation in the area of early childhood support is focused predominantly on expenditure. Public expenditure is moderately coercive, with distribution of government funds to achieve particular policy objectives. But the small expenditure allocated to 'healthy' child policy that is limited to identified demonstration sites with expectations that the program will be shown to be effective before statewide mainstream implementation is overshadowed by a much larger expenditure pool that is not focused on the socio-ecological model. While Best Start signals intent to change the child health policy paradigm, the incentives established through the broader expenditure pool suggest, for the moment, maintenance of the status quo in Victoria.
Guidelines – will enforcement back the regulatory approach in NSW?
In New South Wales the 'Families First' initiative targets families with children 0 to 8 years, with the aim of helping parents give their children a good start in life. Demonstrating the commitment to a whole of government approach, the Office of Children and Young People (OCYP), located within The Cabinet Office, reporting directly to the Premier, has played a lead role in the development and implementation of the Families First strategy. This evidence-based approach is delivered jointly by five NSW government agencies – Area Health Services, Community Services, Education and Training, Housing and Disability, Ageing and Home Care in partnership with parents, community organisations and local government. NSW Health supports 'the ongoing development of partnerships at policy, planning and service delivery levels to enable improved co-ordination and intersectoral collaboration in the delivery of child health services' [[30] pg. 44].
NSW has also successfully translated much of the evidence into coordinated service planning and delivery at the regional level. Paediatric networks, associated with the Area Health Services, were established in 1997 and today provide designated primary, secondary and tertiary level services for families with children aged 0 to 5 years [31]. In addition, the NSW Commission for Children and Young People focuses on increasing the participation of children and young people in decision making that affects their lives, promoting the safety and welfare of children and young people, and strengthening the important relationships in the lives of children and young people and improving their well-being [32].
The implementation of Families First has been guided by a series of policy and practice guidelines. Recently, an independent review of Families First implementation within three regions, (Orana Far West, Illawarra and South West Sydney) found that the system changes required to build and strengthen service networks for families needed more than agreement and goodwill, with considerable effort to develop structures and processes that sustain interagency collaboration [33]. This resulted in a further guide to implementing sustainable and effective child and family service networks.
Regulation involves the imposition of requirements to meet specific obligations. Often regulation is seen to exist within legislation outlining strict rules of behaviour. However, in health policy guidelines are considered effective means of imposing regulation, recognising the inherent uncertainty in safe practice in health care [17]. The implementation of 'healthy' child policy in NSW suggests a strong focus on the socio-ecological approach supported by the research evidence. This is apparent in the whole of government approach with leadership from the Premier's Office, backed by regulation to effect the necessary changes in the delivery system.
However, it is yet to be seen whether the policy will be supported with the necessary resources for compliance. While regulation involves shifting costs of compliance from government to other participants, enforcement and monitoring can be expensive and difficult [21]. Without adequate enforcement the potential for inequality and inequity in access to the proposed service model is high.
Conclusions
A variety of instruments have been used by government to change the child health policy paradigm from that focused on social welfare to 'healthy' public policy predicated on a socio-ecological foundation. NSW has chosen regulation to implement a child health policy framework that is built upon the international evidence of the effectiveness of integrated, multidisciplinary and intersectoral policy and programs. A little slower to change paradigms, Victoria has established demonstration programs through targeted expenditure, without an overarching whole-of-government child health policy framework. Nationally, there is a move to change the paradigm to a broader definition of child health almost exclusively through exhortation.
Instrument choice is influenced by a variety of factors. The use of exhortation by the Commonwealth Government is a relatively risk-free easy approach, which can counteract the divided accountabilities among federal and state governments in the area of health and social services. Exhortation is easy to implement; it is the least coercive and relies on voluntary goodwill. While it may be successful in building a common understanding, and even this is debatable, an independent review of the Families First implementation found that the system changes required to build and strengthen service networks for families needed more than agreement and goodwill to develop the necessary structures and processes – a suggestion that without other approaches to structural reform, exhortation is unlikely to be successful.
The expenditure policy of Victoria illustrates an approach that is compromised by the lack of an underlying agreed evidence-based policy framework. This lack of coordinated government approach is reflected in conflicting expenditure policy in this area, with the potential to confound outcomes.
While the regulatory approach of NSW suggests bold steps to change the paradigm, in fact, because regulation is not subjected to the same level of scrutiny of other instruments, such as expenditure, and even exhortation [17], it is a deceptively simple mechanism to implement policy [21]. The strength of the government intent to change the paradigm will only become apparent with visible enforcing of the service delivery directions.
The evidence for a new policy paradigm is strong. But the use of these different policy instruments underscores the lack of shared understanding and policy agenda. Oberklaid suggests that while there are similarities in the rhetoric throughout Australia, there has been relatively little investment in child health [12]. The change in the child health public policy paradigm will only be successful when the governing instrument or combination of instruments induces the appropriate public and private behaviour. Perhaps we should be thankful that child health policy is on the agenda, and even without a strong, coordinated approach built on the evidence, one would agree that 'these developments in early childhood services demonstrate the translation of research evidence into policy and practice, even if the implementation may be flawed, belated or under-resourced' [[5] pg.15].
Competing interests
The authors declare that they have no competing interests.
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Aust New Zealand Health PolicyAustralia and New Zealand Health Policy1743-8462BioMed Central London 1743-8462-1-61567993510.1186/1743-8462-1-6ResearchAustralian health system restructuring – what problem is being solved? Dwyer Judith M [email protected] Health Policy and Management Department, La Trobe University, Australia2004 19 11 2004 1 6 6 17 8 2004 19 11 2004 Copyright © 2004 Dwyer; licensee BioMed Central Ltd.2004Dwyer; 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 recent years, Australian state and territory governments have reviewed and restructured the health systems they lead and regulate. This paper examines the outcomes of the most recent official published reviews of systems and structures; identifies the common themes; and addresses two questions: what problems are being addressed? And how would we know if the changes were successful?
Results
In all the broad, systemic reviews, the main health system problems identified were money, hospital utilisation and a weak primary health care system. The solutions are various, but there is a common trend towards centralisation of governance, often at state health authority level, and stronger accountability measures. Other common themes are hospital substitution (services to avoid the need for admission); calls for cooperation across the Commonwealth:state divide, or for its abolition; and the expected range of current efficiency and effectiveness measures (eg amalgamate pathology and support services) and ideas in good currency (eg call centres). The top-down nature of the public review process is noted, along with the political nature of the immediate catalysts for calling on a review.
Conclusion
The long-standing tension between the pull to centralisation of authority and the need for innovation in care models is heightened by recent changes, which may be counterproductive in an era dominated by the burden of chronic disease. I argue that the current reforms will not succeed in achieving the stated goals unless they make a difference for people with chronic illness. And if this is correct, the most useful focus for evaluation of the success of the reforms may be their impact on the system's ability to develop and deliver better models of care for this growing group of patients.
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Background
In recent years, there has been a rolling (and sometimes repetitive) tide of structural change in the way state and territory governments organise to lead and/or provide health care within their jurisdictions, with every state and territory of Australia involved at least once in the last 10 years.
This paper examines the outcomes of the most recent official published reviews of systems and structures; identifies the common themes; and addresses two questions: what problems are being addressed? And how would we know if the changes were successful?
This analysis focuses on those reviews which are 'systemic' in the sense that they examine broadly the structure and performance of a state/territory health system, and/or address governance of the system. The NSW restructure has been included, although it differs from the others in the absence of an independent review process and in the related lack of detailed documentation of the rationale for change.
Review of reviews
The most recent wave of systemic reviews in the Australian public health system saw New South Wales [1], South Australia [2], the Northern Territory [3], Western Australia [4] and the Australian Capital Territory [5] go in for restructuring. Victoria reviewed metropolitan health system governance [6], but pulled back from major structural change, having had a round of it in 2000 [7].
NSW has relinquished its status as an island of relative stability, which had been maintained since 1986 in spite of several reviews, penultimately by IPART [8]. In the aftermath of a scandal at MacArthur Health Service [9], the Minister announced the abolition of all Area Health Service boards, and is restructuring the health services into 8 'super-regions' with CEO's who report directly to the head of the Department [1]. Clinicians and the community will be represented on advisory structures, and a new agency will take over support functions.
Queensland stays with central control (virtually no boards of governance to dilute the Department's authority) while Tasmania is reviewing hospital services only [10], having restructured in 1991 (from 'atomised' to regionalised) and 1997 (from regionalised to centralised) [11].
Results
The pattern of systemic reviews over the last 10 years is summarised in Table 1. One notable trend is that the decision to review is often no longer presented publicly as a matter solely for the health minister. The premier or a financial/regulatory arm of government (mostly in concert with the health minister) commissioned the most recent reviews in Western Australia, South Australia, and the Northern Territory. The NSW restructure arises from a different process than the others undergoing structural change, with a brief booklet announcing and explaining the decision [1], rather than extended public review processes with opportunities for community and health service provider input.
Table 1 Review Dateline
Year States Year States
2004 WA, NSW, Tasmania (hospitals only) 2000 Victoria
2003 NSW, Victoria#, SA, NT 1996 Tasmania, Qland, ACT
2002 ACT, 1995 Victoria, SA
2001 WA
Notes: #No major structural changes recommended – focus on governance
The trend: centralisation of governance
In what has emerged as a strong centralising tendency, 6 of 8 jurisdictions have centralised governance authority for public sector health care agencies at the level of the state or territory health authority. Victoria and South Australia are mixed, with regionalised or 'networked' structures predominating in the capital city; and several different approaches to both regional and institutional governance elsewhere. As Somgen points out, Victoria and South Australia were the states most strongly influenced by the 1990's trend to privatisation, outsourcing and output-based funding [12], with less focus on structures and central planning.
Table 2 summarises the current arrangements by state, in order of population size, with the population shown in brackets in the left-hand column (M = million).
Table 2 Governance of public health care agencies in Australian states/territories
State Current Status Recent changes
NSW 6.64M Centralising by 1 January 2005; regionalised since 1986. Moving from 17 Area Health Services with separate governance authority to 8 Area Health Services within Departmental governance.
Victoria 4.87M Rurals partly regionalised for many years; Melbourne 'networked' since 1995. Melbourne networks restructured from 7 to 12 and names changed in 2000. Rural structures mix of regionalised and atomised.
Q'land 3.71M Centralised at state level since 1996 after 5 years of regionalisation. Long history of centralisation with advisory hospital boards; Regional Health Authorities 1991–1996.
WA 1.93M Centralised at state level in 2001/02. Moved from 'atomised' in Perth to one board in 1997, governance centralised in 2001; state now centralised.
SA 1.52M Regionalised in rural areas since 1995; Adelaide partly regionalising. Moved from atomised to regionalised, with 2 regional and 1 specialised health services in the capital as of July 2004.
Tasmania 0.47M Centralised at state level Moved from atomised to regionalised in 1991; centralised at state level in 1997.
ACT 0.32M Centralised (single city system) Single board for Canberra established in 1996; abolished in 2002.
NT 0.2M Centralised at territory Level Never devolved. Some autonomous Aboriginal Health Services.
The recent NSW decision means that there is now a strong predominance of governance at state health authority level, with two-thirds of the Australian population living in areas served by centralised health services.
The second notable trend is the virtual end of 'atomised' structures – stand-alone, single-service agencies (ie, hospitals, community health, or mental health services) in the public sector. There are of course exceptions (women's and children's hospitals may be the last ones standing in a few years), and the picture is different for non-government organisations (like district nursing) which are less amenable to restructuring.
Common Themes
The most recent reviews in WA and SA are characterised by claims to radical change, based on both financial and health goals:
'...incremental reform is no longer the pathway to a financially sustainable vision for WA. A fundamental re-prioritisation of the public health system is needed, and should be carried out over the next decade in a systematic and integrated way' [4], p v).
'The people of South Australia have a decision to make on what type of health system they need now and for the future generation...there needs to be a significant shift from a system focused on illness to a health system reoriented towards health promotion, illness prevention and early intervention' [2]p xiii.
Western Australia is taking on the tertiary hospitals, and reducing the number of tertiary sites from 5 to 2 (with the women's and children's hospital group to be collocated but organisationally separate). All state-run health services are to report through three metropolitan regions (north, south and Women's and Children's) and one rural region, with the CEO's reporting directly to the Department – there are to be no boards of governance. South Australia has succeeded in amalgamating most of Adelaide's hospital and community health boards to form 2 regional health services and 1 child, youth and women's health service (incorporating the women's and children's hospital). This is a notable achievement for a minority government, after at least five separate attempts in the last 20 years to rebalance power and responsibility had largely failed [13-17].
Not all of the systemic reviews claim to set a bold new vision, but there are strong common themes. The reports tell a familiar story of the need to bring increases in state health spending to sustainable levels, set against the trend of increasing costs due to increasing incidence of chronic disease, and more technologies for intervention, in an ageing population. They all focus on the need to improve quality and safety for patients.
The reviews also find that the health system is too fragmented to meet the needs of patients with long-term complex conditions well. This is seen to be partly because the system was designed for acute illness, with the current funding mechanisms also designed primarily on the pattern of acute interventions. The reviews call for better integration of services, so that navigating the system is easier for patients, their carers and care providers.
The reviews consistently argue that in order to achieve this, the primary care system needs to be more effective in managing or coordinating patients' needs for several different kinds of services when and where they are needed. The inevitable corollary is that inpatient care and hospitals have to become less central in the organisation and funding of the system. What can be done elsewhere should be; and the primary care level must have more of the action and more of the pulling power.
In turn, this will require different facilities for different modes of service delivery; different funding allocations and methods of allocation; and a solution to the atomisation of primary care caused by the Commonwealth/state split and the current model of fee-for-service medicine. The need for changes in the private sector is noted in the reviews, but proposals are not developed, because the states have such a limited role here.
Much is also made of the need for providers to be more accountable to government and the community, and/or better governed and managed. While the reports mostly call for less micro-managing from the head offices of health authorities, and a better separation between the roles of central policy-makers and peripheral service providers (or regional CEO's), there is also a countervailing tendency to recommend tighter engagement and control. For example, the Kibble review of governance in Victoria (2003) notes confusion about relative roles and responsibilities and calls for the Department of Human Services to reduce 'attention to the day-to-day operations of Health Services and monitoring of detailed activities' (p 27) but later recommends 'a standardised reporting template' for internal reports to boards across the system (p 35), along with stronger accountability for the CEOs to the Secretary of the Department. More public reporting of service outcomes and activity levels is a related common theme, intended to inform the public and to underpin attention to safety and quality.
The final major common theme in the reviews is the inclusion of an opportunistic range of technical efficiency and effectiveness measures, picking up ideas in good currency or known productivity opportunities. For example, almost everyone recommends a call centre; a web-based method of sharing innovations; amalgamation of support services where relevant; and improvements in the effectiveness of information systems and the use of information.
There are two other commonalities worth noting. Firstly, it is a fact of organisational and political life that official reviews are a top-down affair, commissioned by one level of the system to examine a lower level. Thus it is not surprising that there are no published official reviews of the roles and responsibilities of the Commonwealth health authority in the last twenty years. When the published reviews do address the roles and responsibilities of state health authorities, it is either because they are the providers (NT, WA, ACT) or because intended changes to the service provider level of the system require changes to the roles of central health authorities.
This is an important limitation in the current environment, when some of the key barriers to improving the effectiveness of health care delivery lie in the system's seldom seen upper reaches. As the reviews note, the way that the Commonwealth/state split of responsibility for health is enacted and managed is probably the single most significant problem in health system design. But it is not the only one. I refer not so much to important policy settings (like funding allocation models and public health priorities) which are studied and articulated in publicly-available documents, but rather to the influence of administrative decisions (like who gets special grants and who doesn't) and the effectiveness of relationships with health care provider organisations (as judged from the bottom up as well as the top down). The administrative actions of health authorities seem to go largely unexamined.
Secondly, while the underlying problems the reviews set out to address are all about money, hospital utilisation and a weak primary health care system, the immediate context is often the election of a new government (Victoria, SA, NT), the appointment of a new minister or health authority CEO (WA), media unrest about health in a state with a looming election (ACT) or scandal (NSW). This observation may simply be another way of saying that the health portfolio is highly politically sensitive as well as complex, and so risky that reference to independent expertise is seen as essential.
Discussion
The main line of logic running through the recent reviews seems sound. The primary care system needs strengthening; what can be done outside hospitals should be; and a continuing focus on safety, performance and accountability is necessary.
The reports also make it clear that this is all about responding to the major challenge for the system: to improve its capacity to prevent, intervene early in, and manage chronic disease, the main driver of increased demand. Such a focus is clearly justified. Chronic disease is responsible for approximately 80% of the total burden of disease, with an estimated three million Australians suffering from one or more chronic illnesses [18]. About 40% of total health expenditure, or $12.6 billion, was spent on chronic illness in 1993/94, just less than half of it in hospitals [19]. The system must be able to deliver the kind of care needed by people with (or at risk of) chronic disease, including older people and Indigenous people, and thereby enhance the system's effectiveness and perhaps even reduce the slope of the increasing cost curve.
If this is the imperative, the trend away from atomised governance structures, and towards bringing multiple agencies which serve (at least some) common patients together, seems like the right direction. But there is another important requirement which may not be served by these moves – that is, a stronger focus on innovation in care models. While recommendations abound, we don't yet really know what will work best for the new pattern of illness – how do you best coordinate care around the needs of the chronicly ill?
Uncertainty about care models, and the institutional and policy arrangements needed to support them, can only be resolved through the continued development and testing of innovative approaches, on the ground in health care delivery, as happened most notably with the Coordinated Care Trials [20]. As many of the reviews argue, the engagement of clinicians is critical to this endeavour.
This reality implies that there is a secondary criterion by which the effectiveness of health system structural changes might be judged: do the changes enhance or inhibit the system's ability to innovate? The requirement for innovation and experimentation may not sit comfortably with government requirements for standardisation of known good practice. However in an area where best practice is not known, innovation is critical, and must be supported.
Unfortunately, the Commonwealth:state responsibility split, the one structural barrier most central to the systemic weakness of Australian primary care (and therefore most important for the capacity to develop and support new models of care for chronic diseases), is one that a state can't address, at least not alone. The Productivity Commission's recent call for an independent public review of the whole health system [21], focused on overlapping roles and responsibilities for funding, offers grounds to hope for movement in this otherwise intractable problem.
The other pessimistic sign is the trend to more direct control of health care provision by state governments, related no doubt to the twin problems of increasing demand (and therefore cost) and increasing disclosure of safety and quality problems, both of which can only politicise the system more. Research on innovation, in relation to quality and safety as well as other performance measures, indicates that micro-management from above is not helpful [22,23].
Local evidence to support this view is scant. While the effectiveness of the new arrangements during Queensland's brief period of devolved governance was judged harshly when it came time to re-centralise, some commentators suggest that this period also allowed Queensland to catch up to other states in areas like accreditation, casemix, IT and 'attention to performance management and outcomes' [24].
This may be a critical problem. While recognising that perspectives on this question are highly related to one's place in the structure, I would suggest that real innovation in public hospitals and health services is less likely to be driven by clinicians who are more tightly controlled, staff who have learnt to be risk-averse, or managers who are increasingly frightened of tomorrow's headlines, and whose planning horizon extends to next month's financial and activity data. This problem is only compounded while hospitals and community health services on the one hand, and GPs on the other, continue to work with so little in the way of common incentives.
Conclusion
The recent reviews were established largely to address financial imperatives in an environment of upward pressure on demand for services, and accountability concerns (in relation to quality and safety, and general good governance), mostly in a highly political context. The reviewers rightly sought to take a longer-term strategic perspective. They attempted (with varying degrees of success) to focus on good system design and capacity to meet the broad and complex purposes of public health systems, recognizing the growing challenges the systems face.
Structural reform is hardly ever evaluated, other than when its weaknesses are articulated by those proposing the next round of changes, as part of the rationale for their efforts. There are many reasons for this failure, some of them political. One pertinent reason is that outcomes like containing the pressure of future growth in demand, or improving health outcomes for the population, cannot be judged within a realistic time frame.
However, in the current environment, with strong convergence in the themes addressed by a fairly comprehensive round of reviews of Australian health systems, an argument can be made for evaluation 'at the pointy end' of the changes. Given the challenges the reviews were intended to address, there are grounds to suggest that the current reforms will not succeed in achieving the stated (shorter- and longer-term) goals unless they make a difference for people with chronic illness. And if this is correct, the most useful immediate focus for evaluation of the success of the reforms may be their impact on the system's ability to develop and deliver better models of care for this growing group of patients.
Methods
The 'data' for this project were the published reports of systemic reviews of the health systems, and related material published on departmental websites and in the professional and academic literature.
These sources were analysed to generate an understanding of the recommended governance authority structures; and the common themes emerging from the reasoning on which the recommendations were based. The themes underpinning the recommendations were then assessed in the light of the overarching goals of reform.
Competing Interests
I was a member of the South Australian 'Generational Health Review' Steering Committee, chaired its Governance and Funding Task Force and continue to consult to the SA Department of Human Services. I worked for twenty years in health care agencies, and only one in a central health authority.
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Aust New Zealand Health PolicyAustralia and New Zealand Health Policy1743-8462BioMed Central London 1743-8462-1-71567992810.1186/1743-8462-1-7CommentaryManaging emerging infectious diseases: Is a federal system an impediment to effective laws? Howse Genevieve [email protected] Centre for Public Health Law, School of Public Health, La Trobe University, Vic, Australia2004 19 11 2004 1 7 7 23 8 2004 19 11 2004 Copyright © 2004 Howse; licensee BioMed Central Ltd.2004Howse; 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 the 1980's and 1990's HIV/AIDS was the emerging infectious disease. In 2003–2004 we saw the emergence of SARS, Avian influenza and Anthrax in a man made form used for bioterrorism. Emergency powers legislation in Australia is a patchwork of Commonwealth quarantine laws and State and Territory based emergency powers in public health legislation. It is time for a review of such legislation and time for consideration of the efficacy of such legislation from a country wide perspective in an age when we have to consider the possibility of mass outbreaks of communicable diseases which ignore jurisdictional boundaries.
==== Body
The management of infectious diseases in an increasingly complex world of mass international travel, globalization and terrorism heightens challenges for Federal, State and Territory Governments in ensuring that Australia's laws are sufficiently flexible to address the types of problems that may emerge.
In the 1980's and 1990's HIV/AIDS was the latest "emerging infectious disease". Considerable thought was put into the legislative response by a number of Australian jurisdictions. Particular attention had to be given to the unique features of the disease such as the method of transmission, the kinds of people who were at risk, and the protections needed by the community and the infected population to best manage the care of those infected and to minimize new infections. Health workers and researchers began to find that "the most effective strategies that we have so far found to help promote reduction of the spread of HIV involve the adoption of laws and policies which protect the rights of people most at risk of infection" [1]. A good example of a legislative response which adopts this approach is found in section 119 and 120 of the Victorian Health Act 1958. These sections emphasize the need to protect the privacy of the infected individual and to undertake a staged response which is proportional to the risk presented by the infected individual. The legislation has been very effective with HIV and has been praised for its progressive approach [2].
In 2003 the community has been faced with the emergence of two new infectious diseases, SARS and Anthrax. Whilst there were no cases of either disease in Australia, the threat of a possible outbreak had to be acknowledged and a response planned. Anthrax is not a new infectious disease. Humans can become infected with anthrax by handling products from infected animals or by breathing in anthrax spores from infected animal products (like wool, for example). People also can become infected with gastrointestinal anthrax by eating undercooked meat from infected animals. However, its manufacture and use as a weapon for bioterrorism forces us to rethink its management in a new context.
These two infectious diseases have very different features from HIV which spreads only via transmission of infected bodily fluids such as blood or semen. SARS, by contrast is transmitted via droplets from infected cases which, as a result of coughing, carry the virus to close contacts [3] Thus, the infection profile of SARS requires planning for the possible overrun of Intensive Care Units and the likely infection of a number of ICU staff affecting both morale and capacity to cope. Anthrax raised different problems. These include the possible investigation of terrorist suspects alongside investigation of the outbreak of the infectious disease. Difficulties are also raised by likelihood of public panic, and the flooding of public health officials with reports of suspicious white powder.
In early 2004 the media reported the spread of avian influenza across South East Asia. This disease has different features from HIV/AIDS and SARS and an approach to an Australian outbreak would also be different. The main difference is in the source of transmission of the virus, that is, from infected birds to humans. There is very little difference [from ordinary influenza] in the symptoms (though these may vary in severity) or treatment of the virus [4] It is too early to predict whether this may be the next "emerging infectious disease", but its current spread has given rise to concern about such a possibility [5]
Australia is a federal system. There are two parallel sets of laws in operation. The Commonwealth Constitution sets out the legislative powers of the Commonwealth. Specific powers are listed in the Commonwealth constitution but State constitutions have broad powers covering matters such as peace, order and good governance. As the Commonwealth has no specific power to legislate with respect to health, other than the quarantine power, national legislative schemes in public health which rely upon a cooperative approach from all States and Territories are cumbersome and difficult.
Without a specific head of power, the Commonwealth has limited ability to legislate with respect to health. "That is, the legislative powers of the Commonwealth are specified in the Constitution and do not include expressly most of the activities that together comprise the field of public health"[6] For this reason, there are no Commonwealth emergency health powers except quarantine powers. Quarantine powers are currently restricted to isolation at the border of the country of people, plants, and animals to prevent the spread of disease. There is a real possibility that quarantine laws could have a broader scope. It depends on how widely the High Court would interpret section 51(x) of the Commonwealth Constitution. A quarantine law could override state laws as long as it remained a law "with respect to quarantine". However, "the power is potentially a colossus so far as the expansion of legislative authority in the fields of public health is concerned". [6]
The quarantine power would be the most likely candidate for a head of power on which to base development of commonwealth laws for the management of public health emergencies. Another possibility may be the external affairs power, if there was a relevant treaty or international agreement which could be given effect to in domestic law. However the legislation would have to be limited to laws giving effect to the treaty.
States and territories have a range of emergency powers available to them in their existing public health legislation. Some are relatively old. For example, the Health Act 1911 (WA), Public Health Act 1952 (NT) based on an 1898 Ordinance (Both these Acts are currently under review). Health emergency powers vary from one jurisdiction to another, but include powers to support disease surveillance, contact tracing and orders to restrict behavior or movement of individuals with an infectious disease in certain circumstances. There are also powers to recall food, search premises and seize property, close buildings and a range of other substantial and intrusive powers.
It is suggested that it is time to consider whether state and territory public health legislation contains sufficient measures to manage the outbreak of an infectious disease in a modern environment which includes mass travel, swift spread of infection and additional complexity raised by fears of bioterrorism.
Currently, in a public health emergency caused by the spread of an emerging infectious disease, Australia could need to rely on a patchwork of legislative measures to assist it to cope. Commonwealth quarantine laws and State and Territory powers in public health legislation may all be needed to address the problem. If an outbreak occurred on a border, or in some area where jurisdiction may be in doubt such as airspace or offshore and a state or territory response was required in addition to any quarantine measures, there could be confusion over jurisdiction for the application of State and Territory powers. State and Territory public health acts do not adequately provide for interjurisdictional communication and cooperation. There could also be difficulties if an infectious disease caused overseas deaths of people from more than one State or Territory in circumstances where an Australian coronial investigation was considered desirable. In such a situation, the jurisdiction of more than one Australian coroner would be triggered. Several State and Territory coronial laws could apply and there could be different inquests under different laws undertaken by different coroners into the same incident.
It is suggested that it is time to look at the efficiency of the emergency powers laws of Australia as a whole: to map the laws in each jurisdiction and the Commonwealth quarantine laws and to consider their effectiveness in the face of the outbreak of a fast moving, easily spread infectious disease. The efficacy of Australia's laws should also be considered in relation to bioterrorism. While there were no infections from anthrax in 2003 despite a great deal of media coverage and infections and deaths in the US, a responsible legislature ought to acknowledge the possibility and ensure that the law is ready to support a swift and effective response.
It is not enough to consider whether the individual pieces of legislation are up to the task of managing outbreaks of newly emerging infectious diseases. Indeed many of the jurisdictions are currently reviewing their public health legislation and will no doubt give proper consideration to this issue as part of the review. But who is thinking about how the legislation of all jurisdictions and the Commonwealth quarantine fits together? What powers enable communication and cooperation between jurisdictions about the outbreak of infectious disease? What kind of opportunity is there for a coordinated response? Can public health orders made in one jurisdiction travel to another jurisdiction when the infected individual travels? What arrangements can be made if an outbreak occurs on or close to a interstate border? What if there is an outbreak on a bus carrying passengers from Victoria, through South Australia to the Northern Territory?
It is encouraging to note that, even without specific legislation, there has been a mechanism to achieve communication and cooperation between jurisdictions through the Communicable Disease Network of Australia (CDNA). This Network has in fact been quite successful in fostering regular communication between the Communicable Disease Units across the country and has been involved in coordinated actions during a number of multistate outbreaks.
Despite the existence of this network and other good working relationships between government officials and various agencies in different jurisdictions, a serious outbreak of communicable disease would require the existence of legislative powers. Public health emergencies generate confusion, even panic. Clarity of powers and the way those powers interact with each other would be crucial in an emergency. It became apparent after the Bali tragedy in 2002 that coroner's jurisdiction was triggered differently in different jurisdictions and some acts did not support communication and cooperation when inquests might be needed for deaths of people ordinarily resident in several jurisdictions. The time to find the shortcomings in the legislation is well before the crisis.
A review of the efficacy of how these laws work together to protect the public health of all Australians should be undertaken. It has been possible to overcome the hangovers of federation for the betterment of all Australians in relation to corporations law. When doubts were recently raised about the constitutional basis of the corporations law scheme, the States and Territories were able to cooperate and refer the necessary powers to the Commonwealth to provide certainty about the laws which govern our corporations. Is our public health any less important than governance of our corporations? Could we cooperate to give ourselves certainty, flexibility and a consistent approach which protects the rights of those subject to some very broad powers?
The States and Territories are generally reluctant to refer powers to the Commonwealth. It may be time to seriously discuss referral of powers in the context of health emergency powers. At the very least, it is time that the Commonwealth, States and Territories recognised the need for the laws to work as a set of laws to protect the whole country, not simply individual laws to protect individual jurisdictions.
There has been work done internationally in this area. A model State Emergency Health Powers Act has been developed in the US in 2001 [7] In the preamble to this Act a rationale for its development is set out: "In the wake of the tragic events of September 11, 2001, our nation realizes that the Government's foremost responsibility is to protect the health, safety and wellbeing of its citizens. New and emerging dangers including emergent and resurgent infectious diseases and incidents of civilian mass casualties – pose serious and immediate threats to the population. A renewed focus on the prevention, detection, management and containment of public health emergencies is thus called for." The US, like Australia, is a Federal system. The model was intended to be taken up by those US states which wished to do so. To date, it has been passed in over half the US states. This bill would be an excellent starting point for development of an Australian model. There are a number of legislative mechanisms which could be used to support a nationally uniform approach to health emergency powers legislation in Australia.
The development and adoption of the model food legislation provides a useful model of a cooperative uniform approach. A model act was developed in consultation with all jurisdictions. It covered areas agreed to be core areas of the Act which ought to be the subject of a national approach and other provisions which were considered to be administrative and were to be adopted at the discretion of each jurisdiction. An intergovernmental agreement was signed as a mechanism to protect the uniformity of the legislation. The agreement sets up a Ministerial Council, supported by a Food Regulation Standing Committee. The Council has responsibility for deciding on proposals to amend the model [8] If a decision is made in favor of amendment, States and Territories will use their best endeavors to submit to their respective Parliaments, legislation which gives effect to the amendment.
The law is an important tool in supporting the management of the outbreak of infectious diseases. The existence of our Federal system has meant that we have a different approach in each State and Territory together with Commonwealth control of quarantine. Newly emerging infectious diseases creating real threats to public health in an era of easy mass travel, and the present threat of bioterrorism mean that it is time Australia examined all laws to contain and manage infectious disease outbreak. The laws should be examined both for their effectiveness in the areas they cover, and as part of a whole which ought enable a response which protects the health of all Australians, and crosses borders as easily as SARS or avian influenza.
==== Refs
Kirby Justice Michael Human Rights and the HIV Paradox Lancet 1996 348 1217 8 8898041 10.1016/S0140-6736(96)05468-2
Reynolds Chris 'Forms of Public Health Law – A General Outline' in The Australian Institute of Health Law and Ethics (ed) Public Health Law in Australia – New Perspectives 1998
Australian Government Department of Health and Ageing Guidelines on SARS
Australian Government Department of Health and Ageing Guidelines to Avian Influenza
A number of websites provide information updates on the spread of the disease In particular see Commonwealth Department of Health and Ageing , World Health Organization , Centres for Disease Control , Department of Foreign Affairs and Trade
McMillan John 'The Constitutional Power of the Commonwealth in Public Health' in The Australian Institute of Health Law and Ethics (ed): Public Health Law in Australia – New Perspectives 1998
Model State Emergency Health Powers Act (USA)
Council of Australian Governments (COAG) Food Regulation Agreement
The Centre for Comparative Constitutional Studies Implementation Options for National Legislative Schemes 1999
| 15679928 | PMC544965 | CC BY | 2021-01-04 16:38:25 | no | Aust New Zealand Health Policy. 2004 Nov 19; 1:7 | utf-8 | Aust New Zealand Health Policy | 2,004 | 10.1186/1743-8462-1-7 | oa_comm |
==== Front
Aust New Zealand Health PolicyAustralia and New Zealand Health Policy1743-8462BioMed Central London 1743-8462-1-71567992810.1186/1743-8462-1-7CommentaryManaging emerging infectious diseases: Is a federal system an impediment to effective laws? Howse Genevieve [email protected] Centre for Public Health Law, School of Public Health, La Trobe University, Vic, Australia2004 19 11 2004 1 7 7 23 8 2004 19 11 2004 Copyright © 2004 Howse; licensee BioMed Central Ltd.2004Howse; 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 the 1980's and 1990's HIV/AIDS was the emerging infectious disease. In 2003–2004 we saw the emergence of SARS, Avian influenza and Anthrax in a man made form used for bioterrorism. Emergency powers legislation in Australia is a patchwork of Commonwealth quarantine laws and State and Territory based emergency powers in public health legislation. It is time for a review of such legislation and time for consideration of the efficacy of such legislation from a country wide perspective in an age when we have to consider the possibility of mass outbreaks of communicable diseases which ignore jurisdictional boundaries.
==== Body
The management of infectious diseases in an increasingly complex world of mass international travel, globalization and terrorism heightens challenges for Federal, State and Territory Governments in ensuring that Australia's laws are sufficiently flexible to address the types of problems that may emerge.
In the 1980's and 1990's HIV/AIDS was the latest "emerging infectious disease". Considerable thought was put into the legislative response by a number of Australian jurisdictions. Particular attention had to be given to the unique features of the disease such as the method of transmission, the kinds of people who were at risk, and the protections needed by the community and the infected population to best manage the care of those infected and to minimize new infections. Health workers and researchers began to find that "the most effective strategies that we have so far found to help promote reduction of the spread of HIV involve the adoption of laws and policies which protect the rights of people most at risk of infection" [1]. A good example of a legislative response which adopts this approach is found in section 119 and 120 of the Victorian Health Act 1958. These sections emphasize the need to protect the privacy of the infected individual and to undertake a staged response which is proportional to the risk presented by the infected individual. The legislation has been very effective with HIV and has been praised for its progressive approach [2].
In 2003 the community has been faced with the emergence of two new infectious diseases, SARS and Anthrax. Whilst there were no cases of either disease in Australia, the threat of a possible outbreak had to be acknowledged and a response planned. Anthrax is not a new infectious disease. Humans can become infected with anthrax by handling products from infected animals or by breathing in anthrax spores from infected animal products (like wool, for example). People also can become infected with gastrointestinal anthrax by eating undercooked meat from infected animals. However, its manufacture and use as a weapon for bioterrorism forces us to rethink its management in a new context.
These two infectious diseases have very different features from HIV which spreads only via transmission of infected bodily fluids such as blood or semen. SARS, by contrast is transmitted via droplets from infected cases which, as a result of coughing, carry the virus to close contacts [3] Thus, the infection profile of SARS requires planning for the possible overrun of Intensive Care Units and the likely infection of a number of ICU staff affecting both morale and capacity to cope. Anthrax raised different problems. These include the possible investigation of terrorist suspects alongside investigation of the outbreak of the infectious disease. Difficulties are also raised by likelihood of public panic, and the flooding of public health officials with reports of suspicious white powder.
In early 2004 the media reported the spread of avian influenza across South East Asia. This disease has different features from HIV/AIDS and SARS and an approach to an Australian outbreak would also be different. The main difference is in the source of transmission of the virus, that is, from infected birds to humans. There is very little difference [from ordinary influenza] in the symptoms (though these may vary in severity) or treatment of the virus [4] It is too early to predict whether this may be the next "emerging infectious disease", but its current spread has given rise to concern about such a possibility [5]
Australia is a federal system. There are two parallel sets of laws in operation. The Commonwealth Constitution sets out the legislative powers of the Commonwealth. Specific powers are listed in the Commonwealth constitution but State constitutions have broad powers covering matters such as peace, order and good governance. As the Commonwealth has no specific power to legislate with respect to health, other than the quarantine power, national legislative schemes in public health which rely upon a cooperative approach from all States and Territories are cumbersome and difficult.
Without a specific head of power, the Commonwealth has limited ability to legislate with respect to health. "That is, the legislative powers of the Commonwealth are specified in the Constitution and do not include expressly most of the activities that together comprise the field of public health"[6] For this reason, there are no Commonwealth emergency health powers except quarantine powers. Quarantine powers are currently restricted to isolation at the border of the country of people, plants, and animals to prevent the spread of disease. There is a real possibility that quarantine laws could have a broader scope. It depends on how widely the High Court would interpret section 51(x) of the Commonwealth Constitution. A quarantine law could override state laws as long as it remained a law "with respect to quarantine". However, "the power is potentially a colossus so far as the expansion of legislative authority in the fields of public health is concerned". [6]
The quarantine power would be the most likely candidate for a head of power on which to base development of commonwealth laws for the management of public health emergencies. Another possibility may be the external affairs power, if there was a relevant treaty or international agreement which could be given effect to in domestic law. However the legislation would have to be limited to laws giving effect to the treaty.
States and territories have a range of emergency powers available to them in their existing public health legislation. Some are relatively old. For example, the Health Act 1911 (WA), Public Health Act 1952 (NT) based on an 1898 Ordinance (Both these Acts are currently under review). Health emergency powers vary from one jurisdiction to another, but include powers to support disease surveillance, contact tracing and orders to restrict behavior or movement of individuals with an infectious disease in certain circumstances. There are also powers to recall food, search premises and seize property, close buildings and a range of other substantial and intrusive powers.
It is suggested that it is time to consider whether state and territory public health legislation contains sufficient measures to manage the outbreak of an infectious disease in a modern environment which includes mass travel, swift spread of infection and additional complexity raised by fears of bioterrorism.
Currently, in a public health emergency caused by the spread of an emerging infectious disease, Australia could need to rely on a patchwork of legislative measures to assist it to cope. Commonwealth quarantine laws and State and Territory powers in public health legislation may all be needed to address the problem. If an outbreak occurred on a border, or in some area where jurisdiction may be in doubt such as airspace or offshore and a state or territory response was required in addition to any quarantine measures, there could be confusion over jurisdiction for the application of State and Territory powers. State and Territory public health acts do not adequately provide for interjurisdictional communication and cooperation. There could also be difficulties if an infectious disease caused overseas deaths of people from more than one State or Territory in circumstances where an Australian coronial investigation was considered desirable. In such a situation, the jurisdiction of more than one Australian coroner would be triggered. Several State and Territory coronial laws could apply and there could be different inquests under different laws undertaken by different coroners into the same incident.
It is suggested that it is time to look at the efficiency of the emergency powers laws of Australia as a whole: to map the laws in each jurisdiction and the Commonwealth quarantine laws and to consider their effectiveness in the face of the outbreak of a fast moving, easily spread infectious disease. The efficacy of Australia's laws should also be considered in relation to bioterrorism. While there were no infections from anthrax in 2003 despite a great deal of media coverage and infections and deaths in the US, a responsible legislature ought to acknowledge the possibility and ensure that the law is ready to support a swift and effective response.
It is not enough to consider whether the individual pieces of legislation are up to the task of managing outbreaks of newly emerging infectious diseases. Indeed many of the jurisdictions are currently reviewing their public health legislation and will no doubt give proper consideration to this issue as part of the review. But who is thinking about how the legislation of all jurisdictions and the Commonwealth quarantine fits together? What powers enable communication and cooperation between jurisdictions about the outbreak of infectious disease? What kind of opportunity is there for a coordinated response? Can public health orders made in one jurisdiction travel to another jurisdiction when the infected individual travels? What arrangements can be made if an outbreak occurs on or close to a interstate border? What if there is an outbreak on a bus carrying passengers from Victoria, through South Australia to the Northern Territory?
It is encouraging to note that, even without specific legislation, there has been a mechanism to achieve communication and cooperation between jurisdictions through the Communicable Disease Network of Australia (CDNA). This Network has in fact been quite successful in fostering regular communication between the Communicable Disease Units across the country and has been involved in coordinated actions during a number of multistate outbreaks.
Despite the existence of this network and other good working relationships between government officials and various agencies in different jurisdictions, a serious outbreak of communicable disease would require the existence of legislative powers. Public health emergencies generate confusion, even panic. Clarity of powers and the way those powers interact with each other would be crucial in an emergency. It became apparent after the Bali tragedy in 2002 that coroner's jurisdiction was triggered differently in different jurisdictions and some acts did not support communication and cooperation when inquests might be needed for deaths of people ordinarily resident in several jurisdictions. The time to find the shortcomings in the legislation is well before the crisis.
A review of the efficacy of how these laws work together to protect the public health of all Australians should be undertaken. It has been possible to overcome the hangovers of federation for the betterment of all Australians in relation to corporations law. When doubts were recently raised about the constitutional basis of the corporations law scheme, the States and Territories were able to cooperate and refer the necessary powers to the Commonwealth to provide certainty about the laws which govern our corporations. Is our public health any less important than governance of our corporations? Could we cooperate to give ourselves certainty, flexibility and a consistent approach which protects the rights of those subject to some very broad powers?
The States and Territories are generally reluctant to refer powers to the Commonwealth. It may be time to seriously discuss referral of powers in the context of health emergency powers. At the very least, it is time that the Commonwealth, States and Territories recognised the need for the laws to work as a set of laws to protect the whole country, not simply individual laws to protect individual jurisdictions.
There has been work done internationally in this area. A model State Emergency Health Powers Act has been developed in the US in 2001 [7] In the preamble to this Act a rationale for its development is set out: "In the wake of the tragic events of September 11, 2001, our nation realizes that the Government's foremost responsibility is to protect the health, safety and wellbeing of its citizens. New and emerging dangers including emergent and resurgent infectious diseases and incidents of civilian mass casualties – pose serious and immediate threats to the population. A renewed focus on the prevention, detection, management and containment of public health emergencies is thus called for." The US, like Australia, is a Federal system. The model was intended to be taken up by those US states which wished to do so. To date, it has been passed in over half the US states. This bill would be an excellent starting point for development of an Australian model. There are a number of legislative mechanisms which could be used to support a nationally uniform approach to health emergency powers legislation in Australia.
The development and adoption of the model food legislation provides a useful model of a cooperative uniform approach. A model act was developed in consultation with all jurisdictions. It covered areas agreed to be core areas of the Act which ought to be the subject of a national approach and other provisions which were considered to be administrative and were to be adopted at the discretion of each jurisdiction. An intergovernmental agreement was signed as a mechanism to protect the uniformity of the legislation. The agreement sets up a Ministerial Council, supported by a Food Regulation Standing Committee. The Council has responsibility for deciding on proposals to amend the model [8] If a decision is made in favor of amendment, States and Territories will use their best endeavors to submit to their respective Parliaments, legislation which gives effect to the amendment.
The law is an important tool in supporting the management of the outbreak of infectious diseases. The existence of our Federal system has meant that we have a different approach in each State and Territory together with Commonwealth control of quarantine. Newly emerging infectious diseases creating real threats to public health in an era of easy mass travel, and the present threat of bioterrorism mean that it is time Australia examined all laws to contain and manage infectious disease outbreak. The laws should be examined both for their effectiveness in the areas they cover, and as part of a whole which ought enable a response which protects the health of all Australians, and crosses borders as easily as SARS or avian influenza.
==== Refs
Kirby Justice Michael Human Rights and the HIV Paradox Lancet 1996 348 1217 8 8898041 10.1016/S0140-6736(96)05468-2
Reynolds Chris 'Forms of Public Health Law – A General Outline' in The Australian Institute of Health Law and Ethics (ed) Public Health Law in Australia – New Perspectives 1998
Australian Government Department of Health and Ageing Guidelines on SARS
Australian Government Department of Health and Ageing Guidelines to Avian Influenza
A number of websites provide information updates on the spread of the disease In particular see Commonwealth Department of Health and Ageing , World Health Organization , Centres for Disease Control , Department of Foreign Affairs and Trade
McMillan John 'The Constitutional Power of the Commonwealth in Public Health' in The Australian Institute of Health Law and Ethics (ed): Public Health Law in Australia – New Perspectives 1998
Model State Emergency Health Powers Act (USA)
Council of Australian Governments (COAG) Food Regulation Agreement
The Centre for Comparative Constitutional Studies Implementation Options for National Legislative Schemes 1999
| 15679934 | PMC544966 | CC BY | 2021-01-04 16:37:38 | no | Cerebrospinal Fluid Res. 2004 Dec 10; 1:1 | latin-1 | Cerebrospinal Fluid Res | 2,004 | 10.1186/1743-8454-1-1 | oa_comm |
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Respir ResRespiratory Research1465-99211465-993XBioMed Central 1465-9921-5-291561324310.1186/1465-9921-5-29ResearchEffects of PM10 in human peripheral blood monocytes and J774 macrophages Brown DM [email protected] K [email protected] V [email protected] School of Life Sciences, Napier University, Edinburgh, UK2 ELEGI Laboratory, Wilkie Building, University of Edinburgh, UK2004 21 12 2004 5 1 29 29 29 9 2004 21 12 2004 Copyright © 2004 Brown et al; licensee BioMed Central Ltd.2004Brown 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 effects of PM10, one of the components of particulate air pollution, was investigated using human monocytes and a mouse macrophage cell line (J774). The study aimed to investigate the role of these nanoparticles on the release of the pro-inflammatory cytokine TNF-α and IL-1α gene expression. We also investigated the role of intracellular calcium signalling events and oxidative stress in control of these cytokines and the effect of the particles on the functioning of the cell cytoskeleton. We showed that there was an increase in intracellular calcium concentration in J774 cells on treatment with PM10 particles which could be significantly reduced with concomitant treatment with the calcium antagonists verapamil, the intracellular calcium chelator BAPTA-AM but not with the antioxidant nacystelyn or the calmodulin inhibitor W-7. In human monocytes, PM10 stimulated an increase in intracellular calcium which was reduced by verapamil, BAPTA-AM and nacystelyn. TNF-α release was increased with particle treatment in human monocytes and reduced by only verapamil and BAPTA-AM. IL-1α gene expression was increased with particle treatment and reduced by all of the inhibitors. There was increased F-actin staining in J774 cells after treatment with PM10 particles, which was significantly reduced to control levels with all the antagonists tested. The present study has shown that PM10 particles may exert their pro-inflammatory effects by modulating intracellular calcium signalling in macrophages leading to expression of pro-inflammatory cytokines. Impaired motility and phagocytic ability as shown by changes in the F-actin cytoskeleton is likely to play a key role in particle clearance from the lung.
MacrophageNanoparticleCytokineCytoskeletonPM10
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Introduction
Increased exposure to PM10 particles is associated with adverse health effects [1,2]. Much of the mass of PM10 is low in toxicity and it has been suggested that, combustion-derived nanoparticles (ultrafine particles) [3-5] are a key component that drives these effects, especially inflammation. In individuals with pre-existing lung disease, inhalation of nanoparticles may induce inflammation and exacerbate respiratory and cardiovascular effects through the induction of oxidative stress and inflammation [4,6,7]. Rat inhalation studies using nanoparticles of various types, at high exposure, have demonstrated pulmonary fibrosis, lung tumours, epithelial cell hyperplasia, inflammation and increased cytokine expression [8-11].
The alveolar macrophage plays an important role in particle-mediated inflammation by phagocytosing particles and release of pro-inflammatory mediators such as the cytokine tumor necrosis factor-alpha (TNF-α) [12]. The signalling mechanisms for transcription of the TNFα gene includes calcium-related pathways in diseases such as sepsis [13-15]. Calcium is released from the endoplasmic reticulum stores on stimulation of the cell, leading to a calcium influx across the plasma membrane via calcium channels [16]. Various pathogenic particles have been shown to produce such changes in calcium flux within the cell [17,18] and a large number of pathological responses could be stimulated via such calcium signalling.
In order for macrophages to migrate and phagocytose foreign material, an intact functional cytoskeleton is necessary. The cytoskeleton is sensitive to ROS and oxidative stress, due to the presence of thiol groups located on the actin microfilaments. On oxidation, these filaments cross-link, leading to reduced cell motility, impaired phagocytosis and hence clearance of foreign material from the lung. The cytoskeleton mediates several basic cell functions: chemotaxis, migration, phagocytosis, phagosome-lysosome fusion, and intracellular signalling [19-21]. Several lines of evidence suggest that changes in actin filament organisation play an important role in macrophage motility, adherence to surfaces and phagocytosis. Cellular dysfunctions associated with the cytoskeleton can cause retarded phagocytosis [22] and impaired phagosome-lysosome fusion [23], which may result in a diminished cellular killing and clearance of particles and pathogens from the lung.
The pro-inflammatory cytokine interleukin 1 (IL-1) is not normally produced by the cells of healthy individuals, exceptions being skin keratinocytes, some epithelial cells and some cells of the central nervous system. In response to inflammatory stimuli, however, there is a dramatic increase in the production of IL-1 by macrophages and other cell types [24]. There are two distinct proteins, IL-1α and IL-1β which are the products of two distinct genes but which recognise the same cell surface receptors [25]. IL-1 possesses a wide variety of biological activities. As well as inducing its own synthesis, IL-1 stimulates the secretion of TNF-α and IL-6 from macrophages/monocytes [26,27]. Normal production of IL-1 is vital for host responses to injury and infection, while prolonged secretion has been linked with a number of pathological conditions [28,29].
Our hypothesis in this study was that PM10 particles produce cytokine release and cytokine gene expression in macrophages by a process which involves calcium signalling and reactive oxygen species (ROS). Furthermore, we hypothesise that other effects of PM10, such as alterations in the cytoskeleton, are also mediated via signalling processes involving both ROS and calcium.
Materials and Methods
Particle Characteristics
Collection of PM10 samples was co-ordinated by Casella Stanger, London, England. Particles were collected onto TEOM filters in Marylebone Road, London, a site which had particularly high levels of traffic and therefore high levels of primary, combustion-derived nanoparticles. Ultrafine carbon black (UfCB) was obtained from Degussa (Printex 90), the average particle size was 14 nm. The characteristics and details of UfCB particles have been published previously [30].
Particle Quantification
A single PM10 filter was placed into a bijou bottle and 0.5 ml phosphate buffered saline (PBS) added. The bottle was vortexed for 4 minutes to remove the particles from the filter and the resulting suspension transferred to a clean bijou bottle. The mass of particles was assessed by densitometry. As standards, a series of dilutions of UfCB particles were made, ranging from 15.625 μg/ml to 1 mg/ml in saline, sonicated for 5 minutes, and 75 μl of each concentration was added into triplicate groups of wells in a 96-well plate. Seventy-five microlitres of PM10 sample were added into a separate triplicate group of wells. The samples and standards were then read on a plate reader at 340 nm and the mass of particles calculated from a linear regression of the UfCB standards.
J774.A1 Cell Culture
The mouse macrophage cell line J774.A1 (a kind gift from Dr W Muller GSF, Gauting, Germany) was routinely cultured in RPMI medium (Sigma) containing 5% foetal calf serum (FCS) and Penicillin/Streptomycin. Cells were cultured until confluency was reached and then scraped from the surface of the flasks using a cell scraper. The cells were counted and adjusted to 5 × 105/ml in RPMI plus 5% FCS. Sterile 10 mm glass cover slips were placed in each well of a 24-well plate and 1 ml of cell suspension added to each well. Cells were incubated at 37°C for 24 hours prior to particle treatment.
Isolation of Human Peripheral Blood Mononuclear Cells
Human peripheral blood mononuclear cells were prepared according to the protocol of Dransfield et al, [31]. In brief, two separate volumes of 40 ml of blood were withdrawn from healthy consenting volunteers and transferred to 50 ml sterile Falcon tubes containing 4 ml of 3.8% sodium citrate solution. Tubes were gently inverted and centrifuged at 250 g for 20 minutes, the plasma removed from each tube and pooled without disturbing the cell pellet. Dextran (Pharmacia), prepared as a 6% solution in saline was warmed to 37°C, before adding to the cell pellet (2.5 ml/10 ml cell pellet) and the volume made up to 50 ml with sterile saline. Tubes were gently mixed and the cells allowed to sediment at room temperature for 30 minutes. In order to prepare autologous serum, calcium chloride solution (220 μl 1 M/10 ml), was gently mixed with the plasma and incubated in a glass tube at 37°C until the clot retracted. Percoll (Pharmacia) gradients were made from a stock solution of 90% (18 ml Percoll + 2 ml 10x PBS, (Life Technologies, Paisley) without calcium or magnesium) to give final concentrations of 81%, 70% and 55% using 1x PBS. The separating gradient was prepared by layering 2.5 ml of 70% percoll over 2.5 ml 81% percoll. The leukocyte-rich fraction from the dextran sedimentation was transferred to sterile falcon tubes, 0.9% saline added to give a final volume of 50 ml and the tubes centrifuged at 250 g for 6 minutes. The pellet was resuspended in 55% percoll and 2.5 ml layered over the previously prepared separating gradients. Tubes were centrifuged at 290 g for 20 minutes and the mononuclear cells collected from the 55/70 layer. Cells were washed twice with PBS, counted, and resuspended in RPMI medium at a concentration of 5 × 106 cells/ml and 1 ml added to each well of a 24 well plate. For calcium imaging, cells were also set up in 6-well plates containing a 26 mm diameter sterile glass coverslip. The cells were incubated for 1 hour at 37°C, the medium removed and replaced with RPMI plus 10% autologous serum and incubated for 48 hours at 37°C. After the second incubation, the medium was replaced and the cells incubated for a further 72 hours prior to treatment.
Cell Treatments
PM10 particles were diluted to give a final concentrations ranging from 5 μg/ml to 40 μg/ml in RPMI medium without serum and the suspension was sonicated for 5 minutes to disperse the particles. Cells which had been set up as described above, were washed twice with sterile PBS and 250 μl of particle suspension added to appropriate wells. UfCB particles were quantified as described for the PM10 and set up in parallel with PM10 particles at similar mass concentrations with J774 cells to investigate TNF-α release. One well received medium only (-ve control) and one received 250 μl of 1 μg/ml LPS (+ve control). The calcium antagonists were added concomitantly with the particles to give final concentrations of verapamil (100 μM), BAPTA-AM (50 μM), W-7 (250 μM), trolox (25 μM), and nacystelyn (5 mM). The cells were then incubated at 37°C for 4 hours and the supernatants removed and stored at -80°C until required. The cells cultured on 10 mm cover slips were fixed by the addition of 3% formaldehyde.
J774.A1 Intracellular Calcium Measurements
J774.A1 cells were cultured and removed from flasks as described above. Cells were pooled into a single tube, adjusted to 4.5 × 106 cells/ml in RPMI plus 10% FCS and incubated at 37°C until required for the assay. One millilitre of cell suspension was transferred to an Eppendorf tube, centrifuged at 145 g for 2 minutes, the medium removed, the cell pellet resuspended in 1 ml PBS and again centrifuged at 145 g for 2 minutes. The PBS was removed and cells resuspended in serum-free RPMI medium containing 23 mM Hepes buffer. Cells were loaded with 1 μg/μl Fura 2-AM (Sigma) in DMSO, 2 μl/ml cell suspension, the tube wrapped in foil and incubated in a shaking water bath for 20 minutes at 34°C. After incubation, the tube was centrifuged at 145 g for 2 minutes at 4°C, the medium removed and replaced with 1.5 ml fresh RPMI without serum. The Fura 2-AM-loaded cells were transferred to a quartz cuvette with stirrer and placed immediately into a fluorimeter with heated block and basal fluorescence measurements obtained over a 100 second period. The fluorimeter was set up with to give excitation wavelengths of 340 nm and 380 nm, emission 510 nm and excitation and emission slit widths set at 5 nm. During the experiments, the cuvette temperature was kept constant at 37°C. After 100 seconds, 10 μl appropriate treatment in RPMI medium was added to the cuvette and the experiment allowed to run for a further 1700 seconds. Treatments consisted of PM10 to give a final concentration of 10 μg/ml with and without the calcium antagonists at the concentrations described above. Twenty microlitres of 5% Triton solution were added to the cuvette to lyse the cells to give the maximum fluorescence (Rmax) and the experiment continued for 500 seconds. To give the minimum fluorescence value (Rmin), 15 μl of 0.5 M EGTA in 3 M Tris buffer were added to the cuvette. The experiment was terminated after a further 500 seconds. The ratio of the fluorescence measurements at excitation wavelengths of 340 and 380 nm were converted to calcium concentration values using the method of Grynkiewicz et al, [32].
Human and mouse TNF-α ELISA
The supernatants previously prepared were assayed for TNF-α protein content using a commercially available human TNF-α kit (Biosource) or mouse TNF-α kit (R&D Systems) according to the manufacturer's instructions. Briefly, each well of a 96-well plate was coated overnight with capture antibody, before washing with PBS containing 0.05% tween, and then adding test supernatant to the appropriate wells in triplicate groups. After incubation for 2 hours at room temperature, the wells were washed, a detection antibody added and incubated for a further hour at room temperature. The wells were then washed with PBS/tween before addition of Horseradish peroxidase (HRP)-conjugated streptavidin and incubated for 45 minutes at room temperature. Finally, the colour was developed by adding peroxidase substrate to each well, before reading the absorbance at 450 nm using a Dynatec plate reader.
mRNA Extraction
The experiments described above were also used to generate cells for total RNA extraction. After removal of the supernatant, 400 μl Tri reagent (Sigma) was added to each well. The lysed cells were then scraped from the surface of the plate using a cell scraper and transferred to Eppendorf tubes. Two hundred microlitres of chloroform were added to each Eppendorf, vortexed for 15 seconds and allowed to stand at room temperature for 15 minutes. The resulting mixture was centrifuged at 12000 g for 15 minutes at 4°C. The colourless upper phase was transferred to a fresh Eppendorf, before adding 450 μl isopropanol. The mixed samples were allowed to stand for a further 10 minutes at room temperature. Again the tubes were centrifuged at 12000 g for 10 minutes at 4°C, the supernatant removed and the RNA pellet washed in 1 ml of 75% ethanol. The resulting samples were then vortexed briefly, centrifuged at 7500 g for 5 minutes at 4°C and the RNA pellet air-dried for 10 minutes. The RNA was then suspended in 50 μl diethylpyrocarbonate (DEPC)-treated water and stored at -70°C until required for quantification and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR).
RT-PCR
The RT-PCR procedure was carried out using the Promega Access Kit. Briefly, a master mix of the kit reagents was prepared according to the manufacturers instructions. Ten microlitres of RNA at 0.03 μg/ml was added to 40 μl of the master mix, containing 10 μl of the appropriate human primers Glyceraldehyde Phosphate Dehydrogenase (GAPDH) or IL-1-α (MWG AG Biotech, Ebersberg, Germany). Tubes were placed in a thermal cycler which was programmed for the following temperatures and times. Following an initial 45 minute incubation at 48°, samples were cycled as follows: 94°C for 2 minutes, 95°C for 30 seconds, 60°C for 1 minute, 68°C for 2 minutes. This cycle was repeated 25 times for GAPDH and 30 times for IL-1 alpha. To conclude, the sample was incubated at 68°C for 7 minutes and then cooled to 4°C. The resulting RT-PCR products were separated by electrophoresis using a 2% agarose gel containing 1 μg/ml ethidium bromide and viewed under UV light. The RT-PCR bands were quantified by densitometry using Syngene software and the IL-1α band intensity was expressed as a percentage of the corresponding GAPDH band. These results were then expressed as a percentage of the untreated control.
Calcium Imaging
Human mononuclear cells were isolated from blood as previously described followed by adhesion onto 26 mm glass coverslips contained in 6-well plates. Cells were seeded in RPMI medium containing 0.1% BSA and penicillin/streptomycin at a density of 5 × 105 cells/ ml and incubated at 37°C, 5% CO2 for 1 hour before washing with 1 ml PBS. Prior to particle treatment and digital enhanced video microscopy (Roper scientific), cells were loaded with the calcium-sensitive dye, Fura 2-AM (2 μg/ml in RPMI) (Sigma) for 30 minutes at 37°C. The coverslips were then washed with PBS, assembled into the microscope holder and 400 μl RPMI medium without phenol red (Sigma) added. The fluorescence ratio was observed (excitation 340 and 380 nm, emission 510 nm) at a magnification of 63× (Zeiss Axiovert microscope). Images were captured every 2 seconds by a Coolsnap fx Photometrics (Roper Scientific) camera controlled by Metafluor software. After 100 seconds particle treatment was added to the cells (100 μl of a 250 μg/ml stock solution of particles to give a final concentration of 50 μg/ml) contained in phenol red-free RPMI medium.
F-Actin Staining
The cells cultured on cover slips and fixed with formaldehyde were washed three times with PBS and permeablised with 0.1%Triton for 4 minutes. Cells were then washed three times with PBS and the F-actin stained using 33 ng/ml Phalloidin-FITC (Sigma) in PBS for 30 minutes at room temperature. Cells were washed three times with PBS before staining with propidium iodide (10 μg/ml in PBS) for 5 seconds. Cells were further washed three times with PBS before being mounted on glass slides using Citifluor mounting medium. The cells were then examined using an Axiofluor fluorescence microscope. Images were captured and quantified using Metamorph software (Universal Imaging Corporation). Seven fields of view were captured from each treatment and the images deconvolved using the image software. The staining intensity of each cell was then measured using the analysis software.
Statistical Analysis
Data from all of the experiments were analysed using analysis of variance with Tukey or Fishers multiple comparison test. Significance was set at p < 0.05.
Results
Intracellular Calcium Concentration in PM10-Treated J774.A1 Cells
The effects of PM10 on J774 murine macrophages was investigated at final concentrations of 5, 10 or 25 μg/ml PM10. Treatment of the cells with these particles produced a dose-dependent increase in cytosolic calcium concentration [Ca2+]c up to a concentration of 10 μg/ml. At 25 μg/ml the [Ca2+]c decreased to a value similar to the 5 μg/ml particle concentration. (Figure 1a). Subsequent treatment with thapsigargin to release the endoplasmic reticulum calcium store produced a further increase in cytosolic Ca2+ indicating that the cells remained viable and confirming previous studies [33]. There was a statistically significant difference between control and PM10 treatments at 10 μg/ml (p < 0.05). The [Ca2+]c following concomitant treatment of cells with particles and calcium antagonists was reduced (figure 1b). Both the calcium channel blocker verapamil, and the calcium chelator BAPTA-AM significantly reduced (p,0.05) the intracellular calcium compared with PM10 alone. In contrast, the antioxidant Nacystelyn did not significantly reduce the PM10-stimulated [Ca2+] increase, with Ca2+ concentration remaining significantly greater than the control value (p < 0.05). In our previous studies [34,35] we demonstrated that the antagonists used at the same concentrations used here caused no toxic effects to cells and that the drug treatment produced similar results to the untreated controls.
Figure 1 The cytosolic calcium concentration (nM) in J774 cells on treatment with 5–25 μg/ml PM10 particles for 1700 seconds (a) and with 10 μg/ml PM10 particles plus calcium antagonists (b). There was a significant difference only at the 10 μg/ml particle dose from the control (p < 0.05). With calcium antagonist treatment, there was a significant difference between all of the treatments and the control (p < 0.05). Data represents the mean ± SEM of the intracellular calcium concentration (nM). (n = 3).
Intracellular Calcium Concentration in PM10-Treated Human Monocytes
PM10 also induced a significant increase in cytosolic calcium in the primary human monocytes (p < 0.05). The dose of 10 μg/ml final concentration was chosen as the dose at which a significant increase in [Ca2+]c had previously been observed (figure 1a). At time points from 600 to 800 seconds after the addition of particle/antagonist treatment, there was a rapid increase in [Ca2+]c with particles alone compared with the antagonists. In contrast to the antagonist treatment effects reported in figure 1, the antioxidant nacystelyn significantly inhibited the [Ca2+]c changes induced in the human monocytes treated with 10 μg/ml PM10 (figure 2). Both the intracellular calcium chelator BAPTA-AM and the calcium channel blocker verapamil also significantly inhibited the [Ca2+]c rise compared with particles alone (p < 0.05).
Figure 2 The intracellular calcium concentration (nM) in human monocytes on treatment with PM10 at a concentration of 10 μg/ml. Particles and calcium antagonists were added at zero time and the experiment run for 800 seconds in total (first 800 seconds shown). There was a significant difference between PM10-treated cells and PM10 and calcium antagonist treatment at each time point tested (p < 0.05). Data represents the mean ± SEM of the ratio of 340/380 nm. (n = 3).
Effect of UfCB and PM10 particles on TNF-α release by J774 cells
A comparison of the gram for gram dose effect of PM10 and UfCB particles on TNF-α release by J774 macrophages is shown in figure 3. The data show that PM10 particles caused significantly more TNF-α release as the same mass of UfCB particles by the mouse macrophage cell line.
Figure 3 TNF-α protein release in J774 cells treated with UfCB or PM10 for 4 hours. There was significantly more TNF-α release in PM10 treated cells compared with an equal mass of UfCB. Data represents the mean ± SEM pg/ml TNF-α release (n = 3).
TNF-α release in PM10-treated Human Monocytes
The release of TNF-α protein by human monocytes after treatment with varying concentrations of PM10 is shown in figure 4. The dose of particles ranged from 5 μg/ml to 40 μg/ml with concomitant treatment with calcium antagonists. There was a clear dose response of particle treatment from 10 μg/ml to 40 μg/ml. Within this range, the TNF-α concentration was approximately 170 pg/ml to 1000 pg/ml. At higher particle doses, the calcium antagonists reduced TNF-α release only marginally with the most dramatic and significant effect being seen with a particle concentration of 10 μg/ml with verapamil (V) and BAPTA-AM (B) treatments which reduced TNF-α release to 29 pg/ml and 7 pg/ml respectively (p < 0.05). There was no reduction in PM10 induced TNF-α release with W-7 (W), Trolox (T) or Nacystelyn (N) at any particle dose tested.
Figure 4 TNF-α release by human monocytes after treatment with PM10 (5–40 μg/ml) and with concomitant treatment with calcium antagonists for 4 hours verapamil (V), BAPTA-AM (B), W-7 (W), trolox (T), and nacystelyn (N). There was a significant difference between the untreated control and PM10 treatment only for the 10 μg/ml dose (p < 0.05). Data represents the mean ± SEM pg/ml TNF-α release (n = 5).
IL-1 mRNA Expression
Treatment of human peripheral blood monocytes with 10 μg/ml PM10 for 4 hours produced a significant increase in IL-1α mRNA content compared with unstimulated cells (p < 0.05) (figure 5). The IL-1α band intensities were expressed as a percentage of the GAPDH band intensities and then normalised to the unstimulated control. PM10 induced a five fold increase in IL-1α mRNA expression compared with the control and on treatment with the calcium antagonists, this was reduced to values similar to the control. There was a significant difference between the PM10 exposed cells and concomitant treatment with all of the calcium antagonists and antioxidants tested (p < 0.05).
Figure 5 IL-1α mRNA expression in human monocytes treated with 10 μg/ml PM10 particles with and without calcium antagonists for 4 hours. The top panel shows a typical gel. The graph shows the IL-1α expression as a percentage of the GAPDH and normalised to the control. There was significantly greater expression of IL-1α mRNA in the PM10 treatment which was reduced to control levels with calcium antagonist treatment. Data represents the mean ± SEM of the mRNA intensity. (n = 3).
F-Actin Staining
The fluorescence intensity of cells stained for F-actin after treatment with PM10 particles and calcium antagonists is shown in figure 6. Particles alone significantly increased the phalloidin-FITC fluorescence and hence the F-actin content of the cells compared with untreated cells. All of the calcium antagonists tested inhibited the PM10 induced increase in F-actin intensity to control levels and this was significantly different from particle only treatment (p < 0.05), although the increase in the fluorescence intensity of the PM10-treated cells was modest (a 5% difference).
Figure 6 The fluorescence intensity of F-actin stained J774 cells after 10 μg/ml PM10 treatment and with calcium antagonist treatment for 4 hours. There was a significant difference in the intensity of PM10-treated cells compared with the untreated control (p < 0.05). There was no significant difference between the control and any other treatment. Data represents the mean ± SEM of the fluorescence intensity of the cells. (n = 3).
Discussion
There is evidence that increases in particulate air pollution correlate with increased morbidity and mortality from respiratory and cardiovascular causes [1,36-38] and the pro-inflammatory effects of PM10 are considered to drive these effects [39,40]. The present study aimed to investigate the effect of PM10 particles on oxidative stress- and calcium-related cytokine regulation in human monocytes and on the cytoskeleton in mouse J774 cells.
We have previously shown that ultrafine or nanoparticles enhanced the calcium influx into cells of a monocytic cell line (MM6) [19,34] and that these [Ca2+]c changes lead to production of the proinflammatory cytokine TNF-α [35]. We demonstrate here using calcium imaging, that PM10 particles can also stimulate entry of extracellular calcium into both J774 macrophages and human macrophage derived monocytes, and that this process is inhibited by a calcium channel blocker suggesting that the PM10, in a similar fashion to UfCB induces opening of plasma membrane calcium channels leading to a calcium influx. The results obtained using the antioxidant nacystelyn were confusing. In the J774 macrophages nacystelyn was unable to inhibit PM10 induced increases in cytosolic calcium concentration, whereas the same antioxidant was very effective in the human monocyte derived macrophages. This difference could be due to a species difference or a comparison between a cell line and primary cells. A number of cell lines have been demonstrated to exhibit aberrant calcium signalling pathways. Our previous studies using human macrophages suggest that ultrafine particle-induced increases in cytosolic calcium can be mediated by ROS [35] and since a large proportion of the particles within PM10 are ultrafine, it is conceivable that much of the calcium increase is ROS mediated, at least in part. However, PM10 also contains other substances, such as metals, that could influence this pathway. Metals would in fact be expected to increase the ROS production by the PM10 particles [41].
The present study clearly shows that the same dose of PM10 (10 μg/ml) that induces calcium elevation also stimulates significant increases in both TNF-α protein release and IL-1α mRNA production by macrophages. The calcium channel blocker verapamil and the intracellular calcium chelator BAPTA-AM reduced the calcium increase, TNF-α protein release and IL-1 mRNA expression by human monocytes when stimulated with PM10 particles. This is strong evidence to suggest that influx of extracellular calcium plays a key role in upregulating the proinflammatory response induced by PM10 that could lead to disease. However, the calmodulin inhibitor W-7 had little effect on TNF-α release, while it did inhibit IL-1 mRNA expression. The antioxidants also had variable abilities to block cytokine expression, inhibiting IL-1 mRNA production but not TNF-α protein release. These differences could be explained either by divergent pathways controlling expression of the two cytokines, or that TNF-α protein was measured in comparison to IL-1 mRNA. However, clearly both calcium and ROS are important in the regulation of IL-1α mRNA expression while only calcium is important in controlling TNF-α expression in macrophages exposed to PM10.
These studies indicate that on an equal mass basis PM10 is far more potent that UfCB in terms of its ability to induce TNF-α protein release. This is likely to be due to other components, such as metals and organic compounds other than the carbon core, within PM10 that can promote inflammation. It is also possible that components such as the UF particles and metals could interact to enhance toxicity as has been shown for ROS production in vitro and inflammation in vivro [41]. Our previous studies have failed to detect LPS in the PM10 particles, therefore it is unlikely that cytokine release, changes in intracellular calcium, and IL-1α gene expression can be explained solely by endotoxin.
As explained previously, the cytoskeleton is the scaffold of cells, and in the case of motile cells such as macrophages it is responsible for controlling movement. Disruption of the cytoskeleton, particularly via oxidative stress, is thought to disrupt cellular structure and hence function [42]. We have previously demonstrated that PM10 generates ROS [43]. The ability of the antioxidants trolox and nacystelyn to prevent the PM10 induced increase in F-actin staining in this study demonstrates that particle-derived ROS impact on the macrophage cytoskeleton. Our previous studies also demonstrate that Uf particle-induced ROS play a role in elevating the cytosolic calcium concentration of macrophages leading to increased TNF-α production [35]. The results of this study also suggest that both calcium signalling and ROS are important in modulating the F-actin cytoskeleton in response to PM10 exposure. As has been shown by other workers [44,45], in both the macrophage cell line and primary cells that F-actin is distributed as microfilaments around the cell, with special prominence at the leading edge of the cells. The microtubules in contrast are situated throughout the cell. Microtubules and actin filaments have previously been studied as targets of antitumour drugs [46] which mainly work by acting on microtubules and alter the dynamics of actin filaments. Changes in the distribution of actin filaments and their expression compared with normal cells may indicate alterations in the phagocytic ability of macrophages which may eventually lead to impaired particle clearance from the lungs. We show here that treatment of macrophages with PM10 particles increased the F-actin fluorescence signal in cells stained with FITC-labelled phalloidin, although changes in the distribution of actin filaments was not apparent from microscope analysis there appeared to be more cortical staining. In accord with the role of calcium and ROS in the induction of IL-1 expression, both of these factors appeared to play an important role in modulating the F-actin cytoskeleton.
The present study has shown that PM10 particles may exert their increased pro-inflammatory effects by modulating intracellular calcium signalling in macrophages leading to expression of proinflammatory cytokines. An additional consideration is the effects of particles on the cytoskeleton of the cell. Impaired cellular motility and phagocytic ability is likely to play a key role in particle clearance from the lung, thus perpetuating the effects of PM10. The role of calcium and ROS in other cellular responses are under investigation.
Acknowledgements
This study was generously funded by the Colt Foundation.
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| 15613243 | PMC545043 | CC BY | 2021-01-04 16:47:22 | no | Respir Res. 2004 Dec 21; 5(1):29 | utf-8 | Respir Res | 2,004 | 10.1186/1465-9921-5-29 | oa_comm |
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Respir ResRespiratory Research1465-99211465-993XBioMed Central London 1465-9921-6-51563894310.1186/1465-9921-6-5ResearchDynamic changes of serum SARS-Coronavirus IgG, pulmonary function and radiography in patients recovering from SARS after hospital discharge Xie Lixin [email protected] Youning [email protected] Baoxing [email protected] Yueyong [email protected] Qing [email protected] Liangan [email protected] Hong [email protected] Weijun [email protected] Department of Respiratory Medicine, Chinese PLA General Hospital, 28 Fuxing Road, Beijing, 100853, P.R. China2 Department of Radiology, Chinese PLA General Hospital, 28 Fuxing Road, Beijing, 100853, P.R. China3 BGI-GBI Biotech Company, Beijing, P.R. China2005 8 1 2005 6 1 5 5 18 11 2004 8 1 2005 Copyright © 2005 Xie et al; licensee BioMed Central Ltd.2005Xie 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.
Objective
The intent of this study was to examine the recovery of individuals who had been hospitalized for severe acute respiratory syndrome (SARS) in the year following their discharge from the hospital. Parameters studied included serum levels of SARS coronavirus (SARS-CoV) IgG antibody, tests of lung function, and imaging data to evaluate changes in lung fibrosis. In addition, we explored the incidence of femoral head necrosis in some of the individuals recovering from SARS.
Methods
The subjects of this study were 383 clinically diagnosed SARS patients in Beijing, China. They were tested regularly for serum levels of SARS-CoV IgG antibody and lung function and were given chest X-rays and/or high resolution computerized tomography (HRCT) examinations at the Chinese PLA General Hospital during the 12 months that followed their release from the hospital. Those individuals who were found to have lung diffusion abnormities (transfer coefficient for carbon monoxide [DLCO] < 80% of predicted value [pred]) received regular lung function tests and HRCT examinations in the follow-up phase in order to document the changes in their lung condition. Some patients who complained of joint pain were given magnetic resonance imaging (MRI) examinations of their femoral heads.
Findings
Of all the subjects, 81.2% (311 of 383 patients) tested positive for serum SARS-CoV IgG. Of those testing positive, 27.3% (85 of 311 patients) were suffering from lung diffusion abnormities (DLCO < 80% pred) and 21.5% (67 of 311 patients) exhibited lung fibrotic changes. In the 12 month duration of this study, all of the 40 patients with lung diffusion abnormities who were examined exhibited some improvement of lung function and fibrosis detected by radiography. Of the individuals receiving MRI examinations, 23.1% (18 of 78 patients) showed signs of femoral head necrosis.
Interpretation
The lack of sero-positive SARS-CoV in some individuals suggests that there may have been some misdiagnosed cases among the subjects included in this study. Of those testing positive, the serum levels of SARS-CoV IgG antibody decreased significantly during the 12 months after hospital discharge. Additionally, we found that the individuals who had lung fibrosis showed some spontaneous recovery. Finally, some of the subjects developed femoral head necrosis.
Severe acute respiratory syndrome (SARS)SARS-CoV IgG antibodyPulmonary functionPulmonary fibrosisAvascular necrosis of femoral head
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Introduction
Severe acute respiratory syndrome (SARS) is a new infectious disease in humans. The first victim of SARS to be diagnosed was a businessman from the city of Foshan in Guangdong Province, China. SARS patients may present with a spectrum of symptoms and signs, ranging from relatively asymptomatic to fulminant pneumonitis and death [1]. Lung injury caused by the SARS coronavirus (SARS-CoV) is one of the main clinical manifestations in SARS patients, significantly affecting their prognosis. A regular follow-up survey of SARS patients in the convalescent phase would be helpful to evaluate any changes in acquired immune function, pulmonary function, bones and joints over the course of time. At present, there have been few reports about the relationship between the prognosis for recovery and the degree of lung injury caused by the SARS-CoV. In addition, a study of the serum levels of the specific IgG antibody against SARS-CoV is needed because it is the major immunologic protection to aid in recovery and is essential to avoid repeated infection with SARS-CoV. It has been 14 months since the World Health Organization officially declared the global outbreak of SARS to be under control [2]. The present study focused on the dynamic changes in the IgG antibody levels against SARS-CoV and in lung lesions in the discharged but recovering SARS patients as measured by lung function and imaging tests. The phenomenon of femoral head necrosis was also investigated in those SARS patients who complained of chronic bone and joint pain during the one year follow-up after discharge from the hospital.
Methods
All of the subjects of this study were discharged from Beijing Xiaotangshan Hospital, Beijing Armed Police Hospital, and Chinese 309 PLA Hospital, and all gave their informed consent.
Study Protocol
The subjects of our investigation were 383 clinically diagnosed SARS patients in the convalescent phase (160 male and 223 female, average age 38.2 ± 13.6 years) undergoing testing from May, 2003 to June, 2004. Each clinical diagnosis was based on the Clinical Diagnosis Standard for SARS Patients issued by the Ministry of Chinese Public Health [3]. All participants in the study had met the specified criteria for discharge from the hospital [4]. On the first visit, each patient was given a routine pulmonary function test (ventilation and diffusion function: SensorMedics 2200 pulmonary function test apparatus, U.S.A.), a chest X-ray examination and serum SARS-CoV specific antibody (SARS-CoV IgG) test at the Chinese PLA General Hospital, Beijing, P.R. China. Those individuals suspected of having pulmonary fibrotic changes received high resolution computerized tomography (HRCT) examination of their lungs. Individuals who complained of chronic pain in their bones and joints or who had difficulty walking received femoral head magnetic resonance imaging (MRI) examinations.
Each patient returned a month after the first visit followed by one visit every 3 months. Serum SARS-CoV IgG was tested at each return visit. If negative results were obtained twice consecutively, the case was regarded as a misdiagnosis and the patient did not undergo a follow-up survey. Patients with positive SARS-CoV IgG and abnormal pulmonary diffusion received regular pulmonary function tests and those showing pulmonary fibrosis in imaging examinations received further regular HRCT examinations. Some individuals observed to have avascular necrosis of the femoral head received MRI examinations 3–6 months later.
Clinical Diagnostic Criteria for the Patients with SARS Disease in Mainland China [3]
(1) Epidemiological history
(1.1) The individual has a history of close contact with SARS patients or is part of a cluster of cases of SARS or there is clinical evidence of having infected other patients.
(1.2) The individual has a history of recent travel to an area where SARS cases have been reported within 2 weeks and secondary infected SARS cases have been found.
(2) Symptoms and signs
Acute onset of SARS generally begins with a prodrome of fever with a temperature >38°C, sometimes accompanied by chills, myalgia and anthralgia, headaches, and fatigue. Upper respiratory tract symptoms of catarrh are not prominent, although cough may be present. If present, it is mainly a dry cough, occasionally with blood streak sputum. Some individuals have chest discomfort, and severe cases may present with tachypnea, panting, and even respiratory distress.
Generally, there are no obvious pulmonary signs among SARS patients. Wet rales and signs of lung consolidation, as well as decreased respiration and other signs of pleural effusion can occasionally be found.
Note: Some patients do not show initial symptoms of fever, especially those who have had recent surgery or those having chronic diseases.
(3) Routine laboratory examinations
White blood cell counts are generally normal or below normal, with decreased absolute lymphocyte counts.
(4) Chest radiological examinations
The typical imaging profile of SARS is of multiple patchy opacities with bilateral distribution. The opacities are usually ground-glass in appearance, sometimes with air space consolidation, evolving progressively over the course of the disease. The evolution is very rapid in some cases, resulting in the confluence of lesions and large areas of opacification in a short time. If the chest radiological examination is negative, reexamination after 1 to 2 days should be done.
(5) Antibiotic therapy is ineffective
Suspected cases: In accordance with 1.1+2+3, 1.2+2+4 or 2+3+4.
Clinically diagnosed cases: In accordance with 1.1+2+4, 1.2+2+4+5, or 1.2+2+3+4.
SARS-CoV IgG Antibody Test
The SARS-CoV IgG antibody in serum specimens from recovering SARS patients was assayed by the BGI-GBI Biotech Company with an enzyme-linked immunosorbent assay (ELISA) kit (No. S20030003, BGI-GBI Biotech Company, Beijing, P.R. China). The wells containing polystyrene microplate strips were coated with two recombinant SARS-CoV antigens that are well-characterized. Recovering SARS patients' serum samples in the diluent buffer (1:10) were incubated in the coated wells for 30 min at 37°C and then the wells were washed 5 times with the washing buffer. The dilutedenzyme-labeled anti-human IgG (100 μl) was added to the wells and incubated for 20 min at 37°C. The wells were washed 5 times with the washing buffer. A tetramethyl-benzidine substrate was then added to each well. The presence of specific antibodies was indicated by a yellow color developing after substrate addition. The reaction was terminated by addition of hydrochloric acid. The intensity of the color was measured spectrophotometrically at 450 nm to quantify the amount of antibody in the specimen. The optical density (OD) measured was compared with a standard calibration curve constructed for each lot, yielding concentration values for the samples. The OD values of both the positive and negative controls were determined. The threshold value for IgG was 0.18 OD units, calculated as the mean + 2 standard deviation (SD) levels of the readings given by 1000 control blood donor sera samples. If the OD was above the threshold value, the sample was considered to be positive for SARS-CoV IgG [5].
Pulmonary Function Test
Each recovering SARS patient underwent a standard pulmonary function test (SensorMedics 2200, Yorba Linda, U.S.A) for forced expiratory volume in 1 second (FEV1), vital capacity (VC), forced vital capacity (FVC), total lung capacity (TLC), transfer coefficient for carbon monoxide (DLCO), and carbon monoxide diffusion constant (DLCO/VA) measured by means of the single-breath test. The hemoglobin level was also measured to adjust the DLCO value. The results were compared with those of age- and sex-matched controls and expressed as a percentage of predicted values. Pulmonary function was regarded as abnormal when the DLCO was less than 80% of predicted values (pred). This was considered a diffusion deficit [6].
Chest Radiography and Evaluation
Frontal chest X-ray radiographs (CXR) were obtained at the first follow-up visit for each recovering SARS patient. If abnormities were found in the CXR or if the DLCO was <80% pred despite a normal CXR, the patient was sent for HRCT scanning (GE Light Speed, GE, U.S.A. 1-mm section in thickness with a 10-mm gap, supine position, scanning during inspiration, 1 second per scan, 140 kv, 200 mA). All CXR and HRCT images were assessed by three radiologists via a viewing console. The three radiologists were aware of the patients' clinical diagnosis at the time of their review of the radiographs. The final conclusions were established by consensus. Each segment of the lung was reviewed for ground-glass opacification, interstitial thickening, bronchiectasis, and architectural distortion. Abnormalities were magnified by means of a zoom function and were examined for intralobular interstitial, interlobular septal, or peribronchovascular interstitial thickening. Attention was also paid to the presence or absence of nodules or masses, cavitation or calcification, and emphysema. The presence of parenchymal bands, irregular interfaces (bronchovascular, pleural, or mediastinal), thickened interstitium, and traction bronchiectasis were considered as evidence of fibrotic changes [7].
Magnetic Resonance Imaging (MRI) Examination
All MRI examinations were done using a 1.5 T Signa CVi imager (GE Medical Systems, Milwaukee, WI, U.S.A.). For the patients who complained of chronic bone and joint pain, coronal T1-weighted (spin echo; time to repetition [TR], 440–500; time to echo [TE) 11–14] scans of the hips were done. If there were any abnormalities noted in the T1-weighted images, further T1-weighted sagittal images and coronal short tau inversion recovery (inversion time 145, TR 3500–5000, TE 80–120) or turbo-spin-echo T2-weighted images with fat suppression (TR 2500–3000, TE 80–120) were obtained. Images 3 mm thick were obtained for the coronal studies, and 4 mm thick images were obtained for the sagittal studies. Osteonecrosis was diagnosed by the presence of a band of low signal intensity in T1-weighted images [8].
Statistical Analysis
All data were expressed as the ± SD unless otherwise indicated. Statistical analyses were done by one-way analysis of variance (ANOVA), Student-Newman-Keuls, and Chi-square test for multiple comparisons. We used the STATA™ 7.0 statistical analysis software for Windows (STATA Statistical Software, Inc., U.S.A.) for evaluating the results of our study. With each statistical test, the criterion for significance was a p value of less than 0.05.
Results
The interval from hospital discharge to the first follow-up visit was 45.0 ± 20.7 days (Range: 11–104 days). Of the 383 individuals participating in our study, 311 patients (81.2%) tested positive for SARS-CoV IgG and 72 (18.8%) tested negative. (All patientswere tested twice for SARS-CoV IgG.) Of these, 33 patients (13 male and 20 female, average age 35.7 ± 12.1) with positive SARS-CoV IgG and abnormal pulmonary diffusion received regular follow-up examinations each month, from June to December in 2003, and every two months, from January to June in 2004. Tables 1 and 2 show the dynamic changes of SARS-CoV IgG in patients with positive tests for SARS-CoV IgG within the year after discharge, indicating that the serum SARS-CoV IgG remained at high levels, although it decreased significantly over the course of time.
Table 1 Dynamic changes of serum SARS-CoV IgG antibody levels in patients recovering from SARS
Samples (n) ± SD (OD units)
May, 2003 35 1.240 ± 0.350
June, 2003 74 1.087 ± 0.284
July, 2003 172 1.203 ± 0.306
Aug., 2003 152 1.061 ± 0.376
Sept., 2003 123 1.105 ± 0.378
Oct., 2003 35 1.097 ± 0.282
Nov., 2003 77 0.835 ± 0.327†‡§¶*
Dec., 2003 35 0.829 ± 0.232†§*
Jan.–Feb., 2004 67 0.737 ± 0.169†‡§¶*#
Mar–Apr, 2004 34 0.678 ± 0.179†‡§¶*#
May–June, 2004 46 0.621 ± 0.181†‡§¶*#
F value 30.62
p value 0.0000
Note: Statistical analyses were done by one-way analysis of variance (ANOVA) and Student-Newman-Keuls for multiple comparisons, and values are given as mean ± SD;
† p < 0.05 vs. SARS-CoV IgG antibody results in May, 2003.
‡ p < 0.05 vs. SARS-CoV IgG antibody results in June, 2003.
§ p < 0.05 vs. SARS-CoV IgG antibody results in July, 2003.
¶ p < 0.05 vs. SARS-CoV IgG antibody results in August, 2003.
* p < 0.05 vs. SARS-CoV IgG antibody results in Sepember, 2003.
# p < 0.05 vs. SARS-CoV IgG antibody results in October, 2003.
Table 2 Dynamic changes of serum SARS-CoV IgG antibody levels in 33 regular follow-up examinations of patients recovering from SARS
Samples (n) ± SD (OD units)
June, 2003 33 1.104 ± 0.267
July, 2003 33 1.325 ± 0.357
Aug., 2003 33 1.092 ± 0.249
Sept., 2003 33 1.121 ± 0.432
Oct., 2003 33 1.056 ± 0.309
Nov., 2003 33 0.895 ± 0.203‡¶
Dec., 2003 33 0.800 ± 0.170†‡§¶
Jan.–Feb., 2004 33 0.726 ± 0.163†‡§¶*
Mar–Apr, 2004 33 0.675 ± 0.181†‡§¶*
May–June, 2004 33 0.610 ± 0.167†‡§¶*#
F value 25.69
p value 0.0000
Note: Statistical analyses were done by one-way analysis of variance (ANOVA) and Student-Newman-Keuls for multiple comparisons, and values are given as mean ± SD;
† p < 0.05 vs. SARS-CoV IgG antibody results in June, 2003.
‡ p < 0.05 vs. SARS-CoV IgG antibody results in July, 2003.
§ p < 0.05 vs. SARS-CoV IgG antibody results in August, 2003.
¶ p < 0.05 vs. SARS-CoV IgG antibody results in September, 2003.
* p < 0.05 vs. SARS-CoV IgG antibody results in October, 2003.
# p < 0.05 vs. SARS-CoV IgG antibody results in November, 2003.
There were 88 individuals (23.0%) with abnormal DLCO among the 383 patients participating in our study. Of the 311 individuals testing positive for SARS-CoV IgG, there were 85 with abnormal DLCO (27.3%, 85/311), in contrast to just 3 cases with abnormal DLCO among the 72 subjects testing negative for SARS-CoV IgG (4.2%, 3/72). There was a statistically significant difference between positive and negative SARS-CoV IgG groups in DLCO values (table 3).
Among the 85 patients (29 male and 56 female, average age 42.2 ± 11.9 years) with abnormal DLCO and positive SARS-CoV IgG, 40 individuals received pulmonary function tests 4 times within the year at 42.0 ± 10.4, 70.0 ± 11.8 and 155.1 ± 42.9 day intervals. Among these 40 patients, there were 23 who exhibited abnormal DLCO at their second pulmonary function examination, 23 at the third examination, and 20 at the fourth examination (table 4).
.
Pulmonary fibrosis was detected by CXR and confirmed by HRCT examination in 72 SARS patients in the convalescent phase. Among these, there were 4 individuals with negative and 67 with positive SARS-CoV IgG. Of the 40 patients who received HRCT examinations at least 4 times, all showed improvement in the fibrotic condition (Figure 1).
Figure 1 The results of chest HRCT examination in a SARS patient in the convalescent phase, showing marked reversal of pulmonary fibrosis.
Of the 311 convalescent SARS patients with sero-positive SARS-CoV IgG, 78 received femoral head MRI examinations. The Imaging showed that 18 of these patients (23.1%, 18/78) had avascular necrosis of the femoral head. Of these 18 individuals, 8 had avascular necrosis of both femoral heads and 10 had avascular necrosis of one femoral head. Ten of the 18 patients showed first stage changes and 8 had secondary changes. During the 3–6 month follow-up visits for these individuals, there were no obvious changes in the avascular necrosis for these patient.
Discussion
Since the outbreak of SARS at the end of 2002, despite the great efforts that have been extended, the mechanisms, clinical characteristics, prognosis and effective therapeutics for this disease have not been adequately clarified. Both the SARS virus itself and the anti-viral therapy (such as high-dose glucocorticoids) used in treatment can cause various degrees of toxicity and side effects, including pulmonary fibrosis and avascular necrosis of the femoral head, even in the convalescent phase. Follow-up surveys of SARS patients in the convalescent phase are needed for recognizing the clinical characteristics of this disease and reevaluation of the therapeutic treatments [2,7].
In our study, 72 individuals (18.8%) showed negative results in the SARS-CoV IgG antibody test for at least two tests, suggesting that there may have been misdiagnosis of some clinically diagnosed SARS patients. Comparison of the Chinese clinical diagnosis standard (published April, 2003) [3] to the Center for Disease Control (CDC) SARS case definition (published April 30, 2003) [9], indicates that both of them emphasize the importance of epidemiological history, clinical manifestations and chest radiological changes for the clinical diagnosis of SARS disease. The CDC SARS case definition especially emphasizes the importance of laboratory criteria for the confirmation of a SARS diagnosis. This is accomplished by detecting the dynamic changes in the titration of specific antibodies against SARS CoV and positive detection of SARS-CoV RNA by PCR. In contrast, the Chinese clinical diagnosis standard did not mention the importance of a laboratory SARS-CoV test for the confirmation of a SARS diagnosis. This might have resulted in the misdiagnosis of SARS in some cases. During follow-up examinations, we found that those individuals with positive SARS-CoV IgG remained positive for a year, although the level of the antibody decreased gradually. Therefore, those inoculated with a SARS vaccine or infected by the SARS virus might not receive lifetime immunity, but only immunity for a limited duration. Certainly, our findings must be confirmed by further studies [7,8].
By regular examination of pulmonary function and CXR, we found that those with pulmonary fibrotic changes were able to heal on their own. The fibrotic tissue was absorbed and pulmonary diffusion and VC improved with time, suggesting that the mechanism of lung injury and lung fibrosis caused by the SARS-CoV may have a different pathophysiological process compared to other lung diseases, such as idiopathic pulmonary fibrosis or pulmonary fibrosis secondary to adult respiratory stress syndrome. The reason is not clear. However, in our follow-up study, we found some ground-glass-like changes in the HRCT images from SARS patients one year after discharge. This result shows that changes in the lung can still be observed in convalescents [7,9].
Recent concern has focused on a complication of SARS in the convalescent phase, when avascular necrosis develops on the femoral head. The morbidity of this condition is reported to be 15% to 30% in some SARS patients in Mainland China [8,10]. Among the 78 patients receiving an MRI examination, there were 18 cases of complicated necrosis of the femoral head to different degrees. The causes of this complication include SARS itself and the drugs (such as glucocorticoids) used in treatment, with the latter being more important than the former [11-13]. We didn't find any worsening or improvement of the avascular necrosis of the femoral head in these patients during our follow-up examinations. Although most patients received magnetotherapy, hyperbaric oxygen chamber therapy, local kerotherapy and Chinese traditional medicine to promote local blood circulation, there was no apparent short-term therapeutic effectiveness of these methods for recovery of the femoral head.
In conclusion, SARS, as a new disease, remains unfamiliar to mankind. It has high rates of morbidity and mortality in the acute phase. A significant proportion of patients surviving the acute illness have impairment in their overall functional capacity and health status in the convalescent phase after discharge from the hospital. Follow-up surveys of SARS patients in the convalescent phase are needed to understand the clinical characteristics of this disease. Our findings suggest that follow up studies of these patients are required for a longer duration, including comprehensive assessments for detection and appropriate management of any persistent or emerging sequelae. These types of investigation may facilitate the search for effective therapeutics and aid in ultimately conquering this disease.
Table 3 DLCO results for the convalescent SARS patients with sero-positive or sero-negative SARS-CoV IgG
Positive Negative Total X2 P value
DLCO normal cases 226 69 295
DLCO abnormal cases 85 3 88
Total 311 72 383 17.7269 0.000
Note: Analyzed with Chi-square test.
Table 4 Pulmonary function test results from the 4 follow-up examinations of 40 convalescent SARS patients ( ± SD)
Follow-up* VC(% pred) FEV1(% pred) DLCO(% pred) DLCO/VA(% pred)
Two months 87 ± 15 (51~114) 83 ± 13 (60~108) 69 ± 9 (47~79) 95 ± 14 (58~123)
Four months 94 ± 14 (61~123) 90 ± 13 (65~121) 76 ± 11 (48~94) 99 ± 14 (67~126)
Six months 100 ± 15† (66~136) 93 ± 12† (66~114) 76 ± 11† (52~98) 97 ± 14 (62~129)
Eleven months 103 ± 15† (66~142) 96 ± 11† (67~115) 79 ± 12† (56~98) 97 ± 14 (59~128)
F value 9.23 7.84 6.15 0.63
P value 0.0000 0.0001 0.0006 0.5936
Note: *: Indicating as time after discharge from acute illness.
Statistical analyses were done by one-way analysis of variance (ANOVA) and Student-Newman-Keuls for multiple comparisons, and values are given as mean ± SD;
†: Compared to those in the first follow-up exam, p < 0.05.
Acknowledgments
We are thankful for the research funding from the National High Technology Research and Development Program of China (863 Program) 2003AA208107 from Ministry of Science and Technology, the People's Republic of China.
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| 15638943 | PMC545044 | CC BY | 2021-01-04 16:36:27 | no | Respir Res. 2005 Jan 8; 6(1):5 | utf-8 | Respir Res | 2,005 | 10.1186/1465-9921-6-5 | oa_comm |
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Int J Health GeogrInternational Journal of Health Geographics1476-072XBioMed Central London 1476-072X-4-31564932010.1186/1476-072X-4-3ReviewCurrent practices in cancer spatial data analysis: a call for guidance Pickle Linda Williams [email protected] Lance A [email protected] Andrew B [email protected] Division of Cancer Control and Population Sciences, National Cancer Institute, 6116 Executive Blvd., Suite 504, Bethesda, MD 20892, USA2 Department of Biostatistics, Rollins School of Public Health, 1518 Clifton Road NE Atlanta, GA 30322, USA3 Department of Biostatistics and Epidemiology, Arnold School of Public Health, University of South Carolina, Columbia, SC, USA2005 13 1 2005 4 3 3 24 9 2004 13 1 2005 Copyright © 2005 Pickle et al; licensee BioMed Central Ltd.2005Pickle 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 has long been a recognition that place matters in health, from recognition of clusters of yellow fever and cholera in the 1800s to modern day analyses of regional and neighborhood effects on cancer patterns. Here we provide a summary of discussions about current practices in the spatial analysis of georeferenced cancer data by a panel of experts recently convened at the National Cancer Institute.
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Review
Background
Recently, the North American Association of Central Cancer Registries (NAACCR) formed a Geographic Information Systems (GIS) Task Force that prepared a handbook to aid cancer registry staff in using GIS for the collection, analysis, and presentation of cancer registry data [1]. The first chapters of the NAACCR handbook provide extensive information on registry data issues, particularly address geocoding and confidentiality. In June, 2002, the National Cancer Institute sponsored a meeting of selected subject matter experts in Bethesda, MD, to expand the analytic overview in the NAACCR effort to focus specifically on spatial data analysis. Invitees (listed in Table 1) include individuals with backgrounds in statistics, epidemiology, and geography so as to balance the points of view expressed.
Table 1 Panel members, home institutions, and self-selected focus areas for break-out discussions. The following lists all panel members, their home institutions, and each member's top choices of topics for break-out discussions. All panel members contributed significantly to the general discussion and to initial break-out discussions. A subset of panel members expanded on initial discussions to create the reports listed in Table 2.
Name Institution Primary topics of collaboration
Luc Anselin University of Illinois, Urbana-Champaign Spatial computing, spatial analysis, and exploratory spatial data analysis
B. Sue Bell National Cancer Institute, (currently, Food and Drug Agency) Communicating the results of spatial health analyses, features of spatial data, and disease surveillance
Francis Boscoe New York State Department of Health Features of spatial data, exploratory data analysis, and limitations of spatial analysis
Barnali Das National Cancer Institute Spatial modeling, exploratory spatial data analysis, and spatial cluster detection.
Carol Gotway Centers for Disease Control and Prevention Exploratory spatial data analysis, spatial modeling, and features of spatial data
William Henriques Agency for Toxic Substances and Disease Registry Features of spatial data, overview of spatial analysis, and communicating results of spatial health analyses
Theodore Holford Yale University Disease surveillance, spatial modeling, and exploratory spatial data analysis
Richard Hoskins Washington State Department of Health Communicating the results of spatial health analyses, overview, and spatial computing
Geoffrey Jacquez Biomedware Limitation of spatial analyses, spatial cluster detection, and overview of spatial analysis.
Martin Kulldorff Harvard Exploratory spatial data analysis, spatial cluster detection, and disease surveillance
Andrew Lawson University of South Carolina Overview of spatial analysis, and spatial cluster detection.
Linda W. Pickle National Cancer Institute Project coordinator, overview of spatial analysis, communication of spatial health analyses, spatial modeling, and exploratory spatial data analysis
Peggy Reynolds Environmental Health Investigations Branch, California State Department of Health Spatial modeling, features of spatial data, and disease surveillance
Gerard Rushton University of Iowa Exploratory spatial data analysis, features of spatial data, and spatial modeling
Lance Waller Emory University Chair of panel, spatial modeling, spatial cluster detection, and overview of panel discussion
Mary Ward Division of Cancer Epidemiology and Genetics, National Cancer Institute Features of spatial data, disease surveillance, and overview of spatial analysis
Dan Wartenberg University of Medicine and Dentistry of New Jersey Spatial cluster detection, exploratory spatial data analysis, and communicating the results of spatial health analyses
Dale Zimmerman University of Iowa Spatial modeling, spatial cluster detection, and exploratory spatial data analysis
The purpose of the meeting was to provide guidance from experts in this field who have experience applying these methods to health data, acknowledging that opinion will change as the field continues to evolve. Consensus of recommendations for any technical field is difficult to achieve, but we have attempted to include contributors with a wide-ranging set of backgrounds and experiences in the hope that what is presented represents, if not clear "best practices", at least sound principles for the analysis of spatial health data. This paper introduces motivating ideas and provides a broad overview of an upcoming series of reports by subgroups of the attendees. A listing of initial reports appears in Table 2, and additional topic-specific reports are in preparation.
Table 2 Titles and authors of initial reports by panel members (drafts available upon request). These reports represent summaries and expansions of initial discussions by the panels. The author team took topics and ideas generated by the panel discussions, conducted literature searches, formalized the presentation structure and composed the report. The final reports represent the collective efforts of each author team, building on selected contributions of panel members.
Title Author Team Topics
Current practices in cancer spatial data analysis: a call for guidance Linda W. Pickle, Lance Waller, Andrew Lawson Introduction to panel discussion and background issues.
Communication: reporting spatial health statistics to policy makers and the general public B. Sue Bell, Richard E. Hoskins, Daniel Wartenberg Review of issues involved in communicating results of spatial analyses of cancer data.
Current practices in spatial analysis of cancer data: data characteristics and data sources for geographic studies of cancer Francis P. Boscoe, Mary H. Ward, Peggy Reynolds Review of characteristics and sources of spatially-referenced health data.
Current practices in the spatial analysis of cancer: flies in the ointment Geoffrey M. Jacquez Unresolved issues lurking behind most spatial analyses of health data.
Motivation
Interest in and use of GIS for health data has grown tremendously during the past decade. The recognition of local geographic influences on health date back at least to the development of spot maps of yellow fever and cholera in the earlier-to-mid 1800's [2]. While what is known today as GIS grew out of developments associated with the Canadian Land Inventory in 1963 [3], there were no articles on GIS and human health included in the National Institutes of Heath's (NIH) MEDLINE bibliographic database as recently as 1993; between 1994 and 2002 the number of GIS articles grew 26% per year, four times the rate of increase for human health articles in general. Consequently, the NIH library first added "Geographic Information Systems" as a MEDLINE indexing term in 2003. What has fueled this increased attention? Most attribute it to the increasing computing power and availability of appropriate software on everyone's desktop, thus moving GIS and other analytic tools from the hands of the geographers and computer specialists to those of the health researcher. For example, when the National Cancer Institute prepared its first cancer mortality atlas in the early 1970s [4], the maps had to be prepared on National Oceanographic and Atmospheric Administration computer systems, since they were one of the few government agencies capable of preparing high quality maps. Now anyone with a standard personal computer can prepare such maps on their desktop in just a few minutes. Similarly, complex statistical analyses of georeferenced health data also can run on the desktop. While anyone with access to desktop computing and georeferenced health data can make maps, there is no guarantee that such maps provide meaningful insight to the underlying disease and social processes due to potential epidemiologic, cartographic, and/or statistical issues (e.g., confounding variables, poor choice of visual variables, and/or very small local sample sizes). As a result, the need remains for thoughtful application and appreciation of data, analytic, and interpretive assumptions commonly encountered in the analysis of spatially-referenced health data.
In addition to the impact of the computer revolution is the increasing recognition that all health data are spatial, i.e., referenced to place. A recent call for more widespread use of GIS in the U.K. National Health Service points out that GIS could "act as powerful evidence-based practice tools for early problem detection and solving" [5]. Many health outcomes are related to an individual's "environment" at both the personal and community levels. Personal environmental factors include not only the obvious water, soil, and air content and exposure to hazardous materials, but also lifestyle factors, such as exposure to tobacco smoke (personal and environmental), occupation, transportation choices, hobbies, and characteristics of the home. Community effects, referred to as "neighborhood social context" in the social sciences literature, have been shown to impact health care policy, delivery, utilization and outcomes [6-10]. Even within a specific geographic area, health care often varies among subgroups of residents, leading to the important study of health disparities. As another example, we are just beginning to realize how characteristics of our built environment, such as sidewalks or green space, impact health through relationships to individual's physical activity level [11].
With the increasing interest in and availability of georeferenced health data comes the need for methods to properly analyze them, taking into account the spatial correlation of outcomes in nearby places. Recognition of spatial influences in statistical inference date back to some of the earliest developments of modern statistical methods leading, for example, to notions of randomized plot designs for agricultural field trials [12].
Development of theoretical methods for spatially referenced data includes point process models [13,14], spatial prediction [15,16], and spatial lattice models [17-19] in fields such as agriculture, entomology, bacteriology, cosmology, mining, and meteorology. Methods for the analysis of measurements taken at fixed point locations as random processes grew from independent developments by Matheron [15] and Gandin [16] for analyzing geologic data. While the areas of spatial statistics, statistical computing, and GIS all developed substantially from the 1960's through today, these developments have been and continue to be largely separate and independent of one another.
Application of spatial statistical methods is more common now that both GIS and spatial statistical software packages are widely available. While there are several texts focused on statistical methods for spatial health data [20-25], and health applications of GIS [26-28], there is a growing need for guidance in the combination of the two areas, in particular the selection and proper use of the appropriate statistical techniques for different types of georeferenced health data.
Complicating factors
We specifically focus on spatial and spatio-temporal statistical methods appropriate for observational human health data, not clinical trials or data from other types of designed experiments. A challenging but common problem with this type of data is the difficulty in obtaining accurate exposure and disease outcome data for the time and place most relevant to that disease. Health consequences are the result of a continuum of multiple and varied exposures which often occur over a long period of time and in various places. How can we capture this complex pattern of exposure over decades and, most important for the topic at hand, to what geographic location do we assign the exposure? A subgroup of meeting attendees discussed the problems of defining "place" and locating appropriate data in detail (see Table 2). A forthcoming article in the International Journal of Health Geographics by Boscoe, Ward, and Reynolds will address these issues.
Another problem with data on human health is that the data required for analysis are typically scattered across many sources and often collected by different groups and agencies. For example, unless we belong to a closed medical system such as a health maintenance organization or military services, each person's medical records are housed in different medical offices. Such records collected for clinical purposes also rarely include demographic information desirable for the data analysis and only include the patient's home address (primarily for billing or other contact purposes), offering no information on previous residences or workplace location(s). Accumulating and validating data required for analysis from these multiple sources usually takes longer than the analysis itself, where data validation plays an essential but time-consuming and often overlooked role.
Of increasing concern in this field is protecting the privacy and confidentiality of the study subjects. While all researchers agree that this is important, it is often difficult to reconcile these needs with data needs for a proper analysis. In particular, spatial data analysis and mapping of results are often hampered by the lack of specific addresses. Data collection agencies and medical facilities are imposing increasingly strict requirements for data release and often only identify a place (usually patient's address) to a broad administrative unit. For example, the recently enacted Health Insurance Portability and Accountability Act (HIPAA) often requires removal of geographic subdivisions smaller than the state, and the National Center for Health Statistics only releases death certificate data aggregated to the county level or for places with large populations. The reportable specificity of location is often not good enough to allow the analysis to answer research questions about the spatial patterns of the disease. Methods are currently being explored that would allow use of specific individual information in the analysis but would mask identifying characteristics in the results reported only at an aggregated level. In addition to such federal reporting restrictions, state and local governments may add additional regulatory requirements.
This said, such regulations need not prohibit spatial analysis of health data completely, rather they change the context within which such analyses may occur. For instance, mutually agreeable memoranda of understanding between analysts and agencies holding data often allow analysis of data with individual-level identifiers provided all reports include only aggregate results. Such memoranda also specify when, if ever, detailed maps of locations may be reported. As an example, the National Cancer Institute's Long Island Breast Cancer Study Project provides a protected environment (on site or remotely accessible) within which registered users may analyze (but not remove) sensitive georeferenced health data (for more information, see ).
In addition to general concerns regarding the analysis of health data, the spatial data analysis of cancer data poses unique challenges. Most cancers develop over a period of 20 to 30 years and are a result of multiple exposures interacting with the individual's genetic susceptibility. Few Americans live in a single place for decades – migration presents the problem of which residential address to use for a case's location. Because latencies differ by cancer type and most likely by an individual's susceptibility, little guidance is available for this question. The rarity of cancer also causes a sparse data problem for analysis, both for detecting clusters in data with high spatial variability and for communication of results without violating confidentiality. John Snow's illustration of his theorized cause of cholera in London via a map of case residences was possible because of the large number of cases in a small geographic area with a single, precisely located exposure [29]. The detection of clusters of a rare disease such as cancer requires sophisticated statistical tools that filter out potentially confounding effects of age, spatially-varying population density, and mobility. As pointed out by Waller and Gotway [25], different statistical methods answer different questions and require care in appropriate application and interpretation. Further discussion of these and other limitations of spatial data analysis are addressed in the accompanying article by Jacquez [30].
Despite such concerns, important discoveries in cancer research do result from spatial data analysis. Although U.S. mortality data had been published in tabular form for many years, it wasn't until mortality rates were mapped in 1975 that spatial patterns emerged, such as the cluster of high oral cancer rates in southeastern states, later found to be due to smokeless tobacco use [4,31]. Later, a number of clusters of childhood leukemia were identified, for example in Seascale, UK, and Toms River, NJ [32,33]. Although environmental, genetic and viral hypotheses have been proposed, the cause of most of these clusters remains unclear [34]. These studies illustrate the potential impact of spatial data analysis on medical research.
Finally, in order to ultimately improve public health, the results of the complex analyses of georeferenced cancer data must be disseminated to those in a position to take action, such as state epidemiologists and local cancer control specialists. This audience often needs to obtain statistical data quickly for rapid response to health problems and cannot be expected to have the technical expertise to understand the statistical detail underlying the methods. Statisticians must consider this audience and design maps and reports in such a way as to be easily accessible by them. A forthcoming article in the International Journal of Health Geographics by Bell, Hoskins, and Wartenberg (Table 2) addresses these issues in further detail
Conclusion
In closing, participants in the NCI workshop addressed a wide range of topics summarized in Tables 1 and 2. Initial reports address data issues, communication of results, and current limitations and areas for further development. Further discussions examined and reviewed several technical aspects of analysis relating to public health surveillance, cluster detection and spatial models, and additional reports are in preparation. We hope that public health professionals, geographers, epidemiologists, environmental scientists, and statisticians faced with the analysis of georeferenced health data find these articles to be useful as an introduction to current methods and concerns in the area.
Acknowledgements
We thank all participants in the June 2002 workshop listed in Table 1. Their input contributed significantly to the material contained in this article and related reports.
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World J Surg OncolWorld Journal of Surgical Oncology1477-7819BioMed Central London 1477-7819-2-461560147010.1186/1477-7819-2-46Case ReportIn-situ and invasive carcinoma within a phyllodes tumor associated with lymph node metastases Parfitt Jeremy R [email protected] Chris [email protected]'Malley Frances [email protected] Joan [email protected] Alan B [email protected] Department of Pathology, London Health Sciences Centre, University of Western Ontario, London, Ontario, Canada2 Department of Pathology and Laboratory Medicine, Mount Sinai Hospital and University of Toronto, Toronto, Ontario, Canada3 Lambton Hospital Group – St. Joseph's, Sarnia, Ontario, Canada2004 15 12 2004 2 46 46 13 10 2004 15 12 2004 Copyright © 2004 Parfitt et al; licensee BioMed Central Ltd.2004Parfitt 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
Phyllodes tumors (cystosarcoma phyllodes) are uncommon lesions in the female breast. Rarely, the occurrence of carcinoma within a phyllodes tumor has been reported in the literature, but has never been associated with lymph node metastases.
Case presentation
A 26-year-old woman presented with a firm, mobile, non-tender mass in the left breast and palpable lymph nodes in the left axilla. The excised lesion appeared well circumscribed and lobulated, with variable fleshy and firm areas. Microscopic examination showed a circumscribed fibroepithelial lesion with a well developed leaf-like architecture, in keeping with a benign phyllodes tumor. The epithelial component showed extensive high grade ductal carcinoma in-situ (DCIS) and invasive carcinoma of no special type, located entirely within the phyllodes tumor. Subsequent axillary lymph node dissection revealed metastatic carcinoma in four lymph nodes.
Conclusions
Although rare, phyllodes tumors may harbor DCIS and invasive carcinoma, with potential for lymph node metastasis.
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Background
Phyllodes tumors constitute less than 1% of breast tumors and 2–3% of fibroepithelial breast tumors [1]. They usually occur in middle-aged to elderly women but can occur at any age. No single feature is reliable in predicting clinical behavior of phyllodes tumors. Several histological parameters should be evaluated, including stromal cellularity, atypia, mitoses, stromal overgrowth, infiltrative borders, and presence or absence of necrosis [1,2]. A mitotic rate of less than 5 per 10 high power field (HPF) suggests benign behavior, while a mitotic figure rate of more than 10 per 10 HPF suggests malignant potential [2,3]. Most of these tumors are benign, but up to 30% show malignant stroma [4]. Metastases, usually hematogenous rather than lymphatic, have been reported to occur at a rate of 13% in 10 years for malignant phyllodes tumors [4]. As malignant phyllodes tumors usually spread by a hematogenous rather than a lymphatic route, axillary lymph node dissection is generally not recommended. Importantly, local recurrences are common even in benign tumors and are seen in up to 8% in 10 years [4].
We describe a case of in-situ and invasive carcinoma occurring within a benign phyllodes tumor in a 26-year-old woman, which, to our knowledge, is the youngest presenting age for this combination tumor. We also report the first case of this combination tumor with documented lymph node metastases. Our case highlights the importance of assessing phyllodes tumors for concurrent carcinomatous involvement, as this may affect both the need for axillary lymph node examination and subsequent treatment options.
Case presentation
A 26-year-old woman presented with a 1 month history of a palpable mass in her left breast. Her mother died at the age of 31 years of breast cancer. Examination revealed a 2 cm firm and mobile mass in the lower outer quadrant of the left breast. The left axilla contained two enlarged palpable lymph nodes.
Mammography showed a 2 cm oval density that was homogenous with sharp margins. Ultrasound characterized the mass as solid but with a complex internal architecture. Fine needle aspiration of the mass was performed. It was positive for carcinoma, showing a very cellular sample with many malignant epithelial cells in cohesive groups, as well as lying singly in a background of necrosis and inflammation. The surgeon subsequently performed an excisional biopsy.
Grossly, the excised mass measured 3.3 cm × 2.3 cm × 2.0 cm and appeared well circumscribed. The cut surface was grey and lobulated with variable fleshy and firm areas. Histological examination revealed a well-circumscribed biphasic neoplasm, consisting of proliferating stroma and compressed glands, displaying a well developed leaf-like architecture. The stroma was moderately cellular, mildly vascular, and lacked cytological atypia, mitoses, and stromal overgrowth. The features were consistent with a benign phyllodes tumor (figures 1 &2). More importantly, the epithelial component of the phyllodes tumor showed extensive ductal carcinoma in-situ, with multiple foci of invasive mammary carcinoma (figure 3). The DCIS was of high nuclear grade, cribriform and micropapillary architecture, with (comedo-type) necrosis (figure 4). The invasive carcinoma was of no special type, SBR Grade III/III (Score 8/9). Multiple tumor emboli were present within endothelial-lined spaces adjacent to the tumor, but the surrounding breast was otherwise unremarkable. Although the carcinomatous component was confined to the phyllodes tumor, the lesion was <1 mm from the nearest resection margin. Immunohistochemistry using smooth muscle myosin heavy chain and muscle specific actin demonstrated absence of the myoepithelial cell layer within areas of invasive carcinoma, and focal loss within areas of DCIS. There was intensely positive staining within 80% of tumor cells for estrogen and progesterone receptors.
Figure 1 Low power photomicrograph of the phyllodes tumor: Histologically, the neoplasm was biphasic, with proliferating stroma, compressed glands, and a well developed leaf-like architecture (H&E, original magnification 40×).
Figure 2 High power photomicrograph of the phyllodes tumor: The stroma lacked cellular atypia and mitoses, consistent with a benign phyllodes tumor (H&E, original magnification 400×).
Figure 3 Photomicrograph of the carcinomatous component: The epithelial component showed DCIS and invasive mammary carcinoma (H&E, original magnification 100×).
Figure 4 Photomicrograph of the DCIS patterns: The DCIS showed cribriform and micropapillary architecture, with necrosis (H&E, original magnification 100×).
The patient went on to have a left axillary lymph node dissection and subsequent pathological examination revealed 4 of 13 nodes positive for metastatic adenocarcinoma. She received adjuvant CEF (Cyclophosphamide, Epirubicin, 5-Fluorouracil) chemotherapy and underwent further local excision to achieve wider margins; pathological examination was negative for residual tumor. She subsequently received local radiation and is currently undergoing tamoxifen therapy. The patient is presently alive and disease free, nearly three years after presentation.
Discussion
The epithelial component of phyllodes tumors (whether benign or malignant) may show a range of metaplastic (apocrine, squamous) and proliferative changes [5,6]. However, carcinoma arising within a phyllodes tumor is decidedly rare, with less than 30 reported cases in the literature. The age of the previously reported patients with coexistent carcinoma and phyllodes tumor ranged from 31–80 years, with most of the patients in the 5th or 6th decades [7,8]. This case represents the youngest age at presentation with this combination tumor. The reported subtypes include in-situ and invasive lobular and ductal (no special type) carcinoma, tubular carcinoma and squamous carcinoma [2-7,9-13]. Some authors have noticed greater atypia in epithelial components of recurrent phyllodes tumors. Squamous carcinoma, typically rare in the breast, seems to occur more often in phyllodes tumors, compared to patients without phyllodes tumors [10].
The prognosis for cases of carcinoma within phyllodes tumors is generally favorable, with no deaths yet reported [9,10]. The stroma is benign in the majority and cancer detection often occurs early because the patient presents with a rapidly enlarging mass [10]. Our case appears to represent the first documentation of lymph node spread of the carcinomatous component from within a phyllodes tumor. Axillary lymph node dissection is not part of the standard treatment for phyllodes tumors as lymph node spread is rare [14]. Several authors have suggested treating carcinoma within phyllodes tumors according to the carcinomatous component [9,14]. Our case supports this view, particularly with respect to the need for assessment of axillary lymph node status in determining prognosis and treatment.
Conclusions
Invasive carcinoma occurring within a phyllodes tumor is rare; occurrence of axillary lymph node metastasis is even rarer. Besides the stromal component, the epithelial component of phyllodes tumors should also be examined in detail, as it may harbor an in situor invasive carcinoma.
Competing Interests
The authors declare that they have no competing interests.
Authors' Contributions
JRP prepared the manuscript.
CA and ABT were involved with initial diagnosis of the pathologic specimen and were involved with manuscript editing.
FO provided consultation for the final diagnostic interpretation and was involved with manuscript editing.
JR provided patient history, obtained patient consent and helped with manuscript editing.
All authors read and approved the final manuscript.
Acknowledgements
Consent was obtained from the patient for publication. We thank Dr. D.K. Driman for contributions in editing the report.
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| 15601470 | PMC545046 | CC BY | 2021-01-04 16:38:38 | no | World J Surg Oncol. 2004 Dec 15; 2:46 | utf-8 | World J Surg Oncol | 2,004 | 10.1186/1477-7819-2-46 | oa_comm |
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World J Surg OncolWorld Journal of Surgical Oncology1477-7819BioMed Central London 1477-7819-3-21563664310.1186/1477-7819-3-2ResearchExpression of vascular endothelial growth factor (VEGF)-C in preoperative biopsy specimens and metastatic foci of regional lymph nodes in submucosal gastric carcinoma Ishikawa Makoto [email protected] Joji [email protected] Shinsuke [email protected] Hirokazu [email protected] Department of Surgery, Division of Surgical Oncology, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan2005 7 1 2005 3 2 2 6 10 2004 7 1 2005 Copyright © 2005 Ishikawa et al; licensee BioMed Central Ltd.2005Ishikawa 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
Vascular endothelial growth factor (VEGF)-C is implicated in lymphangiogenesis, however the exact role of VEGF-C in promoting lymphatic spread of cancer cells remains largely unknown.
Methods
The expression of VEGF-C was immunohistochemically determined in 97 endoscopic biopsy specimens from 46 patients with submucosal gastric carcinoma (SGC). Nodal metastases including micrometastasis and isolated tumor cells (ITC) were evaluated by immunohistochemical staining for cytokeratin in 1650 lymph nodes, and tumor cells in these metastatic nodes were also examined for VEGF-C expression.
Results
In biopsy samples, VEGF-C was positively detected in 21 (47%) patients. Metastases were identified in 46 (2.8%) nodes from 15 (33%) patients. Metastases were detected in 39 nodes by hematoxylin-eosin (H&E) staining and in additional 7 nodes as ITC by immunohistochemical staining. The rate of lymph node metastases was significantly correlated with VEGF-C expression in biopsy samples (p < 0.05). The positive and negative predictive values of VEGF-C in biopsy specimens for nodal metastasis were 44 %(10/21) and 80% (20/25), respectively. Among the 46 metastatic nodes, tumor cells in 29 (63%) nodes positive patients expressed VEGF-C, whereas those in 17 (37%) nodes did not. VEGF-C expression was high in macronodular foci in medullary areas, whereas more than half of ITC or micrometastasis located in peripheral sinus lacked the expression of VEGF-C.
Conclusions
Despite the significant correlation, immunodetcetion of VEGF-C in endoscopic biopsy specimens could not accurately predict the nodal status, and thus cannot be applied for the decision of the treatment for SGC. VEGF-C may not be essential for lymphatic transport, but rather important to develop the macronodular lesion in metastatic nodes.
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Background
The incidence of early gastric carcinoma defined as being confined to the mucosa or submucosal layer has increased. In Japan, endoscopic mucosal resection (EMR) is now generally accepted for intramucosal cancers that are associated with a minimal risk of regional lymph node (LN) metastasis [1-4]. For submucosal gastric carcinoma (SGC), however, conventional gastrectomy with complete lymph node dissection has been performed as standard treatment as the frequency of lymph node metastasis is 10–20%, which cannot be ignored clinically [5-8]. This indicates that the conventional operative procedure provides no benefit for the majority of patients with SGC, and thus criteria to safely avoid unnecessary lymphadenectomy for submucosal cancer need to be determined.
VECF-C is known to bind VEGFR-3, which is specifically expressed on lymphatic vessels and stimulates lymphangiogenesis [9,10]. Many previous reports have shown that expression of VEGF-C in cancer tissues has a positive correlation with the risk of lymphatic metastasis in breast [11,12], lung [13], colorectal [14-17], pancreatic [18], prostate [19], esophageal [14,20] and head and neck cancers [21,22]. A similar tendency has been reported for gastric cancer, although a significant correlation between VEGF-C expression and the frequency of nodal metastasis is not always found [23-27].
Recently, small metastatic lesions have been detected genetically or immunohistochemically in various cancers, even though they were diagnosed as negative by conventional examination with H&E staining. Such lesions are designated as micrometastasis or isolated tumor cells (ITC). The biological and clinical significance of such minute nodal invasion of carcinoma cells is still controversial [28-38]. In this study we performed immunohistochemical staining and extensively examined VEGF-C expression in biopsy samples and metastatic lymph nodes including micrometastasis and ITC. From these data, we attempted to determine whether the detection of VEGF-C in biopsy samples could be a clinical predictor of accurate nodal status in SGC. Then, we discussed how the VEGF-C expressed in tumor cells functions in the metastatic process to regional lymph nodes.
Patients and methods
Forty-six patients with SGC diagnosed and treated by curative gastrectomy with standard lymph node dissection at the First Department of Surgery, Tokyo University Hospital, Tokyo, between 1994 and 2002 were included in this study. These patients were examined endoscopically prior to surgery; several pieces of tissue specimens were then sampled with routine biopsy forceps from various portions of the tumor. Formalin-fixed and paraffin-embedded sections of 97 biopsy specimens and 1650 dissected lymph nodes derived from these 46 patients were evaluated in this study. Additionally, all the resected primary tumors were histologically examined with H&E staining according to the Japanese Classification of Gastric Carcinoma [39]. Tumors were histologically classified into two types based on the predominant features: differentiated type (well and moderately differentiated adenocarcinoma) and undifferentiated type (poorly differentiated adenocarcinoma and signet ring cell carcinoma). Several discrete histological parameters, including lymphatic invasion, venous invasion and lymph node metastasis, were also evaluated.
Immunohistochemical study of VEGF-C and Cytokeratin
The expression of VEGF-C was investigated with immunohistochemical staining using affinity purified goat polyclonal antibodies against VEGF-C (IBL, Fujioka, Japan). Sections (3-μm thick) of biopsy samples were deparaffinized in xylene, hydrated through a graded series of ethanol, and then immersed in 3% hydrogen peroxide in 100% methanol for 30 min to inhibit endogenous peroxidase activity. To activate the antigens, the sections were boiled in 10 mM citrate buffer, pH 6.0 for 30 minutes. After being rinsed in phosphate-buffered saline (PBS), the sections were incubated with normal rabbit serum for 10 min, and then incubated overnight at 4°C in humid chambers with the primary antibody to VEGF-C at 1/30 dilution. After three washes with PBS, the sections were incubated with biotinylated rabbit anti-goat immunoglobulin for 20 minute. After washing again with PBS, the slides were treated with peroxidase-conjugated streptavidin for 20 minutes, and developed by immersion in 0.01% H2O2 and 0.05% diaminobenzidine tetrahydrochloride for 3 minute. Light counterstaining with Mayer's hematoxylin was performed. The 46 lymph nodes that showed the presence of carcinoma were also evaluated for the expression of VEGF-C with the same immunostaining method.
The dissected lymph nodes were fixed in 10% formalin and embedded in paraffin. From each node, one 3-μm-thick section was prepared for H&E staining, and another three serial 5-μm sections were prepared for immunohistochemical staining with CAM 5.2 (Becton Dickinson, San Jose, CA), a mouse monoclonal antibody that reacts with human cytokeratin numbers 8 and 18 [40]. The streptavidin biotin immunoperoxidase technique was used. Deparaffinized and rehydrated sections were trypsinized with 1% calcium chloride solution at 37°C for 20 minutes. After nonspecific reactions were blocked with 10% normal rabbit serum, the sections were incubated with CAM 5.2 diluted 1/6, biotinylated rabbit anti-mouse immunoglobulin, and streptavidin peroxidase. Between each two incubation steps, sections were washed carefully in phosphate buffered saline.
Definition of lymph node metastasis
Metastasis was defined as the presence of tumor cells, whether single or in small clusters detected by H&E or immunohistochemical staining. Metastatic lesions that were more than 2.0 mm in diameter were defined as macrometastasis, while micrometastasis was defined as a tumor deposit larger than 0.2 mm but less than 2.0 mm, and ITC was defined as a tumor deposit less than 0.2 mm in maximum diameter.
Statistical analysis
All statistical calculations were carried out using StatView-J 5.0 statistical software (SAS Institute, USA). The relationship between clinical and pathological characteristics of patients and the expression of VEGF-C was examined by Fisher's exact test. Differences with a p value of less than 0.05 were considered to be statistically significant.
Results
Nodal status including micrometastases and isolated tumor cells (ITC) in SGC
Metastases were observed in 12 patients (26.1%) and 39 lymph nodes (2.4%) by H&E examination (Fig. 1-c, f). Among these nodes, cancer cells were detected as micrometastasis (less than 2.0 mm) in 5 lymph nodes and as ITC (less than 0.2 mm) in another 5 nodes. By immunohistochemical staining with anti-cytokeratin antibody, we additionally identified such small metastatic lesions in 7 nodes (Fig. 1-d, g). All of the 7 metastatic lesions were categorized as ITC. Four nodes were derived from 4 patients who had other metastatic nodes detected by H&E staining. Cytokeratin-positive cells were also identified in other 3 nodes from 3 patients who showed no metastatic nodes by H&E staining and were diagnosed as node negative cases. Thus, the final frequency of lymph node metastasis increased to 15 of 46 patients (33%) and 46 of 1650 lymph nodes (2.8%).
Figure 1 Immunohistochemical staining of VEGF-C in biopsied specimens (upper panel) and hematoxylin-eosin (H&E) staining, immunohistochemical staining of cytokeratin and VEGF-C in metastastic lymph node (middle and lower panel). a, VEGF-C positive type in biopsied specimens. b, VEGF-C negative type in biopsied specimens. c, f, hematoxylin-eosin (H&E) staining in metastastic lymph node. d, g, immunohistochemical staining of cytokeratin in metastastic lymph node. e, VEGF-C positive type in metastastic lymph node. h, VEGF-C negative type in metastastic lymph node.
Table 1 show the relationship between the exact nodal status and other clinical/pathological features. Metastases were frequently observed in tumors with deep submucosal invasion or lymphatic involvement, which were consistent with previous reports.
Table 1 Nodal metastasis, including nicrometastasis/ITC and clinical and/or pathological findings
Patients with lymph node metastasis
positive(15) negative(31) p value
Age
60< 6 16
<59 9 15 0.46
Sex
Male 11 22
Female 4 9 0.87
Tumor size (cm)
3.0< 11 19
2.9> 4 12 0.42
Macroscopic type
elevated 2 1
depressed 13 30 0.19
Depth of invasion
sm1 3 18
sm2,3 12 13 0.02
Histological type
differentiated 8 14
undifferentiated 7 17 0.6
Lymphatic involvement
positive 4 2
negative 11 29 0.05
Venous involvement
positive 2 2
negative 13 29 0.44
Immunohistochemical analysis of VEGF-C in biopsy and surgical specimens
The biopsy specimens were divided into two categories by the staining pattern of VEGF-C, diffuse or focal staining of carcinoma cells as described previously [26]. When distinct staining of the cytoplasm was observed in the majority of tumor cells, whether diffuse or focal, these samples were categorized as VEGF-C positive in this study (Fig. 1-a). Whereas other cases in which only a few carcinoma cells stained faintly were classified as VEGF-C negative (Fig. 1-b).
Among the 97 biopsy samples from 46 patients, carcinoma cells were contained in only one biopsy sample in 14 patients, and in 2, 3 and 4 biopsy samples in 16, 13 and 3 patients, respectively. In all of the latter cases, carcinoma cells in biopsy samples derived from different places showed exactly the same staining pattern of VEGF-C, and thus VEGF-C-positive and -negative tumors could be clearly distinguished.
In biopsy specimens, VEGF-C was positively detected in 21 (46%) cases. As shown in Table 1, the expression of VEGF-C in biopsy samples showed a significant correlation with that in surgically removed specimens (p = 0.005). However, 15 (60%) of 25 cases that were classified as VEGF-C negative in biopsy samples showed positive expression of VEGF-C in surgical specimens. In contrast, 20 of 21 cases with VEGF-C-positive biopsy samples also expressed VEGF-C in surgical specimens, indicating that positive expression of VEGF-C was mostly consistent between biopsy and surgical specimens. Thus, the immunodetection of VEGF-C in biopsy sample showed 95% of positive predictive value and 40% of negative predictive values for VEGF-C expression in primary tumor.
Immunohistochemical detection of VEGF-C in biopsy specimens
Nodal metastasis was detected in 10 (48%) of 21 VEGF-C-positive tumors, and the rate was significantly higher than that in VEGF-C-negative tumors as evaluated by biopsy specimens (5/25, 20%) (p = 0.047). However, the positive and negative predictive values of VEGF-C in biopsy for nodal status were 44% (10/21) and 75% (20/25), respectively, and 5 (20%) of 25 VEGF-C-negative tumors were accompanied with lymph node metastasis.
Expression of VEGF-C in tumor cells in metastatic lymph nodes
Table 3 shows the expression pattern of VEGF-C of tumor cells in 46 metastatic lymph nodes as well as in 23 biopsy samples in 15 patients. Interestingly, the expression of VEGF-C in metastatic tumor cells in lymph nodes was not necessarily correlated with that in biopsy samples. In 15 cases with nodal metastasis, VEGF-C was positively detected in 18 biopsy samples from 10 (67%) patients. On the other hand, VEGF-C was positive in metastatic tumor cells in 29 nodes derived from 7 (47%) patients (Fig. 1-e), while tumor cells metastasized in 17 (37%) lymph nodes derived from 11 (73%) patients were negative for VEGF-C (Fig. 1-h).
Table 3 The location of tumor cells in metastatic nodes and VEGF-C expression in 15 node positive patients.
Case VEGF-C expression definition of metastasis** Detection method* location of tumor cells
biopsy specimens# metastatic nodes
1 - - ITC H.E. marginal
2 - + macrometastasis H.E. medullary
+ ITC H.E. marginal
+ macrometastasis H.E. medullary
3 - - macrometastasis H.E. marginal
- macrometastasis H.E. marginal
- ITC I.H.C. marginal
4 - - macrometastasis H.E. marginal
- macrometastasis H.E. marginal
5 + + + macrometastasis H.E. marginal
- macrometastasis H.E. marginal
- macrometastasis H.E. marginal
6 + + macrometastasis H.E. medullary
+ macrometastasis H.E. medullary
+ macrometastasis H.E. medullary
+ macrometastasis H.E. medullary
+ macrometastasis H.E. medullary
- ITC I.H.C. marginal
7 + + micrometastasis H.E. medullary
+ ITC H.E. marginal
+ ITC H.E. marginal
8 + + + macrometastasis H.E. medullary
+ macrometastasis H.E. medullary
+ macrometastasis H.E. medullary
+ micrometastasis H.E. medullary
+ micrometastasis H.E. medullary
9 + - ITC H.E. marginal
- ITC I.H.C. marginal
10 + + - micrometastasis H.E. marginal
11 + + + + macrometastasis H.E. medullary
- micrometastasis H.E. medullary
- ITC I.H.C. marginal
12 + + + + macrometastasis H.E. medullary
+ macrometastasis H.E. medullary
+ macrometastasis H.E. medullary
+ macrometastasis H.E. medullary
+ macrometastasis H.E. medullary
+ macrometastasis H.E. medullary
+ macrometastasis H.E. medullary
+ macrometastasis H.E. medullary
+ macrometastasis H.E. medullary
+ macrometastasis H.E. medullary
+ macrometastasis H.E. medullary
13 - - ITC I.H.C. marginal
14 + + - ITC I.H.C. marginal
15 + - ITC I.H.C. marginal
*:IHC means immunohistochemical staining with anti-cytochelatin mAb.
**:Micrometastasis is defined as tumor deposit larger than 0.2 mm but less than 2.0 mm, and ITC (isolated tumor cell) is defined as tumor deposit less than 0.2 mm in the maximum diameter. Larger deposits were classified as macrometastasis.
#: The number of +/- means the number of examined biopsy specimens obtained from different parts of the tumor.
Among 36 nodes from 10 patients who were determined as VEGF-C-positive in biopsy samples, tumor cells located in 10 (29%) nodes from 4 (40%) patients totally lacked the expression of VEGF-C. This finding clearly indicates that carcinoma cells that highly express VEGF-C are not always preferentially transported to the regional lymph nodes, even though the expression of this lymphangiogenic factor had a positive correlation with lymph node metastasis.
More interestingly, the expression of VEGF-C was strongly correlated with the size of metastatic foci and the location of carcinoma cells in metastatic nodes (Table 3). When carcinoma cells invaded the medullary area of lymph nodes, most of the tumor cells positively expressed VEGF-C (25/26, 96%). In contrast, the rate of VEGF-C-positive cells was markedly lower (4/20, 20%), when the carcinoma cells remained in the peripheral area of lymph nodes. Also, 22 (79%) of 28 macrometastases expressed VEGF-C, while 3 (60%) of 5 micrometastases and only 3 (25%) of 12 ITC were positive for VEGF-C. It is especially notable that all of the 7 ITC detected by immunostaining lacked expression of VEGF-C.
Discussion
Many cancers metastasize to regional lymph nodes, and a positive nodal status often correlates with a poor prognosis of patients. However, the mechanisms of lymphatic metastasis have not been investigated in detail. Recent studies have demonstrated that the expression of VEGF-C is enhanced in various solid tumors, suggesting the possible contribution of VEGF-C to nodal metastasis, possibly through lymphangiogenesis [41,42]. Number of clinical studies has shown a positive correlation between VEGF-C expression and risk of lymph node metastasis in various cancers including gastric cancer [11,12]. However, all of the data were obtained in surgically resected specimens, and thus can not be used for preoperative information to determine the treatment.
In this study, therefore, we evaluated the expression of VEGF-C in biopsy samples in SGC. Our initial hypothesis was that VEGF-C expression can predict the accurate nodal status including micrometastasis/ITC, and thus may be useful to avoid the unnecessary gastrectomy in some SGC. Our results suggest that VEGF-C expression in biopsy specimens correlate with lymph node metastasis in SGC. However, the positive and negative predictive values were 44% and 80% respectively, and 20% of VEGF-C-negative tumors were node positive. This suggests that the immunodetection of VEGF-C in biopsy samples can not be used as clinical indicator to decide the treatment of SGC.
Present study provides some interesting findings on the possible role of VEGF-C in nodal metastasis. Biopsy samples were obtained at preoperative endoscopy, fixed with formalin immediately after biopsy, and thus appear to reflect the in situ expression level of VEGF-C more precisely than surgically resected specimens. In our results, VEGF-C expression in biopsy samples showed a significant correlation with that in surgical specimens with 57% sensitivity and 91% specificity. However, more than half (60%) of the tumors categorized as VEGF-C negative in biopsy specimens were positive in surgical specimens, although VEGF-C-positive tumors in biopsy samples showed a good consistency with those in surgical specimens. This raises the possibility that VEGF-C expression may be somewhat upregulated by surgical manipulation.
VEGF gene expression is regulated by a variety of stimuli, and hypoxia is known to be one of the most potent inducers of VEGF-A [43,44]. VEGF-C expression has also been reported to be enhanced by hypoxia in some reports [45,46], but not in others [47,48]. Although the detailed regulatory mechanisms of VEGF-C gene activation are not well understood, our results suggest a possibility that VEGF-C in surgical specimen may be induced by hypoxia during gastrectomy at least in some cases. This point should be included for the evaluation of the results in previous studies showing the positive correlation with nodal status.
Nonetheless, our data showed a significant correlation of VEGF-C expression in biopsy specimens with nodal metastasis, supporting a possible role of VEGF-C in lymphatic metastasis. As with hematogeneous metastasis, lymphatic metastasis of cancer cells is considered to be divided into several steps: invasion to lymphatic capillaries, movement into the lymphatic lumen with the lymphatic stream, attachment to the subcapsular sinus of lymph nodes, and invasion into the cortex. Lymphangiogenesis means the development and proliferation of new lymphatics from host vessels, but the ability of tumor cells to induce lymphangiogenesis and the presence of intratumoral lymphatic vessels are controversial. However, most malignant tumors are known to be associated with an increased number of lymphatic vessels in the peripheral area [42]. In fact, in vivo experiments using VEGF-C-transfected tumors have shown the same histological findings [49,50]. Since intratumoral interstitial fluid pressure is known to be higher than that in normal tissues, the hydrostatic pressure difference appears to transport the tumor cells from inside of the tumor to the peritumoral area. Therefore, the metastasis-promoting effect has been attributed to an increase and dilatation of peritumoral lymphatic capillaries. VEGF-C may facilitate metastasis by increasing the surface area of lymphatic vessels in contact with interstitial tumor cells in the area around the primary tumor site, and thus increase the chance of these cells entering the lymphatic system.
In regional lymph nodes, tumor cells are thought to reach the peripheral sinus from afferent lymphatics. In fact, many metastatic cells were detected around the sinus area unless they developed into a macronodular lesion. Recently, small lesions have been divided into two categories; micrometastasis and isolated tumor cells (ITC), which are distinguished based on their size [51]. Micrometastasis is defined as a tumor deposit larger than 0.2 mm but less than 2.0 mm, while ITC is defined as a tumor deposit less than 0.2 mm in maximum diameter. Although the biological features of these categories have not been fully clarified, they are now pathologically defined as pN1m1 and pN0, respectively.
In our study, the metastatic lesions did not always express VEGF-C, and such small metastatic foci often lacked the expression of VEGF-C. Especially, ITC identified only with immunohistochemical staining are totally negative for VEGF-C. In addition, in 4 tumors with lymphatic invasion, none of the tumor cells located in the lymphatic vessels in the primary tumor expressed VEGF-C (data not presented). These unexpected results suggest that expression of VEGF-C in tumor cells is not relevant to the transportation to regional nodes once they enter lymphatic vessels.
In contrast, most of the macrometastases or cancer cells invading the medullary area of metastatic nodes highly expressed VEGF-C. This phenomenon was quite interesting, though not fully explained by today's knowledge. This may suggest that proliferation and invasion in the internal area of metastatic nodes may partially require VEGF-C expression in tumor cells. Thus far, there is no definite report on the effects of VEGF-C on tumor cells. The VEGF-receptor 3 (VEGFR-3), a specific ligand of VEGF-C, was expressed only on certain tumor cells [52-54] and not on others. We tried to examine the expression of VEGF-C receptor in these gastric cancers using a polyclonal antibody to VEGFR-3, but could not detect positive staining in any case (data not shown). Thus, it seems to be unlikely that VEGF-C directly affects the behavior of gastric cancer cells. However, it remains a possibility that VEGF-C secreted from tumor cells may act on intranodal lymphatic endothelial cells or other interstitial cells and create favorable conditions for tumor cell growth or invasion in lymph nodes.
In summary, our retrospective study demonstrated that VEGF-C expression in tumor cells in biopsy specimens was significantly correlated with lymphatic metastasis in SGC, although the accuracy was not high enough to be used for clinical indicator. Metastatic tumor cells in micrometastasis or ITC located in marginal sinus often lacked the expression of VEGF-C, whereas macrometastasis located in the medullary area in metastatic nodes highly express VEGF-C. This suggests a possibility that expression of VEGF-C is not essential for lymphatic transport from primary tumor, but rather important to develop the macronodular lesion in metastatic lymph nodes.
Competing Interests
The author(s) declare that they have no competing interests.
Authors' contributions
MI. Conceived of the study and wrote the original version of the manuscript.
JK. Carried out the literature search and helped in drafting the manuscript.
SK. Collected clinical and pathologic data and participated in manuscript preparation
HN Helped to shape the idea for the study coordinated the study and edited the manuscript.
All authors have read and approved the final manuscript.
Table 2 Relationship between the expression of VEGF-C in surgical and biopsy specimens
Surgical specimens Nodal metastasis
positive (35) negative (11) p value positive (15) negative (31) p value
Biopsy specimens
positive (21) 20 1 10 11
negative (25) 15 10 0.005 5 20 0.047
Table 4 The expression of VEGF-C in surgical specimens and nodal metastasis
Positive (35) Negative (11) p value
Lymph node metastasis
positive 14 1
negative 21 10 0.05
Acknowledgement
We thank K. Amitani; pathological technician for excellent technical assistance.
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| 15636643 | PMC545047 | CC BY | 2021-01-04 16:39:05 | no | World J Surg Oncol. 2005 Jan 7; 3:2 | utf-8 | World J Surg Oncol | 2,005 | 10.1186/1477-7819-3-2 | oa_comm |
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RetrovirologyRetrovirology1742-4690BioMed Central London 1742-4690-1-461562034610.1186/1742-4690-1-46ResearchModulating HIV-1 replication by RNA interference directed against human transcription elongation factor SPT5 Ping Yueh-Hsin [email protected] Chia-ying [email protected] Hong [email protected] Jean-Marc [email protected] Mario [email protected] Tariq M [email protected] Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA2 Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA 01605, USA3 Department and Institute of Pharmacology National Yang-Ming University Shih-Pai, Taipei 11221 Taiwan2004 27 12 2004 1 46 46 17 12 2004 27 12 2004 Copyright © 2004 Ping et al; licensee BioMed Central Ltd.2004Ping 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
Several cellular positive and negative elongation factors are involved in regulating RNA polymerase II processivity during transcription elongation in human cells. In recruiting several of these regulatory factors to the 5' long terminal repeat (LTR) promoter during transcription elongation, HIV-1 modulates replication of its genome in a process mediated by the virus-encoded transactivator Tat. One particular cellular regulatory factor, DSIF subunit human SPT5 (hSpt5), has been implicated in both positively and negatively regulating transcriptional elongation but its role in Tat transactivation in vivo and in HIV-1 replication has not been completely elucidated.
Results
To understand the in vivo function of hSpt5 and define its role in Tat transactivation and HIV-1 replication, we used RNA interference (RNAi) to specifically knockdown hSpt5 expression by degrading hSpt5 mRNA. Short-interfering RNA (siRNA) designed to target hSpt5 for RNAi successfully resulted in knockdown of both hSpt5 mRNA and protein levels, and did not significantly affect cell viability. In contrast to hSpt5 knockdown, siRNA-mediated silencing of human mRNA capping enzyme, a functionally important hSpt5-interacting cellular protein, was lethal and showed a significant increase in cell death over the course of the knockdown experiment. In addition, hSpt5 knockdown led to significant decreases in Tat transactivation and inhibited HIV-1 replication, indicating that hSpt5 was required for mediating Tat transactivation and HIV-1 replication.
Conclusions
The findings presented here showed that hSpt5 is a bona fide positive regulator of Tat transactivation and HIV-1 replication in vivo. These results also suggest that hSpt5 function in transcription regulation and mRNA capping is essential for a subset of cellular and viral genes and may not be required for global gene expression.
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Background
The elongation phase of transcription is often a critical juncture for regulating gene expression [1,2] and a number of genes including c-myc, c-fms, hsp70, and those encoded by HIV-1 are regulated at this stage of transcription [3-6]. During transcription elongation, shortly after successful initiation of RNA synthesis, RNA polymerase II (RNA pol II) can pause, arrest, pass through terminator sequences, or terminate transcription. The varying processivity of RNA pol II prior to entering productive elongation is controlled by the action of both negative and positive transcription elongation factors (N-TEFs and P-TEFs, respectively). The function of P-TEFs is to reduce the barrier of N-TEFs and promote the release of RNA pol II from the transition state that can cause termination of transcription [7]. Three elongation regulatory factors, P-TEFb (positive transcription elongation factor b), DSIF (DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole) sensitivity-inducing factor) and NELF (negative elongation factor), have been identified using DRB as a transcription inhibitor [8-10] and function together to regulate transcription elongation.
Modulation of HIV-1 gene expression provides one fundamental example of how transcription elongation can be controlled by such regulatory factors [11-14]. Tat, an HIV-1 regulatory protein, is required for synthesis of viral mRNA and increases the efficiency of transcription elongation from the HIV-1 promoter. In the presence of Tat, the processivity of RNA Pol II complexes that initiate transcription in the HIV-1 5' long terminal repeat (5' LTR) region becomes greatly enhanced. For this increased processivity to occur, Tat binds with a nascent leader RNA element, trans-activation responsive (TAR) RNA, located at the 5' end of all HIV-1 transcripts [15]. Cellular factors in association with Tat and TAR are then recruited to the 5' LTR, stimulating RNA pol II processivity during elongation. More specifically, the C-terminal domain (CTD) of RNA pol II is proposed to be hyperphosphorylated by P-TEFb during Tat transactivation to promote elongation [12-14]. Composed of cyclin-dependent kinase CDK9 and Cyclin T1, P-TEFb has been shown to bind the activation domain of Tat and TAR RNA loop sequence and phosphorylate the CTD of RNA pol II [16-18]. Tat transactivation is postulated to involve Tat-TAR interactions that then give rise to the recruitment of P-TEFb to RNA pol II complexes at the 5' LTR. This recruitment is necessary to enhance the processivity of RNA Pol II from the HIV-1 5' LTR promoter [7,14,17,19]. Thus, TAR RNA provides a scaffold for Tat and P-TEFb to bind and assemble a regulatory switch during HIV replication [20].
Human DSIF consists of subunits hSpt5 and hSPT4 and was originally discovered as a negative elongation factor that binds to RNA pol II [9]. In conjunction with NELF, DSIF represses transcriptional elongation at positions proximal to promoters [9,10]. Escape from transcriptional repression imposed by DSIF and NELF requires P-TEFb, which has been shown in vitro to phosphorylate both hSpt5 and CTD [7,10,21-29]. Interestingly, hSpt5 is conserved among eukaryotes and is a dual transcriptional regulator that can function as both a negative and positive elongation factor [30-32]. Currently, it is postulated that phosphorylation of hSpt5 and RNA pol II by P-TEFb is the key event during which hSpt5 functionally switches from a negative barrier to a positive elongation factor during transcription in human cells. Methylation of SPT5 also has been shown to regulate its interaction with RNA pol II and this posttranslational modification of SPT5 may alter transcriptional elongation functions in response to viral and cellular factors [33].
Although hSpt5's role in transcription regulation in association with P-TEFb has been established, its involvement in Tat transactivation and HIV-1 replication continues to be elucidated. Several in vitro studies have shown that hSpt5 is required for Tat transactivation and that both hSpt5 and RNA pol II phosphorylation is stimulated after recruitment of P-TEFb by Tat [25,29,34]. hSpt5 may also play a positive role in Tat transactivation through its association with human mRNA capping enzyme (HCE), which is a bifunctional triphosphatase-guanylyltransferase required for capping mRNA (reviewed in [1,35]), since SPT5, Tat, and CTD associate with the capping apparatus to stimulate capping [36-43]. However, studies in a recent report have suggested that only P-TEFb hyperphosphorylation of the RNA pol II CTD is directly required for Tat transactivation, precluding a direct role for hSpt5 in RNA pol II processivity during HIV-1 replication [26]. Therefore, hSpt5 role in Tat transactivation and HIV-1 replication in vivo remains unclear.
Here, we used RNA interference (RNAi) to address whether hSpt5 is required for Tat transactivation and thus HIV-1 replication in vivo and further defined hSpt5 cellular functions. RNAi is a remarkably efficient process whereby double-stranded RNA (dsRNA) induces sequence-specific degradation of homologous mRNA in animal and plant cells (reviewed in ref. [44]). In mammalian cells, RNAi can be triggered by 21-nucleotide (nt) small interfering RNA (siRNA) duplexes and a few dsRNA molecules are sufficient to inactivate a continuously transcribed target mRNA for an observable period of time [45,46]. RNAi has recently been used to successfully knockdown the expression of a number of HIV genes, including p24, reverse transcriptase, vif, nef, tat, and rev, and has led to pre- and post-integrative HIV-1 RNA degradation and reduced HIV infectivity [47-52]. These results suggested that targeting viral factors required for the HIV life cycle with siRNAs including those required for HIV replication is a viable method for treating HIV infections. Other groups have targeted cellular factors implicated in supporting the HIV life cycle, including T-cell co-receptors CD4, CXCR4, CCR5, and CD8 [50,52-54] and transcription factor NF-κB [51], which has a role in HIV transcription initiation. Knockdown of the co-receptors reduced HIV infectivity, effectively blocking HIV entry into cells [55]. RNAi has become one of the leading methodologies for studying gene knockdown in human cells. During RNAi, a double-stranded 21-nucleotide (nt) short-interfering RNA (siRNA) targets a specific, complementary mRNA for degradation, resulting in significantly decreased expression, or knockdown, of the targeted gene (reviewed in [56,57]). In this report, siRNA designed to target hSpt5 successfully silenced hSpt5 as observed by decreased hSpt5 mRNA and protein expression. In addition, RNAi directed against hSpt5 did not significantly affect cell viability. hSpt5 knockdown led to significant decreases in Tat transactivation and inhibited HIV replication, indicating that hSpt5 was required for Tat transactivation and HIV replication in vivo. Taken together, silencing of hSpt5 by RNAi firmly established that the regulation of HIV-1 gene expression requires both Tat-TAR-P-TEFb interactions and interactions between RNA pol II transcription complexes and hSpt5.
Results
Specific silencing of hSpt5 expression by siRNA in HeLa cells
To inhibit hSpt5 expression in a cultured human cell line using RNAi, siRNA targeting an hSpt5 sequence from position 407 to 427 relative to the start codon was designed (Figure 1A). Magi cells were transfected with this hSpt5 duplex siRNA using Lipofectamine (Invitrogen). To evaluate the effects of hSpt5 RNAi, total cell lysates were prepared from siRNA-treated cells harvested at various time points after transfection. hSpt5 mRNA or protein levels were analyzed by RT-PCR or western blot using anti-hSpt5 antibodies, respectively. Cells transfected with hSpt5 siRNA had significantly lowered hSpt5 mRNA (Figure 1B, lane 3) and protein expression (Figure 1C, lane 3), indicating that siRNA-mediated silencing of hSpt5 had occurred successfully. hSpt5 knockdown was consistently between ~85–90%. This knockdown effect was dependent on the presence of the 21-nt siRNA duplex harboring a sequence complementary to the mRNA target. As shown in Figures 1B and 1C, mock-treated (no siRNA) (lane 1), single-stranded antisense hSpt5 siRNA (lane 2), or mismatched hSpt5 duplex siRNA (lane 4) containing two nucleotide mismatches between the target mRNA and siRNA antisense strand at the putative cleavage site of the target mRNA (Figure 1A) did not affect hSpt5 mRNA or protein levels. These results showed that hSpt5 knockdown was specific to duplex siRNA exactly complementary to the hSpt5 mRNA target. In evaluating either mRNA or protein levels, human Cyclin T1 (hCycT1) was used as an internal control, showing that the effects of hSpt5 siRNA were specific to hSpt5 and did not effect hCycT1 mRNA or protein expression (Figure 1B and 1C, lower panel). Taken together, these results demonstrated that hSpt5 knockdown was sequence specific and led to significantly decreased hSpt5 mRNA and protein levels.
Figure 1 Specific silencing of hSpt5 expression by RNAi. (A) hSpt5 mRNA is 3261 nucleotides in length. siRNA targeting sequence for hSpt5 was selected from position 407 to 427 relative to the start codon. As a specific control, mutant siRNA containing 2 nucleotide mismatches (underline) between the target mRNA and the antisense of siRNA at the hypothetical cleavage sites of the mRNA was generated. (B) Evaluation of specific hSpt5 siRNA activity by RT-PCR. Total cellular mRNA was prepared from HeLa cells transfected without siRNA or with hSpt5 duplex or control siRNAs and was followed by RT-PCR, as described in Material and Methods. Each RT-PCR reaction included 100 ng total cellular mRNA, gene-specific primer sets for hSpt5 and hCycT1 amplification (0.5 μM for each primer), 200 μM dNTP, 1.2 mM MgSO4 and 1U of RT/platinum Taq mix. Primer sets for hSpt5 produced 2.6 kb products while hCycT1 produced 1.8 kb products. RT-PCR products were resolved on a 1% agarose gel and viewed by ethidium bromide staining. RT-PCR products are shown from cells that were not transfected with siRNA (lane 1), or cells transfected with single-stranded antisense hSpt5 siRNA (hSpt5 (AS), lane 2), hSpt5 duplex siRNA (hSpt5 (DS), lane 3), or mismatch hSpt5 duplex siRNA (hSpt5-mm (DS), lane 4). Lane M is a marker lane. (C) Analysis of specific hSpt5 siRNA activity by western blotting. Cell lysates were prepared from HeLa cells mock-transfected without siRNA (lane 1), or transfected with single-stranded antisense hSpt5 siRNA (hSpt5 (AS), lane 2), hSpt5 duplex siRNA (hSpt5 (DS), lane 3), or mismatch hSpt5 duplex siRNA (hSpt5-mm (DS), lane 4). Cell lysates were analyzed by 10% SDS-PAGE. Protein contents were detected by immunoblotting assay with antibodies against hSpt5 (top panel) and hCycT1 (lower panel).
Kinetics of hSpt5 knockdown by RNAi
Having established that hSpt5 could be knocked down using RNAi, the kinetics of hSpt5 knockdown were examined. To perform kinetic experiments, hSpt5 duplex siRNA, single-stranded antisense hSpt5 siRNA, or mismatch duplex hSpt5 siRNA were transfected into Magi cells. Cell lysates were collected at various time points to assay for protein levels during hSpt5 knockdown. Immunoblot analysis using anti-hSpt5 antibodies revealed the timing of gene suppression and persistence of hSpt5 RNAi effects in Magi cells during the time course experiment (Figure 2). hSpt5 knockdown was first observed between 30–42 h post-transfection, with maximum knockdown (~85–90% knockdown) occurring at 42–66 h post transfection (Figure 2, lane 8–14). Protein levels gradually recovered to normal levels between 66–90 h (data not shown), indicating that the effects of hSpt5 siRNA did not last indefinitely. Neither single-stranded antisense siRNA (Figure 2, lanes 1–7) nor mismatched duplex siRNA (Figure 2, lanes 15–21) affected hSpt5 protein levels throughout the duration of the time course. These results indicated that hSpt5 knockdown by RNAi occurred after 30 h and these knockdown effects were specific to duplex siRNA targeting hSpt5.
Figure 2 Kinetics of specific hSpt5 siRNA activity by Western blotting. HeLa cells were transfected with single-stranded antisense hSpt5 siRNA (hSpt5 (AS), lanes 1–7), hSpt5 duplex siRNA (hSpt5 (DS), lanes 8–14), or mismatch hSpt5 duplex siRNA (hSpt5-mm (DS), lanes 15–21) having 2 nucleotide mismatches between the target mRNA and the antisense strand of siRNA at the hypothetical cleavage site of the mRNA. Cells were harvested at various times post transfection. Protein content was resolved on 10% SDS-PAGE, transferred onto PVDF membranes, and immunoblotted with antibodies against hSpt5 (top bands) and hCycT1 as an internal control (lower bands).
Knockdown of hSpt5 is not lethal to human cells
Knowing that the kinetics of hSpt5 peaked at 42–54 h post-transfection, we were able to evaluate the viability of cells during hSpt5 knockdown experiments over varied time intervals. Cell viability was assessed using trypan blue exclusion at various times after a single transfection of various siRNAs. As shown in Figure 3, during the 66 h time course experiment, the number of non-viable hSpt5 knockdown cells (yellow line) observed was comparable to mock-treated cells (no siRNA; dark blue line). Cells transfected with single-stranded antisense hSpt5 siRNA (purple line) or mismatched hSpt5 duplex siRNA (light blue line) that did not show hSpt5 knockdown also showed minimal changes in cell viability.
Figure 3 Analysis of cell viability by counting trypan blue-stained cells. HeLa cells were transfected with Lipofectamine with various siRNAs or no siRNA. Three siRNA duplexes, including hSpt5 siRNA (yellow), mismatch hSpt5 siRNA (light blue) and siRNA targeting human capping enzyme (HCE, red), were used in these experiments. Controls for viability included cells mock-transfected with no siRNA (dark blue) or cells transfected with single-stranded antisense hSpt5 siRNA (purple). At various times after transfection, cells floating in the medium were collected and counted in the presence of 0.2% trypan blue. Cells that took up dye (stained blue) were counted as not viable.
hSpt5 has been shown to interact with the human mRNA capping enzyme (HCE) and this interaction enhances capping enzyme guanylylation and mRNA capping several fold [40]. Since Spt5, Tat, and CTD associate with the capping apparatus to stimulate capping [36,37,39-42], we planned to separately define the role of HCE and hSpt5 in Tat transactivation by using RNAi to specifically knockdown HCE expression. HCE knockdown was confirmed by RT-PCR (data not shown). In contrast to hSpt5 knockdown cells, HCE knockdown cells showed a significant increase in cell death (Figure 3, red line) over the course of the knockdown experiment. These results indicated that HCE is an essential enzyme for cell viability and growth. Simialr findings showing that RNA capping was essential for metazoan viability have also been previously reported using RNAi in C. elegans [58]. These results indicated that hSpt5 knockdown was not lethal to human cells, while a much more stringent requirement for HCE expression was essential for cell viability.
Role of hSpt5 on HIV-1 Tat Transactivation
To examine whether hSpt5 was required for HIV-1 Tat transactivation in vivo, Tat transactivation during hSpt5 knockdown in Magi cells was monitored. Magi cells are a HeLa cell line harboring a stably integrated single copy of the HIV-1 5' LTR-β-galactosidase gene. These cells are also genetically programmed to express the CD4 receptor for HIV-1 infection ([59]; see below). In this experiment, Magi cells were co-transfected with Tat expression plasmid pTat-RFP and hSpt5 duplex siRNA. Co-transfection with Tat siRNA was used as a positive control for inhibition of Tat transactivation while single-stranded antisense hSpt5 siRNA and mismatched siRNA were used as negative controls. Tat transactivation and protein levels were evaluated by harvesting cells 48 h post transfection, which was within the timeframe that hSpt5 knockdown peaked. Expression of HIV-1 Tat-RFP under the control of the CMV early promoter was confirmed by western blot using anti-RFP antibody and by measuring RFP fluorescence using a fluorescence spectrophotometer (data not shown). In addition, immunoblot analysis confirmed that hSpt5 siRNA specifically inhibited hSpt5 protein expression in the absence and presence of HIV-1 Tat protein in Magi cells (data not shown).
Tat-RFP enhances the expression of genes that are under the control of the HIV-1 5' LTR promoter. In this experiment, Tat transactivation was measured by assaying the β-galactosidase activity resulting from expression of the β-galactosidase gene under the HIV-1 5' LTR promoter. To quantify the effects of various siRNAs on HIV-1 Tat transactivation, the ratio between β-galactosidase activity in cells transfected with pTat-RFP (with or without siRNAs) and mock-treated cells not transfected with pTat-RFP was determined. The results of this quantitation are shown in Figure 4. In Magi cells, Tat-RFP strongly stimulates the expression of β-galactosidase, represented by a 13-fold increase in Tat transactivation (Figure 4, lane 1). On the other hand, Tat transactivation was strongly inhibited in cells transfected with Tat siRNA (~90% knockdown; Figure 4, lane 5), as previously shown [51]. Tat transactivation was similarly inhibited when cells were transfected with hSpt5 duplex siRNA, exhibiting only ~30% of the Tat transactivation observed with Tat-RFP alone (Figure 4, lane 3). Neither antisense hSpt5 siRNA nor mismatched hSpt5 siRNA (Figure 4, lane 4) showed any effect on Tat transactivation. These results indicated hSpt5 knockdown caused by siRNA specifically targeting hSpt5 mRNA inhibited HIV-1 Tat transactivation in human cells. These results strongly supported an important role for hSpt5 in Tat transactivation in vivo and suggested that RNAi of hSpt5 had the potential to inhibit HIV-1 replication.
Figure 4 Effect of hSpt5 siRNA on HIV-1 Tat transactivation in Magi cells. Quantified effect of siRNA on HIV-1 Tat transactivation was determined by measuring β-galactosidase activity. Magi cells were co-transfected with pTat-RFP plasmid and various siRNAs targeting hSpt5 or Tat and harvested at 48 h post-transfection. Activity of β-galactosidase was measured using the β-Galactosidase Enzyme Assay System (Promega). Tat transactivation was determined by the ratio of β-galactosidase activity in pTat-RFP transfected cells to activity measured in cells without pTat-RFP. The inhibitory effect of siRNA was determined by normalizing Tat transactivation activity to the amount of Tat-RFP protein. Tat transactivation was measured for Magi cells transfected with pTat-RFP only (lane 1), or Tat-RFP transfected with single-stranded antisense hSpt5 siRNA (hSpt5-AS, lane 2), hSpt5 duplex siRNA (hSpt5-DS, lane 3), mismatch hSpt5 duplex siRNA (hSpt5-mm-DS, lane 4), or Tat siRNA duplex (Tat-DS, lane 5). Results are representative of three independent experiments.
hSpt5 knockdown inhibits HIV-1 replication
To evaluate the effect of hSpt5 knockdown on HIV-1 replication, a double siRNA transfection protocol was used to maximize the knockdown efficiency of hSpt5 during HIV-1 infection. Magi cells were transfected with siRNA directed against hSpt5. Cells mock transfected without siRNA, or transfected with single-stranded antisense hSpt5 siRNA or mismatch hSpt5 siRNA were used as negative controls. Transfection with Vif or Nef siRNAs was used as a positive control [20]. 24 h after the first transfection, a second siRNA transfection identical to the first was performed. 24 h later, doubly transfected cells were infected with various concentrations of HIVNL-GFP, an infectious molecular clone of HIV-1. Knockdown of hSpt5 protein levels was then evaluated 48 h post infection in doubly transfected cells. An even larger decrease in hSpt5 protein levels was observed in doubly transfected cells (~95% knockdown) as compared to singly transfected cells (~85–90% knockdown; Supplementary Figure 1, compare lanes 4 and 10), suggesting that more robust knockdown of gene expression can be achieved using this double transfection method.
HIV-1 Tat-mediated transactivation of the 5' LTR occurring in cells infected with virus led to β-galactosidase production, which was also quantified 48 h post-infection. In this single-cycle replication assay for evaluating HIV-1 replication, β-gal activity reflected the activity of reverse transcriptase and viral replication of varying amounts of viral inoculum. Therefore, changes in β-gal activity could be directly correlated to changes in the efficacy of HIV-1 replication. The positive siRNA control targeting HIV-1 Vif showed decreased levels of β-gal activity and HIV-1 replication, as shown previously (Figure 5; [47]). Double-stranded siRNA directed against hSpt5 showed a similar decrease in β-gal activity when compared with Vif knockdown. This observed decrease was equivalent to the β-gal activity measured when using 32 times less viral inoculum with mock-treated cells (Figure 5), indicating that hSpt5 knockdown had significantly reduced HIV-1 replication. p24 levels were also monitored during these experiments and decreased in the context of hSpt5 knockdown (data not shown), supporting the conclusion that hSpt5 knockdown has a negative effect on the HIV-1 life cycle. Control experiments using hSpt5 single-stranded antisense or mismatched duplex siRNA duplexes showed no antiviral activities. In addition, no significant toxicity or cell death was observed during these experiments, suggesting that hSpt5 knockdown was not lethal even in the context of HIV-1 infection. These results demonstrated that hSpt5 silencing using RNAi modulated HIV-1 replication and firmly established an important role for hSpt5 in Tat transactivation and HIV-1 replication in vivo.
Figure 5 siRNA targeting hSpt5 modulate HIV-1 replication. HeLa-CD4-LTR/β-galactosidase (Magi) cells were mock-transfected (mock), or transfected with single-stranded antisense hSpt5 siRNA (AS), hSpt5 duplex siRNA (siRNA), mismatched hSpt5 duplex siRNA (MM) or Vif duplex siRNA (T98). 24 h after the first transfection, a second siRNA transfection was performed. 24 h later, cells were infected with HIVNL-GFP, an infectious molecular clone of HIV-1. Cells infected with virus and not treated with oligofectamine are shown (mock). HIV-1 Tat-mediated transactivation of the 5' LTR led to β-galactosidase production, which was quantified 48 h post-infection. Cells treated with duplex siRNA targeting Vif (lanes marked T98 [47]) served as a positive control. Serial double dilutions of the viral inoculum (in cpm of RT activity) are consistent with 32-fold decreases in viral replication.
Discussion
hSpt5, as part of the DSIF complex, was originally discovered as a negative elongation factor required for conferring DRB sensitivity to transcription elongation complexes thereby inhibiting transcription [9]. This negative barrier provided by hSpt5 was thought to be relieved through P-TEFb phosphorylation of both hSpt5 and RNA pol II CTD, which results in increased processivity of RNA pol II complexes [7,10,21-29]. Increased processivity has also been linked to the phosphorylated form of hSpt5 conferring a positive effect on transcription elongation [25,29,34]. Recently, however, it has been shown that Tat is able to enhance transcription elongation in vitro in the absence of hSpt5 [26]. These results appeared to indicate that P-TEFb phosphorylation of RNA pol II was the sole event that directly led to Tat transactivation and increased RNA pol II processivity [26]. Thus, from the results of all of these in vitro studies collectively, the requirement for hSpt5 in positively regulating transcription elongation during Tat transactivation has remained unclear.
Here, we studied the role of hSpt5 in vivo using RNAi and established that hSpt5 played a positive role in Tat transactivation and HIV-1 replication. Knockdown of hSpt5 provided insight into several functional aspects of the hSpt5 protein. First, knockdown of hSpt5 was not lethal in Magi cells, indicating that hSpt5 was not required for cell viability. This was an interesting result because studies of SPT5 mutants in yeast and zebrafish and RNAi of SPT5 in C. elegans have shown that SPT5 was essential for growth and/or embryonic development in those organisms [30,31,60]. It seems likely that hSpt5 holds similar essential functions in human cells during embryonic development but may not be absolutely required in adult cells. Alternatively, hSpt5 knockdown may have led to decreased levels of expression that were still sufficient for hSpt5 to carry out its essential functions. Our results support the notion of using RNAi against hSpt5 as a potential therapeutic strategy for fighting HIV-1 infection since there is the potential that HIV-1 functions could be targeted for inhibition without significantly interfering with host cell functions.
The key finding of this study was that hSpt5 knockdown significantly inhibited both Tat transactivation and HIV-1 replication. These results indicated that hSpt5 was a bona fide regulator of Tat transactivation that is required for HIV-1 replication in vivo. Our in vivo results strongly support previous in vitro results recapitulating Tat transactivation that showed immunodepletion of hSpt5 significantly inhibited Tat transactivation [29,34]. However, it is difficult to reconcile our in vivo results with recently published in vitro experiments showing that P-TEFb hyperphosphorylation of the CTD in the absence of hSpt5 still enhanced RNA pol II processivity during Tat transactivation [26]. In reconciling whether P-TEFb hyperphosphorylation was directly required for Tat transactivation to the exclusion of hSpt5, we would like to propose that the required function of P-TEFb hyperphosphorylation may be distinct from the role hSpt5 plays in enhancing RNA pol II processivity during Tat transactivation. In our model (Figure 6), P-TEFb hyperphosphorylation would occur first, triggering enhanced processivity of RNA pol II. hSpt5 presumably is phosphorylated at around the same time as RNA pol II, stimulating hSpt5 to switch from a negative regulator to a positive elongation factor [25]. Phosphorylated hSpt5 may then be important for positively regulating an initial step in Tat transactivation.
Figure 6 Model for Tat transactivation in absence or presence of SPT5. See text for details.
Conceivably, hSpt5 functions in transcription elongation as a stabilization factor that enhances the stability of RNA pol II elongation complexes formed after P-TEFb hyperphosphorylation of the CTD. This type of role would also support hSpt5 function as an antiterminator factor as described previously [61]. Another important positive function for hSpt5 during Tat transactivation may involve hSpt5 and Tat interactions with the capping machinery [40-42]. Phosphorylation of hSpt5 by P-TEFb may stabilize hSpt5 interactions with HCE thereby stabilizing Tat and CTD interactions with the capping machinery to promote capping and successful production of stable HIV transcripts (see model in Figure 6). Due to the highly structured nature of TAR, capping of the 5' end of HIV transcripts is not very efficient in the absence of Tat [41,42] and Tat stimulated capping may require the presence of hSpt5 for greater access to the 5' end or to stabilize and kinetically arrest the elongation complex. Capping of HIV transcripts has also been shown to occur more proficiently when elongation is paused and not continuous [42], suggesting that DSIF/NELF-dependent pausing of early stage elongation complexes is representative of an elongation checkpoint. One function of this checkpoint may be to allow time for the recruitment of capping machinery and subsequent capping of HIV RNA to stabilize nascent transcripts prior to further elongation. In the absence of hSpt5, pausing may no longer occur during elongation since neither NELF nor hSPT4 binds to RNA pol II without hSpt5 [62,63]. Thus, the window for the capping apparatus to be recruited by Tat and/or stimulate capping may be severely shortened or lost altogether without hSpt5. Any resulting uncapped HIV transcripts would be prone to degradation, accounting for the lower level of Tat transactivation and HIV replication observed during hSpt5 knockdown. The potential roles for phosphorylated hSpt5 in stabilizing RNA pol II processive elongation complexes or with respect to capping during Tat transactivation are not mutually exclusive as shown in Figure 6. hSpt5 may indeed have multi-functional roles as a positive regulator during HIV-1 replication.
Conclusions
The in vitro and in vivo approaches taken to address the importance of hSpt5 function all shed light on the multi-faceted nature of Tat transactivation. Accordingly, these studies altogether support important roles for both P-TEFb and hSpt5 in mediating transcription elongation during HIV-1 replication in vivo. The dual function of hSpt5 as a negative and positive transcription elongation factor also demonstrates the complexity associated with transcriptional regulation during transcription elongation and HIV-1 Tat transactivation. It is likely that posttranslational modifications of hSpt5 dictate functions of Spt5 at various promoters. Further studies will be required to elucidate how various modifications of hSpt5 such as phosphorylation and methylation control transcription elongation of both cellular and viral genes.
Methods
siRNA preparation
Twenty-one nucleotide siRNAs were chemically synthesized as 2' bis(acetoxyethoxy)-methyl ether-protected oligos (Dharmacon, Lafayette, CO). Synthetic RNAs were deprotected, annealed and purified using standard protocols provided by the manufacturer. Formation of duplex RNA was confirmed by 20% non-denaturing polyacrylamide gel electrophoresis (PAGE). Sequences of siRNA duplexes were designed as described previously [46] and subjected to a BLAST search against the NCBI EST library to ensure that only the desired genes were targeted.
Culture and transfection of cells
Magi (multinucleate activation of galactosidase indicator) cells carrying the endogenous HIV-5'LTR β-galactosidase gene were maintained at 37°C in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS), 0.2 mg/ml of Geneticin (G418) and 0.1 mg/ml Hygromycin B (Roche Molecular Biochemicals). Cells were passaged at sub-confluence and plated at 70% confluency for transfection. Reporter plasmids and siRNA were co-transfected into Magi cells using Lipofectamine (Invitrogen) in duplicate 6-well plates (Falcon). A transfection mixture containing 25–150 nM siRNA and 9 μl of Lipofectamine in 1 ml of serum-reduced OPTI-MEM (Invitrogen) was added to each well. For high efficiency knockdown experiments, 150 nM siRNA was used. After incubating at 37°C for 6 h, cells were cultured in antibiotic-free DMEM. For further analysis, transfected cells were harvested at various time intervals, washed twice with phosphate buffered saline (PBS, Invitrogen), flash frozen in liquid nitrogen, and stored at -80°C.
RT-PCR for amplification of hSpt5 and hCycT1 mRNA
Total cellular mRNA was prepared from HeLa cells transfected without siRNA or with hSpt5 or control siRNAs using a Qiagen RNA mini kit, followed by an oligotex mRNA mini kit (Qiagen). RT-PCR was performed using a SuperScript One-Step RT-PCR kit with platinum Taq (Invitrogen) and 40 cycles of amplification. Each RT-PCR reaction included 100 ng total cellular mRNA, gene-specific primer sets for hSpt5 and hCycT1 amplification (0.5 μM for each primer), 200 μM dNTP, 1.2 mM MgSO4 and 1U of RT/platinum Taq mix. Primer sets for hSpt5 produced 2.6 kb products while hCycT1 produced 1.8 kb products. RT-PCR products were resolved on a 1% agarose gel and viewed by ethidium bromide staining. Forward and reverse primer sequence for amplifying SPT5 were 5'-ATGTCGGACAGCGAGGACAGC-3' (nts 1–21) and 5'-TGTACATGGCCGGCGTCCC-3' (nts 2638–2656), respectively. Forward and reverse primer sequences for amplifying hCycT1 are 5'-GCAACAAGTTCAAGATCTGGTCAT-3' (nts 381–404) and 5'-CCCGGGGGATCCTTACTTAGGAAGGGGTGGAAGTGG-3' (nts 2158–2200); underlined sequences represent restriction enzyme sites), respectively.
Western blotting
siRNA treated cells were harvested as described above and lysed in 1X reporter lysis buffer (Promega). After centrifugation to remove cellular debris, concentrations of proteins in lysates were determined using a Dc protein assay kit (Bio-Rad). Proteins in 30 μg of total cell lysates were fractionated by 10% SDS-PAGE, transferred onto a polyvinylidene difluoride membrane (PVDF membrane, Bio-Rad), and immunoblotted with antibodies against hSpt5 (Pharmingen) and hCycT1 (Santa Cruz Biotech). Protein content was visualized by a BM chemiluminescence Blotting Kit (Roche Molecular Biochemicals). The blots were exposed to X-ray film (Kodak MR-1) for various times (between 1 s and 5 min).
β-galactosidase enzyme assay
Magi cells were harvested 48 h after transfection with Tat-RFP plasmids in the absence or presence of siRNAs. Cell lysates were prepared and quantified as described above. To perform standard β-galactosidase assays, 120 μg of cell lysates were mixed in 150 μl of 1X reporter lysis buffer and 150 μl of 2X β-galactosidase assay buffer (Promega), and incubated at 37°C for 30 min. To stop the reaction, 500 μl of 1 M sodium carbonate was added to the mixture and mixed well by vortexing briefly. Absorbance of the reaction mixture was read immediately at 420 nm. The amount of Tat-RFP protein was determined using a fluorescence spectrophotometer (Photon Technology International). 300 μg of cell lysates was subjected to the spectrophotometer with slit widths set at 4 nm for both excitation and emission wavelengths as described previously [46,64]. Fluorescence of Tat-RFP in the cell lysate was detected by exciting at 558 nm and recording the emission spectrum from 568 nm to 650 nm; the spectrum peak at 583 nm represents the maximum fluorescence intensity of Tat-RFP. Tat transactivation was determined by calculating the ratio of β-galactosidase activity (absorbance at 420 nm) of the pTat-RFP transfected cells to that of cells without pTat-RFP plasmid treatment. The inhibitory effect of siRNA treatment was determined by normalizing Tat-transactivation activity to the amount of Tat-RFP protein (represented by RFP fluorescence intensity) in the presence and absence of siRNA.
Magi infectivity assay
HeLa-CD4-LTR/β-gal indicator (Magi) cells [59] were plated in 24-well plates (7.5 × 105 cells per well) and transfected with siRNAs as previously described [47]. siRNA (60 pmol) was transfected into cells using oligofectamine (2 μl, Invitrogen) for 3 h in serum-free DMEM (GIBCO). Cells were rinsed twice and top-layered in 500 μl of DMEM-10% FBS. 24 h after the first transfection, a second identical siRNA transfection was performed. 24 h after the second transfection, cells were trypsinized and seeded in 96-well microtiter plates (4 × 104 cells per well), incubated 3 h and infected with HIVNL-GFP, an infectious molecular clone of HIV-1. HIV-1 virions (normalized to RT activity in cpm) were added in doubling dilutions to duplicate wells. 48 h post infection, cells were harvested to quantify β-galactosidase activity and protein levels.
Competing Interests
The author(s) declare that they have no competing interests.
Authors' contribution
Y-HP carried out all the Spt5 silencing experiments, C-yC performed capping enzyme knockdown experiments, HC performed quantitative analysis of the data, J-MJ performed HIV-1 replication assays, and MS analyzed and interpreted HIV inhibition results. TMR conceived the ideas, and participated in the experimental design and in drafting the manuscript.
Acknowledgements
We thank Stewart Shuman, Bryan Cullen, Jon Karn, and B. Matija Peterlin for helpful discussions. We also thank Stewart Shuman for valuable reagents and Tamara J. Richman for editorial assistance, and acknowledge assay support provided by the University of Massachusetts Center for AIDS Research. This work was supported by grants from the NIH to T.M.R. (AI43198) and M.S (AI32890 and RR11489).
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| 15620346 | PMC545048 | CC BY | 2021-01-04 16:36:37 | no | Retrovirology. 2004 Dec 27; 1:46 | utf-8 | Retrovirology | 2,004 | 10.1186/1742-4690-1-46 | oa_comm |
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Mol CancerMolecular Cancer1476-4598BioMed Central London 1476-4598-4-11564414410.1186/1476-4598-4-1ResearchDietary exposure to soy or whey proteins alters colonic global gene expression profiles during rat colon tumorigenesis Xiao Rijin [email protected] Thomas M [email protected] Frank A [email protected] Arkansas Children's Nutrition Center, 1120 Marshall Street, Little Rock, AR, 72202, USA2 Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, 72202, USA2005 11 1 2005 4 1 1 8 9 2004 11 1 2005 Copyright © 2005 Xiao et al; licensee BioMed Central Ltd.2005Xiao 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 previously reported that lifetime consumption of soy proteins or whey proteins reduced the incidence of azoxymethane (AOM)-induced colon tumors in rats. To obtain insights into these effects, global gene expression profiles of colons from rats with lifetime ingestion of casein (CAS, control diet), soy protein isolate (SPI), and whey protein hydrolysate (WPH) diets were determined.
Results
Male Sprague Dawley rats, fed one of the three purified diets, were studied at 40 weeks after AOM injection and when tumors had developed in some animals of each group. Total RNA, purified from non-tumor tissue within the proximal half of each colon, was used to prepare biotinylated probes, which were hybridized to Affymetrix RG_U34A rat microarrays containing probes sets for 8799 rat genes. Microarray data were analyzed using DMT (Affymetrix), SAM (Stanford) and pair-wise comparisons. Differentially expressed genes (SPI and/or WPH vs. CAS) were found. We identified 31 induced and 49 repressed genes in the proximal colons of the SPI-fed group and 44 induced and 119 repressed genes in the proximal colons of the WPH-fed group, relative to CAS. Hierarchical clustering identified the co-induction or co-repression of multiple genes by SPI and WPH. The differential expression of I-FABP (2.92-, 3.97-fold down-regulated in SPI and WPH fed rats; P = 0.023, P = 0.01, respectively), cyclin D1 (1.61-, 2.42-fold down-regulated in SPI and WPH fed rats; P = 0.033, P = 0.001, respectively), and the c-neu proto-oncogene (2.46-, 4.10-fold down-regulated in SPI and WPH fed rats; P < 0.001, P < 0.001, respectively) mRNAs were confirmed by real-time quantitative RT-PCR. SPI and WPH affected colonic neuro-endocrine gene expression: peptide YY (PYY) and glucagon mRNAs were down-regulated in WPH fed rats, whereas somatostatin mRNA and corresponding circulating protein levels, were enhanced by SPI and WPH.
Conclusions
The identification of transcripts co- or differentially-regulated by SPI and WPH diets suggests common as well as unique anti-tumorigenesis mechanisms of action which may involve growth factor, neuroendocrine and immune system genes. SPI and WPH induction of somatostatin, a known anti-proliferative agent for colon cancer cells, would inhibit tumorigenesis.
colon cancersoywheygene expression profilingneuro-endocrinemicroarrayrat
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Background
Colorectal cancer (CRC) is the third most common cancer and the third leading cause of cancer-related mortality in the U.S. [1,2]. Estimated new cases of colon cancer were 79,650 for men and 73,530 for women in 2004 [1]; approximately $6.3 billion is spent in the United States each year on treatment of CRC [2]. Accumulating evidence suggests that diet is an important environmental factor in the etiology of CRC. High consumption of red meats, animal fats, chocolate, alcohol and refined cereals are linked to higher incidence of these cancers in Western societies [3-5], whereas protective effects of fruits, vegetables and whole grains have been suggested [5].
Soy foods and soybean constituents have received considerable attention for their potential role in reducing cancer risk [6,7]. Our laboratories reported the protective effects of lifetime ingestion of soy protein isolate (SPI) on azoxymethane (AOM)-induced colon cancer in rats [8]. Similarly, the effect of whey protein hydrolysate (WPH) in the diet to reduce colon tumor incidence has been reported by us and others [9-11]. Several hypotheses have been proposed to account for soy and whey protein-induced anti-tumorigenesis. For example, soy isoflavones have been proposed to play a key role in soy's anti-cancer functions [12]. Yanagihara et al., among others, reported that genistein inhibits colon cancer cell proliferation and stimulates apoptosis in vitro [13-15]. However, subcutaneous administration of genistein to mice did not confirm these in vitro effects [16]. Holly et al. reported that soy sphingolipids inhibit colonic cell proliferation, and suggested that this may partially account for its anticancer benefits [17]. Other reports indicate that soy diets inhibit tumorigenesis by regulating the synthesis or activities of specific proteins. For example, Rowlands et al. reported that dietary soy and whey proteins down-regulate expression of liver and mammary gland phase I enzymes involved in carcinogen activation [18]. Elevated activities of phase II detoxification enzymes were reported in soy-fed rats [19,20]. Such dietary effects may result in lower tissue concentrations of activated carcinogen. The anticancer properties of whey proteins have been ascribed to their ability to elevate cellular levels of the antioxidant glutathione [21,22]. Moreover, the whey protein, α-lactalbumin, inhibits proliferation of mammary epithelial cells in vitro [11]. The anticancer properties of whey may also relate to its immune system-enhancing actions [23].
Despite extensive research, there is no consensus for anti-cancer mechanism(s) of soy and whey, which will undoubtedly involve multiple interrelated processes, pathways and many components. Many of the same molecular and biochemical changes underlying human colon cancer are observed in the azoxymethane (AOM)-induced rat colon cancer model [24]. Moreover, previous studies suggest a different molecular etiology for tumors of the proximal and distal colon in this model and in human colon [24,25]. Differential dietary effects on proximal vs. distal colon DNA damage were noted [26] and Westernization of the human diet is thought to have favored a shift of tumors from distal to more proximal locations [27]. Thus, region-specific localization of dietary effects on colon tumorigenesis is an important factor to consider in any molecular analysis of CRC. Here, we use Affymetrix high-density oligonucleotide microarrays to determine the expression profiles of non-tumor (i.e., normal) tissue in proximal colons (PC) of rats, subjected to lifetime diets containing casein (CAS, control diet), soy protein isolate (SPI), or whey protein hydrolysate (WPH) and which were administered AOM to induce tumors. We hypothesized that genes whose expression contributes to anti-tumorigenesis would be regulated in parallel by SPI and WPH; in addition, changes unique to each diet might also be apparent.
Results
Validation of the microarray approach
Quality control steps ensured that the RNA used for microarray and real-time RT-PCR analysis was of high quality. These steps included evaluation of the RNA with the RNA 6000 Nano Assay and assessment of the cRNA hybridization to GeneChips by comparison of data obtained for probe sets representative of 5' and 3' ends of control genes. All RNA samples had an A260/280 absorbance ratio between 1.9 and 2.1. The ratio of 28S to 18S rRNA was very close to 2 on RNA electropherograms, and signal ratios below 3 were noted for 3' vs. 5' probe sets for β-actin and glyceraldehyde-3-phosphate dehydrogenase (per Affymetrix user guidelines) after hybridization.
Total false change rates (TFC) were determined following Affymetrix-recommended guidelines [28], except that the inter-chip comparisons used cRNA targets made in parallel starting from the same RNA pool. Inter-chip variability, measured as TFC%, was 0.25% – 0.6% and well below the suggested 2% cutoff (Table 1). These values confirmed the fidelity and reproducibility of the microarray procedures used. Unsupervised nearest-neighbor hierarchical clustering identified differences in proximal colon gene expression profiles of CAS, SPI and WPH groups (Figs. 1 and 2), indicating that the type of dietary protein has a major effect on gene expression in normal proximal colon tissue of AOM-treated rats. Interestingly, the overall gene expression profiles for SPI and WPH groups were more similar to each other than each was to the CAS group (Fig. 1A).
Table 1 Inter-chip variability
Diet group Number of arrays TFC (%)*
CAS 3 0.252 ± 0.138
WPH 3 0.369 ± 0.025
SPI 3 0.570 ± 0.165
*TFC (Total false change) = false change rate (decreased category) + false change rate (increased category), as described in ref. 28; TFC reported as mean ± SEM, TFC should be no more than 2% (Affymetrix).
Figure 1 Hierarchical clustering of proximal colon gene expression profiles. A. Clustering of nine PC global gene expression profiles (8799 genes); n = 3 profiles each for CAS, SPI and WPH. Each cell represents the expression level of an individual gene in each sample (green = low expression, black = middle expression, red = high expression). The dendrogram reflects the extent of relatedness of different profiles; the shorter branch-point of the SPI and WPH trees indicates the greater similarity between these profiles. B. Clustering of 18 global comparative expression profiles including 9 of SPI vs. CAS and 9 of WPH vs. CAS profiles. Each row in the heat map represents the relative expression level of a given gene across all comparisons (red = up regulated, black = unchanged, green = down regulated).
Figure 2 Hierarchical clustering of 211 differentially expressed genes in either SPI or WPH. The differential expression data are taken only from the pairwise comparison analysis, with CAS profiles used as baseline. Each cell in the heat map represents the relative expression level of a given gene in an individual comparison analysis (red = up regulated, black = unchanged, green = down regulated). The dendrogram reflects the relatedness of different profiles.
Differentially expressed genes
Multiple filtering criteria were applied to the microarray data set so as to identify differentially expressed colon transcripts in rats fed SPI, WPH or CAS; results are reported only for transcripts that passed all three analytical filters used: DMT t-test, SAM and pair-wise comparison survival methods. Among the 8799 genes and ESTs examined with the rat U34A array, we identified 31 induced and 49 repressed genes in proximal colons of SPI-fed rats, whereas 44 induced and 119 repressed genes were detected in WPH-fed rats (Tables 2, 3, 4, 5). Interestingly, more down- than up-regulated genes were noted for both SPI and WPH. Additionally, 37 genes were co-repressed, whereas only two were co-induced by SPI and WPH (Table 6). More than 90% of identified genes in WPH and SPI animals showed the same direction of change relative to CAS. This is visually apparent in the hierarchical clustering output (Fig. 2).
Table 2 Down-regulated genes in rats fed with WPH diet*
Category and Gene Name Probe Set GB Accession No. Fold Change P value
Cell adhesion
Embigin AJ009698 -6.57 0
Cadherin 17 L46874 -4.8 0.036
Cadherin X78997 -3.36 0.004
Protein tyrosine phosphatase M60103 -2.64 0.004
Cytokeratin-8 S76054 -2.71 0
Trans-Golgi network integral membrane protein TGN38 X53565 -4.92 0.012
Tumor-associated calcium signal transducer 1 AJ001044 -9.37 0.001
Claudin-3 AJ011656 -7.55 0.02
Claudin-9 AJ011811 -5.12 0
Cell cycle/growth control
Mapk6 M64301 -2.61 0.003
Epithelial membrane protein 1 Z54212 -4.67 0.015
Glucagon K02813 -7.73 0.005
Peptide tyrosine-tyrosine (YY) M17523 -4.56 0.001
Src related tyrosine kinase U09583 -3.31 0.033
FGF receptor activating protein U57715 -4.25 0.002
Cyclin D1 D14014 -1.97 0.001
Neu oncogene X03362 -2.61 0.017
Defense/immunity protein
Seminal vesicle secretion protein iv J00791 -5.35 0.001
Putative cell surface antigen U89744 -5.22 0.008
Decay accelerating factor GPI AF039583 -6.12 0
Beta defensin-1 AF093536 -26.78 0.001
Detoxification/antioxidation
Glutathione S-transferase J02810 -5.17 0
Glutathione S-transferase Yb X04229 -9.33 0
J03914 -2.43 0.002
Glutathione S-transferase, alpha 1 K01932 -3.07 0.002
Glutathione transferase, subunit 8 X62660 -6.42 0.001
Glutathione S-transferase Yc1 S72505 -3.69 0.004
Glutathione S-transferase Yc2 S72506 -21.38 0.008
N-acetyltransferase 1 U01348 -4.64 0.003
Cytochrome P450CMF1b J02869 -8.23 0.001
Cytochrome P450 4F4 U39206 -6.43 0.004
Cytochrome P450 monooxygenase U39943 -2.82 0.011
Cytochrome P450 pseudogene U40004 -2.87 0
Cytochrome P450 3A9 U46118 -6.91 0
Cytochrome P450IVF M94548 -5.78 0.002
Cytochrome P450, subfamily 51 U17697 -2.07 0.005
Alcohol dehydrogenase M15327 -2.06 0.025
Aldehyde dehydrogenase M23995 -10.56 0.035
AF001898 -2.72 0.004
D-amino-acid oxidase AB003400 -13.69 0
3-methylcholanthrene-inducible UDP-glucuronosyltransferase S56937 -9 0
UDP-glucuronosyltransferase D38062 -3.17 0.005
D38065 -3.29 0.002
UDP glycosyltransferase 1 D83796 -6.87 0
J02612 -6.58 0
J05132 -4.03 0
Metabolism
Meprin 1 alpha S43408 -3.82 0.014
Brain serine protease bsp1 AJ005641 -4.42 0.007
Cystathionine gamma-lyase D17370 -3.05 0.002
Cathepsin S L03201 -2.62 0
Meprin beta-subunit M88601 -5 0.004
Disintegrin and metalloprotease domain 7 X66140 -11.91 0
Fucosyltransferase 1 AB006137 -4.96 0.001
Fucosyltransferase 2 AB006138 -7.97 0.017
UDP-glucose:ceramide glycosyltransferase AF047707 -2.86 0.007
Type II Hexokinase D26393 -2.7 0.001
Hexokinase II S56464 -4.45 0.007
CDP-diacylglycerol synthase AB009999 -4.66 0
Carboxylesterase precursor AB010635 -5.29 0.002
Fatty acid Coenzyme A ligase AB012933 -2.5 0.041
3beta-HSD L17138 -3.27 0
11-beta-hydroxylsteroid dehydrogenase type 2 U22424 -3 0.001
Peroxiredoxin 6 AF014009 -3.55 0.01
Platelet phospholipase A2 X51529 -3.25 0.001
Ligand binding/carrier
Carnitine transporter AB017260 -3.95 0.005
Chloride channel (ClC-2) AF005720 -5.69 0.002
Putative potassium channel AF022819 -4.84 0
Mitochondrial dicarboxylate carrier AJ223355 -3.55 0.009
Aquaporin 3 D17695 -7.83 0
Na_H_Exchanger L11236 -9.81 0.003
Angiotensin/vasopressin receptor (AII/AVP) M85183 -3.3 0.002
H+, K+-ATPase M90398 -13.87 0
Intestinal fatty acid binding protein K01180 -7.29 0.001
Apolipoprotein A-I precursor M00001 -3.45 0.023
Apolipoprotein A-I J02597 -2.47 0.003
Sodium-hydrogen exchange protein-isoform 3 M85300 -7.36 0.004
Liver fatty acid binding protein V01235 -2.62 0
Sodium transporter X59677 -3.8 0
Cation transporter X78855 -3.62 0.003
ATP-binding cassette AB010467 -3.89 0.004
Methionine adenosyltransferase II, alpha J05571 -2.91 0.007
Phenylalanine hydroxylase M12337 -7.43 0
Carbonic anhydrase IV S68245 -4.28 0.011
Signal transduction
B7 antigen X76697 -170.95 0.002
CD24 antigen U49062 -3.08 0
Chemokine CX3C AF030358 -5.04 0.011
Itmap1 AF022147 -7.5 0.001
HCNP E05646 -2.5 0.001
Brain glucose-transporter protein M13979 -2.97 0.019
Protein kinase C delta M18330 -2.48 0.002
Guanylate cyclase 2C M55636 -4.58 0.003
A2b-adenosine receptor M91466 -2.8 0.04
Guanylate cyclase activator 2A M95493 -4.18 0.005
Phospholipase C beta-3 M99567 -2.57 0.018
Tm4sf3 Y13275 -3.33 0
Phospholipase D AB000778 -2.71 0.009
BEM-2 D45413 -6.41 0.015
Sgk L01624 -3.93 0
Stress response/apoptosis
Prostaglandin D synthetase J04488 -43.11 0.009
GTP cyclohydrolase I M58364 -3.26 0.014
Structure proteins
Chromogranin B (Chgb) AF019974 -2.56 0.005
Intestinal mucin M76740 -5.09 0.002
Muc3 U76551 -11.07 0.006
Mucin-like protein M81920 -11.97 0.001
Myosin 5B U60416 -3.94 0
Keratin 18 X81448 -3.23 0.004
Keratin 19 X81449 -2.69 0.001
ZG-16p protein Z30584 -4.43 0.002
Plasmolipin Z49858 -7.2 0
Cytokeratin 21 M63665 -4.96 0
Syndecan S61865 -3.3 0.006
Claudin 3 M74067 -6.68 0.01
Transcription factor/regulator
Hepatocyte nuclear factor 3 gamma AB017044 -6.96 0
Apolipoprotein B mRNA editing protein L07114 -2.34 0
DNA-binding inhibitor L23148 -4.1 0.01
Kruppel-like factor 4 (gut) L26292 -3.08 0.017
Others
Prolactin receptor M74152 -3.26 0.014
LOC286964 U89280 -2.96 0.003
Ckmt1 X59737mRNA -2.65 0.025
Arginase II U90887 -23.69 0
Deleted in malignant brain tumors 1 U32681 -3.47 0.002
3' end GAA-triplet repeat L13025 -2.73 0.001
Polymeric immunoglobulin receptor L13235 -2.93 0.004
*Changes in gene expression were determined by t-test (DMT), comparative analysis (MAS 5.0), and SAM (Stanford). Gene expression profiles from CAS animals were used as control. P value and fold change are based on DMT analysis; whereas final genes listed met all of the analytical criteria as described in Methods.
Table 3 Up-regulated genes in rats fed with WPH diet*
Category and Gene Name Probe Set GB Accession No. Fold Change P value
Cell adhesion
Fibronectin X05834 2.3 0
EGF-containing fibulin-like extracellular matrix protein 1 D89730 2.17 0.004
Cell cycle/growth control
Somatostatin M25890 2.72 0.001
Somatostatin-14 K02248 3.87 0.009
APEG-1 U57097 3.24 0.002
Defense/immunity protein
IgG gamma heavy chain M28670 2.21 0.009
T-cell receptor beta chain X14319 2.14 0
Adipsin M92059 3.21 0
Ligand binding/carrier
Angiotensin receptor M86912 2.75 0.017
Calretinin X66974 2.52 0.005
Purkinje cell protein 4 M24852 3.06 0.001
Secretogranin III U02983 2.77 0.005
Secretogranin II M93669 2.84 0.001
Aquaporin 1 X67948 3.4 0.008
Cacna2d1 M86621 2.84 0
Retinol-binding protein M10934 2.17 0.018
Metabolism
Lipoprotein lipase L03294 2.72 0
Ubiquitin carboxyl-terminal hydrolase D10699 3 0.003
Signal transduction
Thy-1 protein X02002 2.89 0.002
CD3 gamma-chain S79711 3.28 0.002
Synapsin M27925 3.94 0.001
Alpha-actinin-2 associated LIM protein AF002281 2.74 0.009
RESP18 L25633 2.74 0.033
T3 delta protein X53430 2.75 0.003
Protein phosphatase inhibitor-1 J05592t 2.6 0.009
CART protein U10071 2.16 0.001
Neuroendrocrine protein M63901 3.7 0.006
Protein kinase C-binding protein Zeta1 U63740 3.14 0.003
cannabinoid receptor 1 X55812 2.17 0.002
Guanylyl cyclase A J05677 3.18 0.007
Tachykinin 1 X56306 2.36 0.036
Protein tyrosine phosphatase L19180 2.47 0.041
Argininosuccinate synthetase X12459 4.69 0.004
Stress response/apoptosis
Small inducible cytokine Y08358 3.35 0.029
Structure proteins
Fast myosin alkali light chain L00088 4.52 0.03
Light molecular-weight neurofilament AF031880 2.41 0
Neurofilament protein middle Z12152 2.97 0.006
Alpha-tubulin V01227 2.25 0
Peripherin AF031878 2.82 0.007
Transcription factor/regulator
snRNP M29293 2.11 0.004
snRNP-associated polypeptide X73411 3.33 0.002
Others
C1-13 gene product X52817 3.17 0
ND5, ND6 S46798 2.31 0.015
Sensory neuron synuclein X86789 2.84 0
*Changes in gene expression were determined by t-test (DMT), comparative analysis (MAS 5.0), and SAM (Stanford). Gene expression profiles from CAS animals were used as control. P value and fold change are based on the DMT analysis; whereas listed genes met all of the analytical criteria as described in Methods.
Table 4 Down-regulated genes in rats fed with SPI diet*
Category and Gene Name Probe Set GB Accession No. Fold Change P value
Cell adhesion
Embigin AJ009698 -5.13 0.001
Cell Cycle/growth control
FGF receptor activating protein 1 U57715 -5.59 0.002
BEST5 protein Y07704 -2.37 0.003
Peptide tyrosine-tyrosine (YY) M17523 -3.91 0.002
Glucagon gene K02813 -6.58 0.002
Epithelial membrane protein-1 Z54212 -3.47 0.017
Neu oncogene X03362 -1.58 0.05
Defense/immunity protein
Beta defensin-1 AF068860 -42.16 0.001
AF093536 -10.2 0
Detoxification/antioxidation
Glutathione S-transferase J02810 -7.14 0
Glutathione S-transferase Yb X04229 -11.71 0.001
Glutathione S-transferase, alpha 1 K01932 -4.18 0.004
Glutathione S-transferase Yc1 S72505 -5.23 0.001
Glutathione S-transferase Yc2 S72506 -5.27 0.012
S82820 -3.45 0.006
Cytochrome P450 4F4 (CYP4F4) U39206 -6.52 0.002
Cytochrome P450CMF1b J02869 -4.12 0.002
Cytochrome P450 (CYP4F1) M94548 -2.88 0.002
1-Cys peroxiredoxin Y17295 -2.55 0.002
Metallothionein M11794 -2.92 0.006
D-amino-acid oxidase AB003400 -5.42 0
Peroxiredoxin 6 AF014009 -3.07 0.008
Phenylalanine hydroxylase M12337 -10.99 0.001
Metabolism
Dipeptidase L07315 -3.08 0.001
Meprin beta-subunit M88601 -3.27 0.001
Disintegrin and metalloprotease domain 7 X66140 -14.03 0
Ligand binding/carrier
Carnitine transporter AB017260 -3.81 0.003
Chloride channel (ClC-2) AF005720 -3.26 0.001
Putative potassium channel AF022819 -2.69 0.001
Mitochondrial dicarboxylate carrier AJ223355 -2.54 0.01
Aquaporin 3 D17695 -4.13 0
Intestinal fatty acid binding protein K01180 -4.43 0.005
Na_H_Exchanger L11236 -4.47 0.002
H+, K+-ATPase M90398 -2.52 0.001
Carbonic anhydrase IV S68245 -4.28 0.005
Sodium transporter X59677 -3.4 0
Phosphatidylethanolamine binding protein X75253 -2.69 0
Signal transduction
B7 antigen X76697 -170.95 0.002
HCNP E05646 -3.38 0
Itmap1 AF022147 -7.97 0.005
Guanylate cyclase activator 2A M95493 -3.28 0.006
Sgk L01624 -2.76 0
Stress response/apoptosis
Prostaglandin D synthetase J04488 -45.8 0.01
Structure proteins
Muc3 U76551 -3.56 0.01
Intestinal mucin M76740 -3.31 0.006
Mucin-like protein M81920 -3 0
Plasmolipin Z49858 -2.92 0.003
Transcription factor/regulator
Testis specific X-linked gene X99797 -6.91 0.003
Others
Arginase II U90887 -3.22 0
3-phosphoglycerate dehydrogenase X97772 -4.15 0.017
Aldehyde dehydrogenase family 1 AF001898 -3.93 0.004
*Changes in gene expression were determined by t-test (DMT), comparative analysis (MAS 5.0), and SAM (Stanford). Gene expression profiles from CAS animals were used as control. P value and fold-change are based on the DMT analysis; whereas genes listed above met all of the analytical criteria as described in Methods.
Table 5 Up-regulated genes in rats fed with SPI diet*
Category and Gene Name Probe Set GB Accession No. Fold Change P value
Cell adhesion
Collagen alpha1 type I Z78279 2.49 0
Secreted phosphoprotein 1 M14656 111.39 0.006
Matrix metalloproteinase 13 M60616 24.34 0.002
Regenerating islet M62930 193.08 0.011
Defense/immunity protein
Ig gamma-2a chain L22654 115.17 0.001
Ig gamma heavy chain M28670 3.22 0
Ig germline kappa-chain C-region M18528 2.48 0.038
Ig light-chain U39609 2.63 0.021
Fc-gamma M32062 4.72 0.017
Detoxification
Glutathione S-transferase 1 J03752 2.86 0
Glutathione-S-transferase,alpha type2 K00136 2.56 0.009
UDP glucuronosyltransferase D38066 2.83 0.014
Metabolism
Matrix metalloproteinase 7 L24374 3.63 0.02
lysozyme rc_AA892775 2.77 0
Matrix metalloproteinase 12 X98517 11.8 0.013
Mitochondrial carbamyl phosphate synthetase I M12335 59.25 0.001
Aldolase B, exon 9 X02291 8.7 0.01
Aldolase B, exon 2 X02284 2.71 0.001
Signal transduction
MHC class II antigen RT1.B-1 beta-chain X56596 2.55 0.001
CD3 gamma-chain S79711 4.51 0.001
Ligand binding/carrier
Intracellular calcium-binding protein L18948 28.29 0.014
Retinol binding protein II M13949 5.11 0.001
Apolipoprotein B M27440 6.47 0.024
Apolipoprotein A-I J02597 2.49 0.004
Iron ion transporter AF008439 18.78 0.008
Stress response/apoptosis
Heme oxygenase J02722 9.66 0.002
JE product X17053 3.52 0.001
Pancreatitis-associated protein M98049 68.39 0.004
Pancreatitis associated protein III L20869 15.35 0
Reg protein E01983 30.25 0.001
Others
Histamine N-tele-methyltransferase S82579 6.17 0.04
*Changes in gene expression were determined by t-test (DMT), comparative analysis (MAS 5.0), and SAM (Stanford). Gene expression profiles from CAS animals were used as control. P value and fold-change are based on the DMT analysis; whereas final listed genes met all of the analytical criteria as described in the Methods.
Table 6 Genes co-regulated with WPH and SPI diet*
Category and Gene Name Probe Set GB Accession No. Fold Change in WPH P value Fold Change in SPI P value
Down-regulated genes
Embigin AJ009698 -6.57 0 -5.13 0.001
Epithelial membrane protein 1 Z54212 -4.67 0.015 -3.47 0.017
Glucagon K02813 -7.73 0.005 -6.58 0.002
Peptide tyrosine-tyrosine (YY) M17523 -4.56 0.001 -3.91 0.002
FGF receptor activating protein U57715 -4.25 0.002 -5.59 0.002
Neu oncogene X03362 -2.61 0.017 -1.58 0.05
CD52 antigen X76697 -170.95 0.002 -170.95 0.002
Beta defensin-1 AF068860 -54.48 0.001 -42.16 0.001
Glutathione S-transferase J02810 -5.17 0 -7.14 0
Glutathione S-transferase Yb X04229 -9.33 0 -11.71 0.001
Glutathione S-transferase, alpha 1 K01932 -3.07 0.002 -4.18 0.004
Glutathione S-transferase Yc1 S72505 -3.69 0.004 -5.23 0.001
Glutathione S-transferase Yc2 S72506 -21.38 0.008 -5.27 0.012
Cytochrome P450CMF1b J02869 -8.23 0.001 -4.12 0.002
Cytochrome P450 4F4 U39206 -6.43 0.004 -6.52 0.002
Cytochrome P450IVF M94548 -5.78 0.002 -2.88 0.002
D-amino-acid oxidase AB003400 -13.69 0 -5.42 0
Meprin beta-subunit M88601 -5 0.004 -3.27 0.001
Disintegrin and metalloprotease domain 7 X66140 -11.91 0 -14.03 0
Carnitine transporter AB017260 -3.95 0.005 -3.81 0.003
Chloride channel (ClC-2) AF005720 -5.69 0.002 -3.26 0.001
Putative potassium channel AF022819 -4.84 0 -2.69 0.001
Mitochondrial dicarboxylate carrier AJ223355 -3.55 0.009 -2.54 0.01
Aquaporin 3 D17695 -7.83 0 -4.13 0
Na_H_Exchanger L11236 -9.81 0.003 -4.47 0.002
H+, K+-ATPase M90398 -13.87 0 -2.52 0.001
Fatty acid binding protein 1 K01180 -7.29 0.001 -4.43 0.005
Sodium transporter X59677 -3.8 0 -3.4 0
Carbonic anhydrase IV S68245 -4.28 0.011 -4.28 0.005
Itmap1 AF022147 -7.5 0.001 -7.97 0.005
HCNP E05646 -2.5 0.001 -3.38 0
Guanylate cyclase activator 2A M95493 -4.18 0.005 -3.28 0.006
Sgk L01624 -3.93 0 -2.76 0
Prostaglandin D synthetase J04488 -43.11 0.009 -45.8 0.01
Mucin 3 M76740 -5.09 0.002 -3.31 0.006
Mucin-like protein M81920 -11.97 0.001 -3 0
Plasmolipin Z49858 -7.2 0 -2.92 0.003
Up-regulated genes
Ig gamma heavy chain M28670 2.21 0.009 3.22 0
CD3 gamma-chain S79711 3.28 0.002 4.51 0.001
*Changes in gene expression were determined by t-test (DMT), comparative analysis (MAS 5.0), and SAM (Stanford). Gene expression profiles from CAS animals were used as control. P value and fold change are based on the DMT analysis; whereas final listed genes met all of the analytical criteria described in Methods.
Gene expression: effects of WPH
As based on Gene Ontology (GO) annotations, the 44 up-regulated and 119 down-regulated genes of the WPH group belong to multiple functional categories including cell adhesion (n = 10), cell cycle and growth control (n = 10), detoxification (n = 17), defense and immunity (n = 7), signal transduction (n = 29), transcriptional regulation (n = 6), metabolism (n = 19), ligands and carriers (n = 27), cell death (n = 3), structural proteins (n = 16), and others (Tables 2 &3). The fold change for up-regulated genes ranged between 2.1 [small nuclear ribonucleoparticle-associated protein (snRNP)] to 4.7 (argininosuccinate synthetase), whereas down-regulated genes exhibited fold changes between 2.0 (cyclin D1) and 171 (CD52 antigen).
Lifetime ingestion of WPH affected the expression of xenobiotic metabolism-related enzymes including several of the cytochrome P450s and glutathione S-transferases, alcohol dehydrogenase (ADH), and UDP-glucuronosyltransferase. Cytochrome P450 enzymes and ADH are considered to play key roles in activation of the proximate carcinogen from AOM [29]. Down-regulation of expression of Phase I detoxification enzymes by WPH might therefore diminish AOM-induced DNA adducts and genomic instability. Consistent with results from a study in which whey proteins inhibited cell proliferation in vitro [11], lifetime feeding of WPH was associated with changes in expression of genes involved in cell cycle control and proliferation; cyclin D1, neu oncogene, mapk6, glucagon, and peptide YY (PYY) genes were down-regulated, whereas the expression of somatostatin, a growth-inhibitory peptide was induced. WPH altered expression of genes involved in cellular defense. Induced genes included Ig gamma heavy chain, adipsin, and T-cell receptor beta chain, whereas expression of the antibacterial peptide beta defensin-1 and seminal vesicle secretion protein IV (SVS IV) were down-regulated. About 20% of WPH-affected genes are involved in cell signaling; these include guanylate cyclase 2C, protein kinase C delta, and synapsin. Additionally, genes encoding ligands or membrane channels [i.e., chloride channel, intestinal fatty acid binding protein (I-FABP), apoliprotein A-I (Apo-AI), Na+, K+-ATPase, and sodium transporter] were down-regulated by WPH, whereas calretinin and retinol binding protein (RBP) levels were increased.
Gene expression: effects of SPI
Colon genes, whose mRNA expression was affected by ingestion of SPI, fell into multiple functional categories including cell adhesion (n = 4), cell cycle and growth control (n = 6), detoxification (n = 18), defense and immunity (n = 6), signal transduction (n = 4), transcriptional regulation (n = 1), metabolism (n = 8), ligands and carrier proteins (n = 17), cell death proteins (n = 5), and structural proteins (n = 3) (Tables 4 &5).
Relative abundance of numerous transcripts was changed in the same direction by WPH and SPI (Fig. 2). However, some exceptions were noted. For example, mRNA encoding Apo-AI was down-regulated by WPH, but elevated by SPI. Apo-AI is the major determinant of the capacity of HDL particles to promote cholesterol efflux and this protein is associated with the inhibition of atherosclerosis [30]. However, the impact of differential response of Apo-AI to WPH and SPI on anti-tumorigenesis is unknown.
Confirmation of differential gene expression
We performed quantitative real-time RT-PCR on selected genes to confirm the microarray results. Based upon known associations with cell proliferation or differentiation, 14 genes were chosen for further study. Included in this group was BTEB2; this gene was not present on the microarrays but was included in RT-PCR analysis due to its significant expression in intestine and involvement in cell proliferation [see discussion]. As shown in Figure 3, eight genes were confirmed to be differentially expressed: these included the gastrointestinal hormone genes PYY (12.9-fold down-regulated in WPH fed rats; P = 0.004), glucagon (17.8-fold down-regulated in WPH fed rats; P = 0.005), and somatostatin (3.92-, 2.65-fold up-regulated in SPI and WPH fed rats; P = 0.05, P = 0.025, respectively); cyclin D1 (1.6-, 2.4-fold down-regulated in SPI and WPH fed rats; P = 0.033, P = 0.001, respectively); BTEB2 (1.9-, 6.7-fold down-regulated in SPI and WPH fed rats; P = 0.024, P < 0.001, respectively); c-neu proto-oncogene (2.5-, 4.1-fold down-regulated in SPI and WPH fed rats; P < 0.001, P < 0.001, respectively); the colonocyte differentiation marker I-FABP (2.9-, 4.0-fold down-regulated in SPI and WPH fed rats; P = 0.023, P = 0.01, respectively); and the mucin, MUC3 (2.78-, 4.05-fold down-regulated in SPI and WPH fed rats; P < 0.001, P < 0.001, respectively). Differential expression of five other genes was not confirmed statistically, due to individual animal variation in the transcript levels; however, the mean-fold changes for mRNA abundance were greater than two and in agreement with the corresponding microarray results for these genes. Only one of the selected genes – retinol binding protein (RBP), failed to exhibit greater than a 2-fold change (in the predicted direction) at the mRNA level by real-time RT-PCR.
Figure 3 Quantitative real-time RT-PCR verification of microarray results. RNA used for real-time RT-PCRs was from the same animals (n = 7 per diet group) whose RNAs comprised the pools for microarray analysis. Values are mean ± SEM and were analyzed by one-way ANOVA, *P < 0.05, SPI or WPH vs. CAS.
Serum somatostatin (Sst)
As shown in Fig. 4, circulating Sst concentration was significantly higher in rats fed WPH and SPI. Colonic Sst protein content in colon homogenates was below the limit of detection of the assay used (data not shown).
Figure 4 Diet effects on serum Sst concentration. Values are mean ± SEM. One-way ANOVA. *P < 0.05, SPI or WPH vs. CAS.
Discussion
The type of dietary protein(s) can markedly affect the onset and/or progression of CRC [31]. Epidemiological and animal studies have found that dietary soy and whey proteins decrease the incidence of certain tumors, including those of the colon and rectum [6,7,32-35]. Using the AOM-treated male Sprague Dawley rat model, we previously found that lifetime feeding of SPI led to a ~ 76% lower incidence of AOM-induced colon tumors compared to rats lifetime-fed CAS [8]. Additionally, in the same studies, a ~ 46% lower incidence of colon tumors was found in WPH-fed compared to CAS-fed rats [9]. The molecular mechanism(s) by which these dietary proteins reduce the incidence of chemically-induced colon tumors is unclear, although several mechanisms and putative bio-active factors have been proposed [11-24]. The present study has now identified genes that are differentially expressed as a function of these diets and which serve to highlight potential pathways for dietary protection from carcinogenesis.
The ability to simultaneously analyze a large number of different mRNAs makes microarrays very appealing for identifying genes and gene families whose expression is altered by diet [36,37]. We focused on the 'normal' colon tissue since we are interested in genes that are differentially regulated by diet and which act in anti-oncogenic fashion in pre-cancerous tissues. We limited our analysis to the proximal colon since several studies have suggested that the molecular etiology of proximal and distal colon tumors differs [25,26] and proximal colon tumors have become more prevalent with Westernization of the diet and aging of the population [27]. We chose to include colonic smooth muscle with the mucosa since: a) the former tissue layer interacts with the latter to influence its growth and function, and b) we could monitor all colonic genes affected by diet. However, one potential caveat to this strategy is the 'dilution' effect that may have been imposed on the more rare mucosal transcripts. Another caveat is that no information is obtained regarding where the differentially expressed transcripts occur. In this regard, however, we have confirmed by immuno-histochemistry that I-FABP is expressed predominantly in the inter-cryptal surface epithelium of colons from AOM-treated rats (Fig. 5). Our study used a sample size of three (per diet group) which balanced the costs for the experimental reagents with the minimum number required for statistical analysis. The quantitative PCR analyses provided confirmation that the filtering strategies used yielded bona-fide differentially expressed transcripts.
Figure 5 Immuno-histochemistry for I-FABP in colons from AOM-treated rats. Panels A and B are sections from CAS and WPH-fed animals, respectively. Arrows point to the strong areas of staining for I-FABP in the inter-cryptal surface epithelium (overall intensity of staining is greater for CAS than for WPH).
Only two transcripts were induced by both SPI and WPH; whereas 37 transcripts were repressed by both SPI and WPH. This suggests that the cancer-protective actions of the two diets are generally associated with repression of colonic genes that facilitate tumorigenesis. An alternative explanation is that CAS induces genes that facilitate colon cancer development when compared to SPI and WPH. It is also likely that SPI and WPH diets act in unique ways to inhibit tumorigenesis. Irregardless, our results indicate that the nature of the dietary protein can profoundly affect colon gene expression profiles. Thus, gene expression profiling studies of colons should account for potential confounding effects of diet.
Dietary factors in SPI or WPH inhibit cell proliferation and induce apoptosis among other biological actions [11,13]. In the present study, we identified cyclin D1 gene and the neu proto-oncogene as being repressed in proximal colon by SPI and WPH. Cyclin D1 is a key regulator of cell cycle progression [38], and a target of β-catenin, a protein whose abnormal accumulation in the nucleus is strongly linked to the development of multiple tumor types, including those of the colon [39]. Aberrantly increased expression of cyclin D1 in colon epithelial cells contributes to their abnormal proliferation and tumorigenicity [40,41]. Similarly, the oncogenic and cellular growth-promoting activities of the HER-2/neu proto-oncogene are well known [42]. HER-2/neu, a tyrosine kinase receptor for neu-differentiation factor, is expressed in normal colonic epithelium and is up-regulated in adenomatous polyps of the colon [43]. The down regulation of cyclin D1 and c-neu mRNA abundance by SPI and WPH may at least partly explain their anti-tumorigenic properties. Similarly, Krüppel-like transcription factors have been linked to cell growth and tumorigenesis. BTEB2 (also known as Krüppel-like factor 5, KLF5, or intestinal KLF) was reported to enhance intestinal epithelia cell colony formation, cyclin D1 transcription, and cell proliferation [44]. Consistent with our results for cyclin D1, colonic BTEB2 mRNA expression was down-regulated by SPI and WPH. Aquaporin 3, a water channel highly expressed in colonic epithelium, was down-regulated by SPI and WPH. Aquaporins are thought to be induced early in colon cancer and to facilitate oncogenesis [45], therefore, dietary repression of such genes may additionally contribute to anti-tumorigenesis. The results for I-FABP and MUC3 indicated 3–4 fold decreases in transcript abundance in proximal colons of rats on SPI or WPH diets. These particular results are not easily reconciled with decreased tumorigenesis in SPI and WPH groups, since both genes are highly expressed in the normal differentiated colonic epithelium and are likely to be under-expressed in adenomas and adenocarcinomas [46]. Perhaps, these represent diet-modulated genes that are not direct participants in anti-tumorigenesis.
Gastrointestinal hormones regulate a myriad of intestinal functions including motility, absorption, digestion, cell proliferation and death, and immune response [47]. The microarray and real-time RT-PCR assays identified inductive effects of SPI and WPH on somatostatin mRNA and protein abundance. These results implicate this gene product in autocrine and paracrine mechanisms underlying colon cancer protection by SPI and WPH since somatostatin is a well-known anti-proliferative agent for colon tumor cells [48,49]. This hormone is also a negative regulator of angiogenesis [50]; this is predicted to counter tumorigenesis. It is possible that the small decrements in rat growth rates observed with lifetime SPI or WPH diets [8,9] are a consequence of this increased circulating somatostatin level. We also found decreased abundance of mRNAs encoding peptide YY (PYY) and glucagon in colons of WPH-fed rats. PYY gene expression in human colon tumors is much reduced relative to the adjacent normal tissue [51]; however, chemically-induced colon tumors in rats generally exhibit higher overall expression of PYY due to increased prevalence of PYY-positive cells, compared to normal mucosa [52,53]. PYY stimulates proliferation of intestinal epithelium [54]; therefore, an inhibition of PYY expression by dietary WPH may contribute to colon cancer-protective actions. A similar scenario might apply to colon glucagon gene expression, as this growth stimulatory peptide for colon cancer cells [55] was inhibited by WPH at the level of colon mRNA abundance.
Our data highlighted other aspects of diet and colon gene expression that warrant further study. For example, the B7 antigen (also known as CD52) mRNA was strongly down-regulated by SPI or WPH. The corresponding protein is normally expressed at high levels on cell membranes of T and B lymphocytes and monocytes; infusion with anti-CD52 antibody leads to systemic depletion of T cells [56]. The lower abundance of this transcript in non-tumor colon tissue of rats on SPI or WPH diets may reflect fewer numbers of immune cells in this tissue, as compared to CAS-fed animals. One possible interpretation of this data is that the 'normal' tissue of the CAS group has manifested an immune response, perhaps in response to increased tumorigenicity relative to SPI or WPH groups. Such an interpretation raises the prospect of a functional immuno-editing mechanism [57] occurring in this model of colon cancer and an indirect effect of diet on lymphocyte populations (via presence of tumors or tumor precursors) in the colon. An alternative mechanism is that dietary protein can directly affect the populations of lymphocytes resident in the colon, which in turn, may affect tumorigenesis. A related observation was the enhanced abundance of CD3 gamma chain transcripts in colons of SPI and WPH animals. The protein encoded by this transcript helps mediate T cell antigen receptor engagement and signaling [58]; its decreased abundance in colonic T cells of CAS-fed animals may indicate a specific immune defect [59] occurring in the CAS-fed animals after exposure to carcinogen and thereby contributing to enhanced tumorigenesis in this group.
Several microarray studies of human paired normal colon vs. colon tumors have been published [60-64]. Comparison of the present results for normal colon tissue of AOM-treated rats on different diets to the published studies for human CRC identified only a small number of common differentially expressed genes and/or gene families in common (data not shown). This small number is probably due to the fact that our study did not examine colon tumors; rather we focused on 'normal' colon tissue. In this regard, it will be interesting to examine the expression profiles of colons from animals not treated with AOM so as to more specifically correlate transcripts with diet and cancer phenotype. This study has illuminated a number of genes and gene families that may act as dietary protein-dependent modulators of oncogenesis in the rat colon. Additional studies that specifically address the functional involvement of these genes in cancer-prevention via dietary means are required to confirm the postulated roles.
Conclusions
We have identified genes in rat colon that are differentially expressed, as a consequence of altered dietary protein, during AOM-induced oncogenesis. These are candidates for genes that sub-serve the anti-cancer effects of dietary SPI and WPH in this tissue.
Methods
Rats, diets and carcinogen treatment
The animals whose colons were used in the present study have been previously described [8,9]. Time-mated [gestation day (GD) 4] Sprague-Dawley rats were purchased from Harlan Industries (Indianapolis, IN), housed individually and allowed ad libitum access to water and pelleted food. Rats were randomly assigned to one of three semi-purified isocaloric diets made according to the AIN-93G diet formula [65] and which differed only by protein source: a) CAS (New Zealand Milk Products, Santa Rosa, CA), b) WPH (New Zealand Milk Products, Santa Rosa, CA) or c) SPI (Dupont Protein Technologies, St. Louis, MO). Offspring were weaned to the same diet as their mothers and were fed the same diets throughout the study. At 90 days of age, male offspring received s.c. injections of 15 mg/kg AOM (Ash Stevens, Detroit, MI) in saline once a week for 2 weeks. Forty weeks later, rats were euthanized, and the colon (cecum to anus) was divided into two equal segments (proximal and distal), opened longitudinally, and examined for tumors. We found that both WPH and SPI significantly decreased the colon tumor incidence [data published in [8,9]]. A representative non-tumor segment of each proximal colon (PC) was frozen in liquid nitrogen and stored at -80°C for later use. Animal care and handling were in accordance with the Institutional Animal Care & Use Committee guidelines of the University of Arkansas for Medical Sciences.
RNA processing
Total RNA was isolated from rat proximal colons (n = 7 for each of CAS, SPI and WPH diets) using TRIzol reagent (Invitrogen, Carlsbad, CA), and further purified with the RNeasy Mini Kit (QIAGEN, Valencia, CA). To remove contaminating DNA, on-column DNA digestion with RNase-Free DNase (QIAGEN) was performed. Integrity of isolated RNAs was confirmed using the RNA 6000 Nano LabChip kit with the Agilent 2100 Bioanalyzer System (Agilent Biotechnologies, Palo Alto, CA). To reduce errors due to biological variability, RNA samples were pooled as proposed by Bakay et al [66]. Pooled RNA (equal amounts of RNA from each of 7 animals; 8 ug total) was used for cDNA synthesis using a T7-(deoxythymidine)24 primer and Superscript II (Life Technologies, Inc., Gaithersburg, MD). The resulting cDNA was used with the ENZO BioArray High Yield RNA Transcript labeling kit (ENZO, Farmingdale, NY) to synthesize biotin-labeled cRNA. The cRNA was purified on RNeasy spin columns (QIAGEN) and subjected to chemical fragmentation (size range of 35 to 200 bp). Three replicate cRNA targets were made in parallel starting from each RNA pool.
Microarray procedures
Ten ug of cRNA was hybridized for 16 hours to an Affymetrix (Santa Clara, CA) rat U34A GeneChip (3 chips used per diet group), followed by incubations with streptavidin-conjugated phycoerythrin, and then with polyclonal anti-streptavidin antibody coupled to phycoerythrin. Following washing, GeneChips were scanned using an Agilent GeneArray laser scanner. Images were analyzed using Affymetrix MAS 5.0 software. Bacterial sequence-derived probes on the arrays served as external controls for hybridization, whereas the housekeeping genes β-actin and GAPDH served as endogenous controls and for monitoring the quality of the RNA target. To compare array data between GeneChips, we scaled the average of the fluorescent intensities of all probes on each array to a constant target intensity of 500.
Bioinformatics
To validate the microarray procedure for our samples, unsupervised nearest-neighbor hierarchical clustering (Spotfire, Somerville, MA) was performed on gene expression data. The inter-chip variability test also was performed as specified in the Affymetrix data analysis manual [28]. To identify colon genes differentially expressed with SPI or WPH (control: CAS diet), multiple criteria were applied; final results are reported only for transcripts that passed all three analytical steps described below. Firstly, the t-test feature of DMT (Affymetrix) was used to identify genes whose expression was regulated (induced/repressed with P < 0.05) by SPI or WPH, and signal fold changes (FC) for these genes were calculated. Secondly, microarray data were analyzed using 'Significance of Analysis of Microarrays' (SAM, Stanford) to identify significant changes in gene expression among diet groups [67], using a false discovery rate (FDR) cutoff of 0.5%. Lastly, a pair-wise comparison survival (3 × 3) method was used to identify differentially expressed transcripts [68]. In brief, the three replicate expression profiles obtained for SPI colons were iteratively compared with the three CAS profiles (latter as baseline) in MAS 5.0 (Affymetrix), generating nine comparisons in total. Transcripts with a log ratio greater than or equal to 1 (≥2 fold change), which increased (I) in nine of nine comparisons, and which were expressed above background (i.e., called as Present) in all three SPI GeneChips, were considered to be up-regulated by SPI. Transcripts with a log ratio less than or equal to -1, were decreased (D) in nine of nine comparisons, and expressed above background (Present) in all three CAS chips were considered to be down-regulated by SPI. WPH-regulated genes were similarly identified. Genes that were independently identified by all three approaches comprised the final reported lists of differentially expressed genes (Tables 2, 3, 4, 5, 6).
Validation of gene expression by quantitative real-time RT-PCR
One μg of total RNA from each of the 21 individual proximal colons (which comprised the original pools for the microarray experiment) was reverse-transcribed using random hexamers and MultiScribe Reverse Transcriptase in a two-step RT-PCR reaction (Applied Biosystems, Foster City, CA). Primers (Table 7) were designed using 'Primer Express' (Applied Biosystems) and were selected to yield a single amplicon; this was verified by dissociation curves and/or analysis in agarose gels. SYBR Green real-time PCR was performed with an ABI Prism 7000 Sequence Detector. Thermal cycling conditions included pre-incubation at 50°C for 2 min, 95°C for 10 min followed by 40 PCR cycles at 95°C for 15 sec and 60°C for 1 min. The relative transcript levels for each gene were calculated using the relative standard curve method (User Bulletin #2, Applied Biosystems) and normalized to the house-keeping gene β-actin. Data are reported as mean ± SEM of n = 7 animals per dietary group. Significant differences between diet groups were determined by one-way ANOVA (P < 0.05).
Table 7 Primer sequences for real-time RT-PCR
Gene Forward primer Reverse primer Accession no.
Beta-actin 5'-GACGGTCAGGTCATCACTATCG-3' 5'-ACGGATGTCAACGTCACACTTC-3' NM_031144
I-FABP 5'-AGGAAGCTTGGAGCTCATGACA-3' 5'-TCCTTCCTGTGTGATCGTCAGTT-3' K01180
Neu Oncogene 5'-GTGGTCGTTGGAATCCTAATCAA-3' 5'-CCTTCCTTAGCTCCGTCTCTTTTA-3' X03362
PYY 5'-AGGAGCTGAGCCGCTACTATGC-3' 5'-TTCTCGCTGTCGTCTGTGAAGA-3' M17523
Glucagon 5'-TGGTGAAAGGCCGAGGAAG-3' 5'-TGGTGGCAAGGTTATCGAGAA-3' K02813
Somatostatin 5'-GGAAACAGGAACTGGCCAAGT-3' 5'-TGCAGCTCCAGCCTCATCTC-3' K02248
PAP III 5'-AAGAGGCCATCAGGACACCTT-3' 5'-CACTCCCATCCACCTCTGTTG-3' L20869
CYP4F1 5'-CCAAGTGGAAACGGTTGATTTC-3' 5'-TCCTGGCAGTTGCTGTCAAAG-3' M94548
GST 5'-ACTTCCCCAATCTGCCCTACTTA-3' 5'-CGAATCCGCTCCTCCTCTGT-3' X04229
Cyclin D1 5'-TCAAGTGTGACCCGGACTGC-3' 5'-ACTTCCCCTTCCTCCTCGGT-3' D14014
Beta defensin-1 5'-TCTTGGACGCAGAACAGATCAATA-3' 5'-TCCTGCAACAGTTGGGCTATC-3' AF093536
H+, K+-ATPase 5'-ATTCCGCATCCCTAGACAACG-3' 5'-TCTTACTAAAGCTGGCCATGATGTT-3' M90398
Prostaglandin D synthetase 5'-CAAGCTGGTTCCGGGAGAAG-3' 5'-TTGGTCTCACACTGGTTTTTCCTTA-3' J04488
RBP 5'-TCGTTTCTCTGGGCTCTGGTAT- 3' 5'-TTCCCAGTTGCTCAGAAGACG-3' M10934
Muc3 5'-AAGGTGTGAGGAAGTGATGGAGA-3' 5'-GCAGAGACCGTCGGCTTTATC-3' U76551
BTEB1 5'-ACACTGGTCACCATCGCCAA-3' 5'-GGACTCGACCCAGATTCGGT-3' NM_057211
BTEB2 5'-CTACTTTCCCCCATCACCACC-3' 5'-GAATCGCCAGTTTCGAAGCA-3' AB096709
Serum Sst
Rat serum Sst content (15 animals from each diet) was determined using the somatostatin-28 EIA kit purchased from Phoenix Pharmaceuticals Corporation (Belmont, California).
Authors' contributions
RX performed the microarray and real-time PCR experiments, conducted the data analysis, and participated in drafting the manuscript. TMB designed and oversaw the animal component of the study. FAS designed the analytical and overall approaches to the study, supervised the project, and drafted the manuscript. All authors read and approved the final manuscript.
Acknowledgements
We thank Dr. Rosalia C.M. Simmen and Dr. Rick Helm for insightful comments on the manuscript and Amanda L. Linz for performing the I-FABP immuno-histochemistry. Supported by USDA CRIS 6251-5100-002-06S.
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| 15644144 | PMC545049 | CC BY | 2021-01-04 16:36:35 | no | Mol Cancer. 2005 Jan 11; 4:1 | utf-8 | Mol Cancer | 2,005 | 10.1186/1476-4598-4-1 | oa_comm |
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Microb Cell FactMicrobial Cell Factories1475-2859BioMed Central London 1475-2859-4-31563893510.1186/1475-2859-4-3ResearchDecreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase Vethanayagam Joe GG [email protected] Ann M [email protected] Department of Microbiology and Immunology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202-9037, USA2 Protein Expression Laboratory/NIAMS, National Institute of Health, Bethesda, MD 20892, USA2005 7 1 2005 4 3 3 15 11 2004 7 1 2005 Copyright © 2005 Vethanayagam and Flower; 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
Protein expression vectors that utilize the bacteriophage T7 polymerase/promoter system are capable of very high levels of protein production. Frequently, however, expression from these vectors does not reliably achieve optimal levels of protein production. Strategies have been proposed previously that successfully maintain high expression levels, however we sought to determine the cause of induction failure.
Results
We demonstrated that decreases in protein overproduction levels are not due to significant plasmid loss nor to mutations arising on the plasmid, but instead largely are attributable to chromosomal mutations that diminish the level of functional T7 RNA polymerase, resulting in decreased expression from the plasmid. Isolation of plasmid DNA from non-expressing strains and reintroduction of the plasmid into a T7 RNA polymerase-producing strain such as BL21(λDE3) reproducibly restored high level protein production.
Conclusions
Our results suggest that a major contributing factor to decreased expression levels in T7 based systems is chromosomal mutation resulting in loss of functional T7 RNA polymerase. Consistent with this hypothesis, we found that optimal protein overproduction was obtained reproducibly from T7 promoters using freshly transformed cells that had not been subjected to outgrowth during which mutations could accumulate.
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Background
Often the first step in protein purification is production of the protein of interest in Escherichia coli using a gene expression system that allows selective overexpression of a cloned gene [1]. In the most desirable situation, expression of the cloned gene will consume the majority of the cellular resources, such that at the time of harvest the target protein makes up the bulk of the cell content. Complete purification of the protein then requires fewer selective extraction procedures and results in a more concentrated product. It is clear that optimizing the overexpression step is an effective method for maximizing and facilitating protein purification.
One commonly used system for achieving high levels of target protein production depends on the extremely selective nature of the bacteriophage T7 RNA polymerase for specific promoters [2-4]. In the pET system, the gene encoding T7 RNA polymerase is usually supplied by the host bacterial cell in the form of a λ lysogen which expresses the polymerase gene under control of the lacUV5 promoter [2,3]. The target protein is encoded on a plasmid in which the gene has been cloned so that transcription will be driven by the T7 RNA polymerase. Thus, expression of the T7 RNA polymerase gene is controlled by addition of IPTG to the growth media, and in turn, production of the polymerase controls expression of the plasmid-borne target gene.
The T7 RNA polymerase system is extremely effective, resulting in very high levels of target protein production, and the controllable promoters allow even the cloning and expression of genes encoding highly toxic proteins in E. coli [2,3]. However, anecdotal evidence has suggested that many investigators observe less than optimal expression from this system and even the lack of detectable protein overproduction. In our hands, the level of synthesis of proteins varied, not only with the specific protein encoded on the plasmid but also from sample to sample of the same protein.
Although poor protein synthesis, or complete lack of protein production, appears to be a common problem, strategies to cope with it are varied. Some protocols suggest "pre-induction" tests to determine specific colonies that will result in overproduction [5]. We found that this test, although simple, is tedious and can fail to identify expressor colonies. However, there are a number of effective strategies that have been proposed over the years [2,3,6-8]. While we support these recommendations, in this work we sought to determine the underlying cause of reduced expression from pET vectors in order to more effectively prevent problems with poor protein production levels.
Results
We initially examined the conditions required for optimal target protein production from pET plasmids because we observed inconsistent results upon induction of plasmid-borne genes. This inconsistency was most apparent with the plasmid that encoded SecB, pJW25. Following the protocol provided [5], we streaked a frozen glycerol stock of BL21(λDE3) containing pJW25 for single colonies on LB plates with ampicillin, picked 4 colonies and grew small cultures to analyze overproduction of SecB. Only 1 of the 4 cultures demonstrated detectable overproduction of the SecB protein, and the level of overproduction from that one was not remarkable. Random selection of a large number of additional colonies (greater than 100 total) resulted in only 25–30% that synthesized levels of SecB that were clearly greater than the uninduced control. Further, if a culture that was determined initially to overproduce SecB was inoculated into a larger volume (500 ml), overproduction of SecB in the larger culture was not always detected. As we intended to grow strains for overproduction in a 5 liter fermentor, it was critical that the culture used reproducibly synthesize high levels of target protein.
While overproduction of SecB was unreliable from frozen stocks, we found that BL21(λDE3) that had been newly transformed with pJW25 overproduced SecB to a much higher level and that every colony examined synthesized SecB to a similar extent. Continued subculturing, however, resulted in a decrease or loss of overproduction (Figure 1A). These results suggested that with time the bacteria were losing the ability to overproduce SecB.
To verify that our observations were not unique to SecB, we examined a variety of other plasmids for overproduction of E. coli proteins in BL21(λDE3); the plasmid pJGGV encodes the secretory protein, proOmpA, pT7SecA produces SecA, the translocation ATPase [9], and pET11Tus encodes the Tus DNA binding protein [10]. Similar results were obtained with each of these plasmids. In all cases, newly transformed BL21(λDE3) gave rise to high levels of target protein production, while repeated subculturing decreased the level of expression, eventually to undetectable levels. The initial level of synthesis varied for each protein as did the severity of the effect of subculturing on overproduction. In all cases, however, the degree of overproduction was optimal immediately after transformation. Results are shown for data obtained with the pJW25 (SecB) and pJGGV (proOmpA) plasmids (Figure 1).
To more accurately determine the effect of subculturing on target protein production, protein levels were monitored as the strains were allowed to grow. Newly transformed BL21(λDE3) were grown as overnight cultures, then repeatedly subcultured as described in Methods. After each subculturing, when cultures reached early log phase (A600 = 0.4), a sample was removed and tested for the ability to overproduce plasmid-encoded protein. The amount of SecB synthesized by pJW25 decreased gradually with each subculturing, so that by the eighth subculture SecB protein production was minimal (Figure 1A). At this point, four of eight cultures no longer made detectable levels of SecB; the other four were greatly reduced in secB expression (data not shown).
The synthesis of proOmpA was even more dramatically affected by subculturing. Induction was abolished very quickly as there was no detectable overproduction in 15 out of 16 cultures after 3 subculturings (Figure 1B). SecA retained reasonable, but low, protein production levels through 11 subculturings, while Tus synthesis was nearly abolished after only 3 subculturings (data not shown). In all cases, cultures that continued to overproduce protein did so at greatly reduced levels. These results indicated that some proteins are more deleterious to the cell and that continued growth, even without induction, results in alterations to the strain such that overproduction is no longer possible.
We considered the following possible explanations for decreased synthesis of target protein with time: 1) the plasmid was not stably maintained in all bacteria, even with the constant presence of selective agent, 2) the plasmid was accumulating mutations that decreased expression levels, or 3) the bacterial strain accumulated mutations that inhibited production of functional T7 RNA polymerase, resulting in decreased or no expression from the plasmid. We examined each of these possibilities.
Previous protocols highly recommend that strains be examined for loss of plasmid prior to induction, and suggest that this is the major contributor to decreased expression levels [2,3]. However, our strains were grown with constant selective pressure for plasmid maintainence and subculturings were always made at high dilutions (1:1000) to minimize transfer of any β-lactamase from lysed cells to the fresh culture. Therefore, we felt that, while plasmid loss may be occurring, it probably would not be sufficient to account for the dramatic decreases observed in protein levels. To address this point, we prepared plasmid DNA from strains that had been newly transformed and overproduced proteins at high levels and also from the same strains that had been subcultured repeatedly and no longer synthesized high levels of target protein. We found that the amount of DNA present as observed by agarose gel electrophoresis was quite similar in both strains, regardless of the plasmid examined (data not shown). While the results we obtained are not quantitative, they indicated that plasmid loss alone could not account for the reduced level of expression.
To address possible plasmid loss more directly, we compared newly transformed strains and ones that had been subcultured repeatedly for their ability to form individual colonies on LB agar or LB agar containing ampicillin. As β-lactamase is a periplasmic enzyme, it is possible that sufficient leakage of the enzyme occurred to inactivate the ampicillin in liquid culture and allow growth of bacteria that no longer retained plasmid DNA despite the high dilution (1:1000) upon subculturing. If that were the case, we would expect the strain that had been subcultured many times to contain fewer plasmid-containing cells and therefore to form fewer colonies on LB+ampicillin. If no significant plasmid loss were occurring, we would expect the number of colonies to be equal whether or not ampicillin was present. We found that the pJW25-containing strain formed equivalent numbers of colonies on LB and on LB-ampicillin, even after subculturing eight times to result in a culture that did not overproduce SecB (data not shown). Thus, plasmid loss is not sufficient to account for the observed decrease in protein overproduction in these strains.
The second hypothesis was that the plasmids had suffered mutations that rendered them unable to overexpress target genes, for example mutations to the T7 RNA polymerase binding site or to the Shine Dalgarno region. To observe such a great decrease in protein production, a mutated plasmid must confer a growth advantage that results in cells containing the mutated plasmid to overtake the culture. Therefore, strains that no longer overproduce significant amounts of target protein should contain mutated plasmid. To test this possibility, we isolated plasmid DNA from strains that no longer synthesized significant amounts of target protein and retransformed BL21(λDE3) with the plasmid DNAs. If plasmid mutations were occurring, we would expect a large number of the transformants to be poor expressors. On the other hand, if plasmid mutations were not responsible for decreased expression, then we would predict that the new transformants would produce high levels of protein. Indeed, this was the result we observed. Every colony examined, from every plasmid tested, synthesized target protein efficiently after retransformation. Results with pJW25 and pJGGV are shown in Figure 1. The levels of protein production in newly transformed cells were similar to that of the original strain. This result indicated that plasmid mutation was not a significant cause of decreased target protein production from these plasmids.
The third hypothesis was that mutation of the bacterial chromosome might lead to decreased production of active T7 RNA polymerase, in turn decreasing expression from the plasmid. We felt this explanation was probable as a single chromosomal mutation that diminished T7 RNA polymerase expression could be sufficient to abolish protein production from all resident plasmids and this mutant could quite likely overtake the culture in a fairly short period of time, even in the presence of antibiotic to maintain the plasmid. We examined this possibility by titering a mutant T7 phage, Δ4107, that requires host production of T7 RNA polymerase for growth [2]. T7 Δ4107 produced very large, clear plaques when grown on BL21(λDE3) (Figure 2A). When the host strain was BL21(λDE3) that had been newly transformed with pJW25, the titer decreased to about 25% the original, and the plaques produced were slightly smaller (Figure 2B). Furthermore, resistant colonies could be seen emerging within the plaques. After BL21(λDE3) containing pJW25 had been subcultured 10 times so that SecB was no longer detectably overproduced, the phage was again titered. The titer was about 25% of the original, but the plaques produced on this strain were very small and cloudy (Figure 2C). These results are consistent with loss of functional T7 RNA polymerase. We would not predict complete loss of plaque forming ability as the strain culture probably consists of a mixed population at the time of plating; that is, while most cells in the population would be derived from a mutant in which there is little or no functional T7 RNA polymerase, there may be some cells that have not suffered a mutation and would therefore support growth of the phage. We conclude therefore, that a major cause of decreased production of proteins from pET plasmids may be a decreased level of functional T7 RNA polymerase due to mutations in the BL21(λDE3) chromosome.
The loss of T7 RNA polymerase activity could be due either to mutation of the polymerase gene or excision of the λ prophage. To distinguish between these possibilities, we performed PCR using primers that amplify an 1100 base pair fragment of the T7 RNA polymerase gene. As template DNA, we used either BL21(λDE3), BL21(λDE3) freshly transformed with pJW25, or BL21(λDE3) containing pJW25 that had been subcultured eight times and no longer produced significant amounts of SecB. In all cases, a DNA product of the correct size was observed, indicating that prophage excision was not the major cause for decreased T7 RNA polymerase activity (data not shown). Therefore, our results suggest that mutation to the prophage, resulting in decreased levels of functional T7 RNA polymerase, was the predominant contributory factor for decreased production of target proteins.
Discussion
Optimal overproduction is an important first step towards high quantity purification of target proteins. It is critical that one be certain that the culture used will synthesize large quantities of the protein of interest, particularly if it will be used for large scale production procedures, such as growth in a fermentor. When used correctly, the T7 RNA polymerase/promoter system is an excellent choice for such overproduction. However, care must be taken to achieve optimal protein production levels.
It had been suggested previously that plasmid loss is the primary cause for decreased expression from target genes in the pET system [2,3]. Our results differed. While plasmid loss may have occurred to a small extent, in the cases analyzed here the principal reason that lowered levels of protein production occurred was that mutations within the lysogen on the host bacterial chromosome resulted in decreased levels of functional T7 RNA polymerase. The fact that strains which no longer produced plasmid-encoded proteins also no longer supported vigorous growth of the mutant T7 phage, Δ4107, indicates that T7 RNA polymerase function was absent or greatly reduced in the majority of the population. Further, PCR amplification of the T7 RNA polymerase gene suggested that the reduction in polymerase function was due to mutation rather than to prophage excision.
Surprisingly, there was no clear growth disadvantage to strains carrying pJW25 as assessed by monitoring growth over time (data not shown). We do not think that this finding negates our proposal that chromosomal mutation occurs, however. Rather, this may explain why we see loss of expression more rapidly with some plasmids than with others; for example, synthesis of proOmpA was drastically decreased after only three subculturings. Apparently, even in the uninduced state, sufficient expression of target protein occurred, so that a detrimental effect resulted. If that target protein is sufficiently detrimental, selection for polymerase mutations will occur. For this reason, the steps that are taken to ensure optimal expression from target genes must be directed at limiting opportunities for mutation to the T7 RNA polymerase and for those mutants to overtake the bacterial culture.
Two methods have been suggested previously to ensure high levels of protein production; either screening individual colonies for protein synthesis [5] or testing the population of bacteria to assess the fraction of cells capable of overproduction [2,3]. The first is tedious and unreliable, while the second may be better used as an indicator of the degree of mutation that has occurred. However, as we found in this study, these precautions alone are not sufficient to ensure that the colony used for expression will be the highest protein producer possible. In particular, if any further growth of the strain occurs between the time of testing and the actual induction, mutations may occur that decrease expression levels. Our findings support previous recommendations for growth and storage of BL21(λDE3) containing pET plasmids [2,3,6,7] as the conditions described would limit mutation of the T7 RNA polymerase gene. Specifically, plasmid-containing BL21 (λDE3) grown to saturation in rich media will allow basal expression of the target gene [3]. Depending on the toxicity of the protein thus produced, these conditions will select for random mutations to the chromosomally encoded T7 RNA polymerase. Our studies also support the use of bacterial strains that contain the T7 lysozyme as the lysozyme is an inhibitor of the T7 RNA polymerase and will thus decrease uninduced expression even further [3].
We found that the quickest and most reliable method for obtaining optimal protein production was to freshly transform BL21(λDE3) with the desired plasmid, and use a colony directly from the transformation plate for expression studies with only a single subculture of an overnight culture. Should decreased levels of target protein production be observed, the problem can be simply remedied by isolating plasmid DNA from poor expressors and retransforming BL21(λDE3). However, we acknowledge that this approach may not be realistic for very large scale production facilities. Nevertheless, understanding the molecular basis for decreased protein production will facilitate development of techniques that will minimize conditions that may lead to selection and outgrowth of mutant strains.
Conclusion
A common difficulty with overproduction of proteins using the T7 RNA polymerase based system is decreased target protein synthesis or even lack of detectable protein production. It is clear that synthesis of proteins, even at low basal levels, leads to the loss of induction capability. Effective strategies have been presented previously to avoid loss of expression, all of which are based on preventing basal expression levels. In this report, we demonstrate that a significant underlying mechanism leading to loss of expression is a decrease in functional T7 RNA polymerase, and selection of mutants unable to express the recombinant gene.
Materials and methods
Bacterial strains and plasmids
Escherichia coli B strain BL21(λDE3) was used for overproduction of proteins from plasmids containing T7 promoters and was obtained from Stratagene. All plasmids are derivatives of pET11 (Stratagene). Plasmids encoding SecB (pJW25) [5], SecA (pT7SecA) [9], and Tus (pET11Tus) [10] were generous gifts from Linda Randall, Bill Wickner, and Thomas Hill, respectively. The plasmid encoding proOmpA (pJGGV) was constructed in this laboratory by PCR amplification of the ompA gene from E. coli K12 strain MC4100 [11] using primers ompA-1 (gacctacccgggcatatgaaaaagacagctatcgc) and ompA-2 (ggtcatcccgggtgatcattaagcctgcggctgagttac, underlining indicates regions of homology to the ompA gene). The PCR product was digested with restriction enzymes NdeI and BclI and cloned into pET11c that had been digested with NdeI and BamHI. Standard protocols were used for PCR, restriction digests, ligations, and transformations [12,13]. Plasmid DNA was recovered from strains using a QiaPrep Spin Miniprep kit (Qiagen) following manufacturer's instructions.
Induction and analysis of protein production
All strains were grown in LB medium [14]. When plasmid was present, ampicillin was added to a concentration of 100 μg/ml. Cultures were induced for protein production at an A600 of 0.4 by addition of IPTG to a final concentration of 1 mM. Growth was allowed to continue for 2 hours after addition of IPTG. Uninduced controls were grown the same except no IPTG was added. Cells were lysed by boiling in SDS [14], and proteins were analyzed by SDS polyacrylamide gel electrophoresis [13].
For experiments using newly transformed BL21(λDE3), colonies were picked directly from the transformation plate and inoculated into 5 ml LB containing ampicillin for overnight growth. The overnight culture was diluted 1:1000 into fresh LB with ampicillin and grown to an A600 of 0.4 for induction. Thus, the bacteria were subcultured only once. For continuous subculturing experiments, samples were removed before addition of IPTG and used to inoculate fresh LB plus ampicillin media at a dilution of 1:1000.
T7 phage titering
Bacteriophage T7 mutant Δ4107 [2], which was a generous gift from Dr. William Studier, was grown for single plaques on BL21(λDE3) containing various plasmids using standard phage protocols [14].
PCR amplification of T7 RNA polymerase
Genomic DNA was isolated using the Qiagen DNeasy kit. Oligonucleotides used as primers (t7pol1 – gattaacatcgctaagaacg and t7pol2 – gattcatgtcgatgtcttcc) were obtained from Midland Certified Reagents, Midland, TX. PCR was performed using the FailSafe PCR kit (Epicentre Technologies, Madison, WI) following manufacturer's recommendations. PCR products were visualized by agarose gel electrophoresis.
Abbreviations used
IPTG – isopropyl-β-D-galactoside
PCR – polymerase chain reaction
LB – Luria-Bertani
SDS – sodium dodecyl sulfate
Authors' contributions
JGGV was responsible for the original observations of inconsistent expression levels and the data for Figure 1, as well as the agarose gel analysis not shown. AMF performed the experiments for Figure 2 and prepared the manuscript. Both authors read and approved the final manuscript.
Acknowledgements
We thank Majda Valjavec-Gratian, Thomas Hill, and Shelley Horne for helpful discussion. We are indebted to Margaret Smith and Edith Green for technical assistance. We are very grateful to John Dunn for helpful discussion. This work was supported by Career Award MCB-9600851 from the National Science Foundation (AMF) and NSF award MCB-0110594 (AMF).
Figures and Tables
Figure 1 Protein production levels under various conditions. SDS PAGE shows protein levels of SecB (A) and proOmpA (B). Gels were stained with Coomassie Blue. A. 15% PAGE of cell lysates from BL21(λDE3) containing pJW25. Arrow indicates SecB migration. Lane 1, molecular weight marker; lanes 2 (uninduced) and 3 (induced) are samples from cells immediately after transformation; lanes 4 (uninduced) and 5 (induced) are the same strain after 3 subculturings; lanes 6 (uninduced) and 7 (induced) after 8 subculturings; lanes 8 (uninduced) and 9 (induced) are BL21(λDE3) retransformed with the plasmid rescued from the strain in lane 6. We note that lane 8 was unintentionally underloaded. B. 10% PAGE of cell lysates from BL21(λDE3) containing pJGGV. Arrow indicates proOmpA migration. Lane 1, molecular weight marker; lanes 2 (uninduced) and 3 (induced) are samples from cells immediately after transformation; lanes 4 (uninduced) and 5 (induced) are the same strain after 3 subculturings; lanes 6 (uninduced) and 7 (induced) are BL21(λDE3) retransformed with the plasmid rescued from the strain in lane 4.
Figure 2 Growth of T7 phage Δ4107. A. Host strain was BL21(λDE3), 0.1 ml of phage from a 10-8 dilution was plated. B. Host strain was BL21(λDE3) freshly transformed with pJW25. 0.1 ml of phage from a 10-7 dilution was plated. C. Host strain was BL21(λDE3) carrying pJW25 after 10 subculturings. 0.1 ml of phage from a 10-6 dilution was plated.
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| 15638935 | PMC545050 | CC BY | 2021-01-04 16:05:47 | no | Microb Cell Fact. 2005 Jan 7; 4:3 | utf-8 | Microb Cell Fact | 2,005 | 10.1186/1475-2859-4-3 | oa_comm |
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Thromb JThrombosis Journal1477-9560BioMed Central London 1477-9560-3-11563893910.1186/1477-9560-3-1Original Clinical InvestigationThe risk of hemorrhagic complications in hospital in-patients who fall while receiving antithrombotic therapy Bond Andrew J [email protected] Frank J [email protected] Marilyn [email protected] Marlene [email protected] Malcolm [email protected] Clinical Epidemiology Program, Ottawa Health Research Institute, Ottawa, Ontario, Canada2 Elisabeth Bruyere Research Institute, Sisters of Charity Ottawa Health Service, Ottawa, Ontario, Canada3 Division of Geriatric Medicine, University of Ottawa, Ottawa, Ontario, Canada4 Department of Nursing Professional Practice, The Ottawa Hospital, Ottawa, Ontario, Canada2005 7 1 2005 3 1 1 17 8 2004 7 1 2005 Copyright © 2005 Bond et al; licensee BioMed Central Ltd.2005Bond 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 use of antithrombotic agents and falls are independently associated with an increased risk of hemorrhagic injury. However, few studies have delineated the risk of fall-related hemorrhagic complications in persons who are taking antithrombotic therapy. The objective of this study was to compare the rates of fall-related hemorrhagic injury in hospital in-patients who are taking and not taking antithrombotic therapy.
Methods
A 4-year retrospective chart review of consecutive patients who fell during admission to a 500-bed tertiary-care teaching hospital was conducted. Major hemorrhagic injuries including subdural hematomas and major bleeding/cuts, patients' use of antithrombotic medication (warfarin, aspirin, clopidogrel and heparin) and their anticoagulation status at the time of their fall were recorded.
Results
A total of 2635 falls in 1861 patients were reviewed. Approximately 10% of falls caused major hemorrhagic injury. One fall resulted in a subdural hematoma. Persons taking warfarin were less likely to suffer a fall-related major hemorrhagic injury compared with persons not taking antithrombotic therapy (warfarin, 6%; no therapy, 11%; p = 0.01). Logistic regression showed that fall-related major hemorrhagic injury was associated with female gender (odds ratio 1.6; 95% CI 1.3, 2.1), use of aspirin (odds ratio 1.4; 95% CI 1.1, 1.8) and use of clopidogrel (odds ratio 2.2; 95% CI 1.1, 4.8), but not with the use of warfarin or heparin, or the intensity of anticoagulation.
Conclusions
In this study, compared with persons taking no antithrombotic therapy, those taking warfarin had lower rates of fall-related hemorrhagic injuries. The absolute rate of the development of fall-related intracranial hemorrhagic injury such as subdural hematomas was low, even in persons taking warfarin. These counter-intuitive results may be due to selection bias, and suggest that physicians are very conservative in selecting patients for warfarin therapy, choosing only those who are sufficiently healthy to be at much lower than average risk of suffering fall-related hemorrhagic injuries. This phenomenon may lead to physicians overestimating the potential for fall-related major hemorrhagic injury in persons taking antithrombotic therapy, with the possible denial of warfarin therapy to many of those who would benefit. This perception may contribute to the care gap between the number of patients who would theoretically derive overall benefit from warfarin therapy and those who are actually receiving it.
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Introduction
Antithrombotic agents such as warfarin, aspirin, clopidogrel and heparin have proven efficacy and are widely prescribed in the prevention and treatment of many cardiovascular and cerebrovascular diseases [1-3]. However, a significant disadvantage of the use of these therapies is an increased incidence of major hemorrhagic episodes [4]. Compared to younger persons, those over the age of 65 years are at higher risk of these antithrombotic-related complications [5]. Falling, which is also more common in older persons, can also lead to an increased risk of hemorrhagic injury [6]. Thus, at least two studies [7,8] have attempted to delineate the risk of fall-related hemorrhagic complications in older persons who are taking antithrombotic therapy. Stein et al [7] examined 400 consecutive falls in stroke patients admitted to a rehabilitation center. They found no excess risk of hemorrhagic complications in those who were taking warfarin compared with those who were not. More recently, a study [8] using decision analytic modeling also determined that the risk of fall-related hemorrhagic complications was low, even in those taking warfarin. It concluded that the risk of falling should not influence the choice of antithrombotic therapy in older persons with atrial fibrillation. However, many clinicians continue to perceive that older persons who are at increased risk of falling have an unacceptably high risk of antithrombotic-related major hemorrhage [9]. Thus, some continue to withhold antithrombotic therapy in patients who could potentially derive significant benefit from treatment [10]. The objective of this retrospective study was to determine whether there is a difference in hemorrhagic complications in hospital in-patients who fall and are taking antithrombotic therapy compared with those who are not.
Methods
The protocol was approved by the Ottawa Hospital Research Ethics Committee and is in compliance with the Helsinki Declaration. Since implementation of a fall prevention program in 1991, the Ottawa Hospital – Civic Campus, a 500-bed tertiary care teaching hospital, has had a formal policy of documenting the pertinent details of all patient-related falls occurring within the hospital. Using its dedicated falls database, the records of consecutive falls occurring over a 4-year period (January 1, 1997 to December 31, 2000) were identified. The hospital policy requires completion of an incident report when patients fall. Information collected includes the time and circumstances of the fall, the type of injury (including head injury) that occurred and a categorization of its severity, and the need for subsequent follow-up. A member of the physician team must eventually review all fall incident reports, follow-up any significant injury from the fall and document this information in the hospital record and incident report.
Hemorrhage (i.e. cuts or bruising) was classified as major if the immediate attention of a physician was required, or a clinically apparent intracranial hemorrhage subsequently occurred. All other hemorrhage was classified as minor.
Since patient falls can be seen as a reflection of sub-optimal nursing care, the hospital has adopted a non-punitive approach to the completion of reports in order to focus on improving the process that led to the falls. The hospital recognizes that the value of the falls database is predicated on maximum completion of incident reports, leading to the assignment of nurse practitioners dedicated to ensuring the completion of the falls reports. Therefore, it is unlikely that falls leading to an injury would not be captured. During the study period, the hospital had no formal policy or guidelines governing the use of antithrombotic therapy in patients.
For all identified falls, the hospital records of the corresponding in-patient admission were retrieved. Data extraction from these charts included pertinent patient demographic information, and the use of antithrombotic therapy (warfarin, aspirin, clopidogrel and/or heparin) at the time of the fall. The indications for the use of the particular antithrombotic therapy were also recorded. Using computerized laboratory reports, the international normalized ratio (INR) and partial thromboplastin time (PTT) values that were closest to the time of the fall were also recorded. Patients' INR values at the time of the fall were determined as follows:
1. If an INR had been done within 12 hours pre- or post-fall, this was accepted as the INR value at the time of the fall. For patients with both 12 hour pre- and post-fall INRs, these values were averaged.
2. For patients not receiving warfarin, if an INR had not been done within 12 hours of the fall and the closest temporal INR was within the normal range (INR<1.2), this was accepted as the INR value occurring at the time of the fall. If the INR values were abnormal, then the pre- and post-INR values temporally closest to the time of the fall were averaged.
3. For persons receiving warfarin at the time of the fall, if no 12 hour pre- and post fall INRs were available, then the pre- and post-fall INR values done temporally closest to the fall were averaged.
4. For persons who did not have an INR while in hospital and were not receiving warfarin, their charts were reviewed for possible reasons to have an elevated INR (e.g. liver disease, coagulapathies). If these were not present, their INR were deemed to be normal (INR 1.0).
For patients taking heparin, an identical approach was used for determining the PTT values at the time of their fall.
The discharge summaries, nursing notes and medical notes of the hospital records were also reviewed for any immediate and subsequent complications due to the fall including head trauma, subdural hematoma, intracerebral hemorrhage, fractures, major and minor hemorrhage. Multiple falls by the same person were considered independent events. Data extractors were not blinded to the exposure status of patients or their outcomes.
Statistical analysis was performed using SPSS software version 10 (SPSS, Chicago, IL). Chi-square testing was performed to determine the relationship between individual demographic and clinical factors, and the occurrence of major hemorrhage. Step-wise forward logistic regression analysis was then performed with variables that had p-values < 0.20 on univariate analysis. Given that multiple statistical comparisons were performed, a p-value < 0.01 was considered statistically significant.
Results
For the 4-year period, there was a total of 2664 recorded falls in 1861 patients. Despite numerous attempts, the corresponding hospital records could not be located for 29 (1.1%) of the falls. Thus, pertinent data were available and extracted regarding 2635 falls. A significant percentage (29.4%) of patients fell more than once during their admission. The average age of the patients was 71.5 years (SD 15.2; range 16–104), with most being male (55.2%).
Antithrombotic use persons who fell
Table 1 (see additional file 1) shows the details of the antithrombotic therapy use of the patients who fell. Approximately 50% of the patients were taking some form of antithrombotic therapy (warfarin, aspirin, clopidogrel or heparin) at the time of their fall. Approximately 20% of patients had INR values that were higher than the normal range, with a similar number having PTT values outside the normal range. The most common reason for patients to be taking warfarin was stroke prevention in atrial fibrillation. The most common reasons for taking aspirin and heparin were prevention of myocardial infarction and deep vein thrombosis prophylaxis respectively. No patients with high INRs due to reasons other than warfarin use (e.g. liver failure) were found.
Fall-related injuries
Table 2 (see additional file 2) shows the subsequent injuries due to the falls. Major hemorrhage (i.e. bruising and/or cuts requiring immediate attention from a physician) occurred with 10.7% of falls (n = 282). Only one fall resulted in the development of a subdural hematoma. This person was an 89 year old male who was taking warfarin 2 mg and aspirin 81 mg daily (both for stroke prevention in atrial fibrillation) at the same time. His INR and PTT at the time of the fall were 4.3 and 77 seconds respectively. It was documented that he suffered head trauma during the fall. The subdural hematoma was confirmed by CT scan of the head, and he subsequently died from his injuries.
There was also one fall possibly resulting in an intracerebral hemorrhage occurring in a 60 year old female. She also suffered head trauma from her fall, but was not taking any antithrombotic therapy, and her INR and PTT values were in the normal range at the time of the fall. She recovered from this injury, with no apparent sequelae.
The absolute rate of major hemorrhagic injury (i.e subdural hematoma, intracerebral hemorrhage and major bruising) was lower in persons taking warfarin, compared with those taking no antithrombotic therapy at all (warfarin, 6.2%; no therapy, 11.3%; p = 0.01). A comparison of major hemorrhagic injury between those with normal INR values (INR = <1.3) and those in the therapeutic range (INR 2–3) showed a strong trend towards fewer complications in the group with therapeutic INRs (normal INR, 10.1% (209/2066); INR 2–3, 6.9% (15/218); odds ratio 0.65, 95% CI; 0.38, 1.13; p = 0.15). A similar comparison between those with normal INRs and those with INRs between 3–5, showed no statistical difference in hemorrhagic complications between the two groups (normal INRs, 10.1% (209/2066); INR 3–5, 11.4% (9/79); odds ratio 1.14, 95% CI; 0.56, 2.32; p = 0.70).
Univariate analysis demonstrated a very strong relationship between gender and the occurrence of fall-related major hemorrhagic injury (i.e. subdural hematomas, intracerebral hemorrhages and major bruising), with females being much more likely to suffer one of these complications compared with males (13.3% (157/1181) versus 8.7% (126/1454); p < 0.001). Univariate analysis also showed that there were trends towards an increase in the occurrence of major hemorrhagic injury with increasing age (p = 0.04), increasing INR values at time of fall (p = 0.04), and the use of clopidogrel (p = 0.05) or aspirin (p = 0.20). There was no relationship between major hemorrhagic injury with PTT values at time of the fall (p = 0.27), the use of warfarin (p = 0.42) or the use of heparin (p = 0.62). Similar analyses using major bruising/cuts alone (and excluding subdural hematomas and intracerebral hemorrhages) yielded almost identical results. This was due to the occurrence of only one subdural hematoma and one intracerebral hemorrhage.
Logistic regression analysis showed that the factors important in the development of major hemorrhagic injury due to falls were female gender (odds ratio 1.6; 95% CI 1.3, 2.1), the use of aspirin (odds ratio 1.4; 95% CI 1.1, 1.8) and the use of clopidogrel (odds ratio 2.2; 95% CI 1.1, 4.8). Of note, increasing age was not an independent risk factor and there was no interaction between warfarin and aspirin use.
Repeating the analyses with exclusion of all recurrent falls in individuals (n = 1861) resulted in no significant differences in the results reported above.
Of note, fractures occurred in 1.4% of falls (n = 38), with 20 of these being hip fractures.
Discussion
Falling is a common phenomenon in both hospitalized [11] and community-dwelling [12] older persons, with many falls leading to major hemorrhagic injury. Many physicians perceive that the concomitant use of antithrombotic therapy (especially warfarin) increases the chance of fall-related major hemorrhagic injury. This study documents the frequency of these injuries due to falling in hospitalized persons taking antithrombotic therapy and compares them to those who are not.
Numerous studies [13-15] have shown that the frequency of hemorrhagic injury is directly proportional to the intensity of anticoagulation. This study found a significant trend (p = 0.04) towards higher intensity anticoagulation status (as measured by INR values) leading to an increasing chance of the development of fall-related major hemorrhagic injury. However, when compared to persons with INRs in the normal range (INR <1.3), there was no trend suggesting that persons with INRs in the therapeutic range (INR 2–3) suffer more frequent major hemorrhagic injury. This suggests that persons with INRs in the therapeutic range are not at increased risk of suffering fall-related major hemorrhagic injury, with excess risk only in those with INRs above the therapeutic range.
The overall results of this study found that persons taking warfarin were less likely to suffer a fall-related hemorrhagic injury, compared to those taking no antithrombotic therapy. This counter-intuitive result may be due to selection bias. That is, physicians were very conservative in selecting patients for warfarin therapy, choosing only those who were robust enough to be at very low risk of suffering a fall-related hemorrhagic injury. Thus, physicians possibly overestimate the potential for major hemorrhagic injury in persons taking antithrombotic therapy, leading to the possible denial of warfarin therapy to many of those in whom warfarin would otherwise be indicated. This practice may contribute to the well-documented care gap between the number of patients who would theoretically derive overall benefit from warfarin therapy and those who are actually receiving it. [16,17] However, it must be remembered that other reasons for this care gap may exist. For example, since the risk of stroke from atrial fibrillation more of a long-term, rather than short-term clinical decision, the in-hospital physicians may have had a tendency to defer decision-making about anticoagulation to the primary care physicians of these patients.
In this study, only 1 SDH occurred as a result of the more than 2500 falls. Therefore, it was not possible to perform meaningful statistical analyses regarding the contributors to this complication. However, the results confirm that fall-related subdural hematomas are not common in older hospitalized persons, even if they are taking antithrombotic agents. That being said, the development of the single SDH found in this study was almost certainly related to the concomitant use of warfarin and aspirin, with an associated INR of greater than 4.0.
The reason(s) for this study finding that female patients have a greater risk of developing fall-related hemorrhagic injury is unclear. Age was not a factor as the mean age of female patients (71.6 years) was similar to male patients (71.5 years). Also, the percentages of female and male patients taking warfarin, heparin, clopidogrel or aspirin in this study were very similar. Other studies [18,19] have shown that females are more likely to suffer fall-induced injuries compared with males, though these studies included non-hemorrhagic injuries such as fractures. The relationship between gender and fall-related fractures is explainable by the higher prevalence of osteoporosis in the older female population.
The use of aspirin or clopidogrel is generally considered to be less likely to lead to major hemorrhagic injury when compared with the use of warfarin or heparin. Therefore, it was surprising to find that there was a weak, but statistically significant association between fall-related major hemorrhagic injuries and the use of aspirin or clopidogrel, but no such relationship with the use of warfarin or heparin. Again, this result may be due to selection bias, with physicians favoring the use of aspirin or clopidogrel over warfarin or heparin in persons who are less healthy and more prone to serious hemorrhagic injury if they fall.
There are a number of limitations to our study. Due to the retrospective design, it was not possible to apply standardized definitions and measures when determining the occurrence, severity and consequences of falls. Also, we examined the injuries related to hospital-based falls. Therefore, it is unclear whether our results are generalizable to other settings. However, the 10.7% rate of major fall-related hemorrhagic injury (SDH, ICH or major bruising/cuts) in this study is similar to previous hospital- and community-based studies [6,12] that found that approximately 5%–10% of falls result in serious injury. In our database, there were fewer falls causing minor hemorrhagic injury compared with major hemorrhagic injury. This suggests that there was likely underreporting of minor hemorrhagic injury due to falls. This may have occurred because, despite hospital policy, nurses were less inclined to complete incident reports for patients whom they believed had no potential sequelae to their falls. Many falls that resulted in little to no injury may not have been captured. Therefore, the results of our study are likely to overestimate, rather than underestimate, the rate of hemorrhagic injury in persons who fall. The methodology of this study would have been strengthened by reviewing all hospital admissions over the study period for the risk of fall-related injuries. The rate of falling in those receiving and not receiving antithrombotic therapy could then be determined. If those receiving warfarin were less likely to fall, then the conclusion that clinicians were reluctant to prescribe antithrombotic agents (especially warfarin) to patients they deemed at risk for falls would be strengthened. Unfortunately, resource considerations prevented us from taking this approach. It also would have been advantageous to have collected further information regarding potential confounders related to bleeding risk such as the presence of previous falls or fractures, and the amount of time patients were in hospital. However, some of this information was not or could not be reliably collected from the charts. This is because most primary care physicians often defer to the specialists to make these decisions. Finally, since there were persons who fell multiple times, one could argue with our assumption that each fall was an independent event. However, reanalysis of the data by including only the first recorded fall from each individual resulted in no significant changes to the results.
The study also has an important strength. Not all fall-related major hemorrhagic injury (especially subdural hematomas) is identifiable immediately after a fall. We were able to follow-up the sequelae of falling throughout the course of the patients' hospital admission. Therefore, it is unlikely that we failed to identify any serious consequences of falling in our study population.
Conclusion
This study provides evidence that the absolute rate of the development of fall-related subdural hematomas is low, even in persons taking warfarin. Also, the lower than expected rate of fall-related hemorrhagic injury in persons taking warfarin suggests that physicians may overestimate the potential for fall-related major hemorrhagic injury in older persons taking antithrombotic therapy, leading to an overly conservative approach to assessing the risk of anticoagulant-related bleeding. This information may help close the care gap between the number of patients who would theoretically benefit from anticoagulant therapy and the number that actually receive it. Further study is necessary to delineate the characteristics of patients who are at high risk of developing fall-related hemorrhagic injury when taking antithrombotic therapy.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
MM and FM proposed the study. All authors participated in the design of the study and contributed to the drafting and revision of the manuscript. AB and ML conducted the chart reviews.
Supplementary Material
Additional File 1
TABLE1Aug04revised.doc : this is Table 1 entitled, "Fall-related antithrombotic use"
Click here for file
Additional File 2
TABLE2Aug04revised.doc : this is Table 2 entitled, "Consequences of falls"
Click here for file
Acknowledgements
We thank Angeline Luteyn and the rest of the staff in Health Records at the Ottawa Hospital – Civic Campus for tirelessly searching for the hospital records needed to complete this study.
We also thank the anonymous reviewer for his/her helpful and constructive comments and suggestions.
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| 15638939 | PMC545051 | CC BY | 2021-01-04 16:36:24 | no | Thromb J. 2005 Jan 7; 3:1 | utf-8 | Thromb J | 2,005 | 10.1186/1477-9560-3-1 | oa_comm |
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J Transl MedJournal of Translational Medicine1479-5876BioMed Central London 1479-5876-3-11564711910.1186/1479-5876-3-1ResearchPhase I vaccination trial of SYT-SSX junction peptide in patients with disseminated synovial sarcoma Kawaguchi Satoshi [email protected] Takuro [email protected] Kazunori [email protected] Yuriko [email protected] Satoshi [email protected] Tomohide [email protected] Sigeharu [email protected] Hiroeki [email protected] Hideyuki [email protected] Kumiko [email protected] Hiroko [email protected] Toshihiko [email protected] Hiroaki [email protected] Takeshi [email protected] Shin-ichiro [email protected] Noriyuki [email protected] Toshihiko [email protected] Department of Orthopaedic Surgery, Sapporo Medical University School of Medicine, Sapporo, Japan2 Department of Pathology, Sapporo Medical University School of Medicine, Sapporo, Japan3 Marine Biomedical Institute, Sapporo Medical University School of Medicine, Rishirifuji, Japan4 Cancer Vaccine Laboratory, Innovation Plaza Hokkaido, Japan Science and Technology Corporation, Sapporo, Japan5 Division of Orthopedics, National Hospital Organization Hokkaido Cancer Center, Sapporo, Japan6 Division of Orthopaedic Surgery, Chiba Cancer Center Hospital, Chiba, Japan2005 12 1 2005 3 1 1 6 12 2004 12 1 2005 Copyright © 2005 Kawaguchi et al; licensee BioMed Central Ltd.2005Kawaguchi 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
Synovial sarcoma is a high-grade malignant tumor of soft tissue, characterized by the specific chromosomal translocation t(X;18), and its resultant SYT-SSX fusion gene. Despite intensive multimodality therapy, the majority of metastatic or relapsed diseases still remain incurable, thus suggesting a need for new therapeutic options. We previously demonstrated the antigenicity of SYT-SSX gene-derived peptides by in vitro analyses. The present study was designed to evaluate in vivo immunological property of a SYT-SSX junction peptide in selected patients with synovial sarcoma.
Methods
A 9-mer peptide (SYT-SSX B: GYDQIMPKK) spanning the SYT-SSX fusion region was synthesized. Eligible patients were those (i) who have histologically and genetically confirmed, unresectable synovial sarcoma (SYT-SSX1 or SYT-SSX2 positive), (ii) HLA-A*2402 positive, (iii) between 20 and 70 years old, (iv) ECOG performance status between 0 and 3, and (v) who gave informed consent. Vaccinations with SYT-SSX B peptide (0.1 mg or 1.0 mg) were given subcutaneously six times at 14-day intervals. These patients were evaluated for DTH skin test, adverse events, tumor size, tetramer staining, and peptide-specific CTL induction.
Results
A total of 16 vaccinations were carried out in six patients. The results were (i) no serious adverse effects or DTH reactions, (ii) suppression of tumor progression in one patient, (iii) increases in the frequency of peptide-specific CTLs in three patients and a decrease in one patient, and (iv) successful induction of peptide-specific CTLs from four patients.
Conclusions
Our findings indicate the safety of the SYT-SSX junction peptide in the use of vaccination and also give support to the property of the peptide to evoke in vivo immunological responses. Modification of both the peptide itself and the related protocol is required to further improve the therapeutic efficacy.
Synovial sarcomaSYT-SSXantigenic peptidevaccinationPhase I trial
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Background
Synovial sarcoma is a relatively rare, high-grade malignant tumor of soft tissue, characterized by biphasic or monophasic histology, specific chromosomal translocation t(X;18), and its resultant SYT-SSX fusion gene[1]. This tumor affects mostly adolescents and young adults. The 5-year survival rates of patients with this localized disease have ranged from 66% to 80% in the current literature [2-5]. However, the majority of metastatic or relapsed diseases still remain incurable despite intensive multimodality therapy. Therefore there is a need for additional new therapeutic options other than conventional surgery, radiotherapy, and chemotherapy.
Vaccination of tumor antigenic peptide serves as a commonly accepted method in anti-cancer immunotherapy[6,7]. This is based on the rationale that T cells recognize antigenic peptide in the context of MHC molecules on the tumor cell or antigen presenting cells through the T cell receptor, which elicits subsequent anti-tumor immune responses. Identification of antigenic peptides recognized T cells enabled us to apply vaccination trials to a variety of tumors, except bone and soft tissue sarcomas[8].
Currently tumor specific chromosomal translocations are defined in leukemias, lymphomas, and sarcomas[9,10]. The fusion regions of translocation products are specifically expressed by its corresponding tumors, thereby serving as targets of great potential for tumor specific therapies, including immunotherapy[11,12]. We[13,14] found that SYT-SSX fusion gene-derived peptides can be recognized by circulating CD8+ T cells in patients with synovial sarcoma and elicit HLA-restricted, tumor specific cytotoxic responses by in vitro stimulations. In this study, we conducted a phase I pilot trial of vaccination of a SYT-SSX-derived junction peptide in elected synovial sarcoma patients.
Methods
Peptide
A 9-mer peptide (SYT-SSX B: GYDQIMPKK) spanning the SYT-SSX fusion region was synthesized under good manufacturing practice (GMP) conditions by Multiple Peptide Systems (San Diego, CA). The identity of the peptide was confirmed by mass spectral analysis, and was shown to have more than 98% purity when assessed by high pressure liquid chromatography analysis. The peptide was delivered us in the form of a freeze-dried, sterile white powder. It was dissolved in 1.0 ml of physiological saline (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) and stored at -80°C until just before usage. The affinity of the peptide to HLA-A24 molecules and its antigenicity were determined in previous studies[13,14].
Eligibility
The study protocol was approved by the Clinical Institutional Ethical Review Board of the Medical Institute of Bioregulation, Sapporo Medical University, Japan. Eligible patients were those (i) who have histologically and genetically confirmed, unresectable synovial sarcoma (SYT-SSX1 or SYT-SSX2 positive), (ii) HLA-A*2402 positive, (iii) between 20 and 70 years old, (iv) ECOG performance status between 0 and 3, and (v) who gave informed consent. Exclusion criteria included (i) prior chemotherapy, steroid therapy, or other immunotherapy within the past 4 weeks, (ii) presence of other cancers that might influence the prognosis, (iii) immunodeficiency or a history of splenectomy, (iv) severe cardiac insufficiency, acute infection, or hematopoietic failure, (v) ongoing breast-feeding, (vi) unsuitability for the trial based on the clinical judgment of the doctors involved. This study was carried out at the Department of Orthopaedic Surgery, Sapporo Medical University Hospital from June 2003 until the end of September 2004.
Vaccination schedule
Vaccinations with SYT-SSX B peptide were administered subcutaneously into the upper arm six times at 14-day intervals. In order to set up a dose-escalation trial, the patients were separated into the two groups. Each group included three patients. Those from group 1 received 0.1 mg and group 2 participants received 1.0 mg.
Delayed-type hypersensitivity (DTH) skin test
Delayed-type hypersensitivity (DTH) skin test was performed at each vaccination. The peptide (10 μg) solution in physiological saline (0.1 ml) or physiological saline alone (0.1 ml) were separately injected intradermally into the forearm. A positive reaction was defined as a diameter of erythema of more than 4 mm, 48 hr after the injection.
Toxicity evaluation
Patients were examined closely for signs of toxicity during and after vaccination. Adverse events were recorded using the National Cancer Institute Common Toxicity Criteria (NCI-CTC).
Clinical response evaluation
Physical examinations and hematological examinations were monitored before and after each vaccination. Tumor size was evaluated by computed tomography (CT) scans before treatment, and again after three vaccinations, and then at the end of the study period. A complete response (CR) was defined as complete disappearance of all measurable diseases. A partial response (PR) was defined as a >= 50% decrease from the baseline in size of all measurable lesions (sum of products of maximal perpendicular diameters) lasting for a period of at least 4 weeks. Progressive disease (PD) was defined as an increase in the sum of the bi-dimensional measurements of all known disease sites by at least 25% or by the appearance of new lesions. No change (NC) was defined as the absence of matched criteria for CR, PR, or PD.
Tetramer staining
HLA-A24/peptide tetramers (HLA-A24/B, HLA-A24/R49.2, and HLA-A24/HIV) were previously constructed [13-15]. Flowcytometric analysis was performed by taking peripheral blood mononuclear cells (PBMCs) from patients. PBMCs were taken at pre-vaccination and again one week after 1st, 3rd, and 6th vaccination. Cells were stained with PE-labeled tetramers at 37°C for 20 min and a FITC-conjugated anti-CD8 mAb (Becton Dickinson) at 4°C for 30 min. Analysis of stained PBMCs was performed using FACScan (Becton Dickinson) and CellQuest software (Becton Dickinson). The frequency of CTL precursors was calculated as the number of tetramer positive cells / the number of CD8+ cells.
CTL induction
Cytotoxic T lymphocytes (CTLs) were induced from the PBMCs of patients using SYT-SSX B peptides according to the method described before[13,14]. The cytotoxic activity was evaluated by 6-h 51Cr release assay[13]. As target cells, synovial sarcoma cell lines (Fuji, HS-SY-II, and SW982), an erythroleukemia cell line (K562), and a T-B Lymphoblast hybrid transfected with HLA-A*2402 (T2-A*2402) were used. Fuji and HS-SY-II were both HLA-A24 and SYT-SSX positive lines. SW982 and K562 were both HLA-A24 and SYT-SSX negative lines used as controls. T2-A*2402 cells were used to determine peptide-specific cytotoxicity by pulsation with SYT-SSX B or HIV peptide before labeling. The stimulated CD8+ T cells were mixed with the labeled target cells. After a 6-h incubation period at 37°C, the release of the 51Cr label was measured by collecting the supernatant, followed by quantification in an automated gamma counter. The percentage of specific cytotoxicity was calculated as the percentage of specific 51Cr release: [(experimental 51Cr release - spontaneous 51Cr release) / (maximum 51Cr release - spontaneous 51Cr release)] × 100. Maximum 51Cr release was measured by incubating the labelled target cells with 2% NP-40, instead of the stimulated CD8+ T cells. CTL induction was determined as successful when specific cyotoxicity of 10% or more was achieved on Fuji, HS-SY-II, and SYT-SSX B peptide-pulsed T2-A*2402 cells.
Results
Patient profiles
Six patients were enrolled in the study (Table 1). There were four men and two women with an average age of 34.7 years old (range 21–69 years). All patients had multiple metastatic lesions of the lung. A six-time vaccination schedule was completed in three patients, while the remaining three discontinued the vaccination regimen because of rapid disease progression. None of the treatment interruptions were due to the adverse effects of the vaccination.
Table 1 Profiles of participants and clinical resoponses
Patient no. Age Gender Dose of peptide (mg) Number of vaccination Adverse events DTH skin test Evaluation of CT images
1 69 M 0.1 1 - - PD
2 32 M 0.1 3 - - PD
3 21 F 0.1 6 - - PD
4 21 M 1.0 6 - - PD
5 39 F 1.0 6 Fever - NC
6 26 M 1.0 4 - - PD
PD: progressive disease, NC: no change
Safety and DTH skin test
One patient (case 5) experienced slight fever (grade 1) after the first vaccination. No other adverse events were observed during vaccination. DTH skin test was performed at each vaccination and assessed 48 hr later. Reactions were determined as negative in all patients.
Clinical response
Recognized disease progression occurred in five out of six patients during the vaccination period (Table 1, Fig. 1). In contrast, one patient (case 5) showed no such rapid progression (Table 1, Fig. 2). These patients, except in case 1, had received systemic multidrug chemotherapy from one to four months before enrolling on this study.
Figure 1 CT scan image of the lung of case 3 patient. A: Before vaccination (May 15, 2003). B: After the third vaccination (June 18, 2003). Rapid growth of the metastatic tumors and pleural effusion were seen.
Figure 2 CT scan image of the lung of case 5 patient. A: Before vaccination (July 8, 2003). B: After the first vaccination (July 22, 2003). C: After the third vaccination (August 19, 2003). D: After the sixth vaccination (September 16, 2003). The metastatic tumors appeared to be dormant after the first vaccination.
Tetramer analysis and CTL induction
Peptide-specific immunological responses were evaluated in five patients by using HLA-A24/peptide tetramer analysis and in vitro CTL induction. As determined by flowcytometric analysis using HLA/peptide tetramers (Table 2), frequencies of CTLs specific for SYT-SSX B peptide were shown to be at background levels (less than 0.1%) in three patients prior to vaccination. Those frequencies increased after the first (cases 4 and 6) and the third vaccination (case 2) (Fig. 3). In the remaining two patients (cases 3 and 5), SYT-SSX B peptide-specific CTLs existed beyond the background levels before vaccination. Of these, B peptide-specific CTL frequencies increased slightly in case 2 upon a series of vaccinations. On the contrary, the CTL frequencies in peripheral blood decreased to the background level after the third vaccination in case 5, whose metastatic diseases remained stable during the vaccination period. For comparison, tetramers with irrelevant peptides were constructed as internal controls and utilized in four patients. Notably, CTL frequencies reacting to those irrelevant tetramers remained under the background level during the course of vaccinations in all patients.
Table 2 HLA-A24/peptide tetramer analysis
case Pre-vaccination After 1st vac. After 3rd vac. After 6th vac.
2 0.02/N.D 0.02/N.D 3.05/N.D N.D
3 0.42/0.02* 0.49/0.02 0.52/0.02 0.62/0.01
4 0.06/0.01 0.41/0.00 0.36/0.01 0.47/0.01
5 0.50/0.06 0.52/0.01 0.09/0.00 0.03/0.02
6 0.02/0.01 0.15/0.01 0.08/0.00 N.D
N.D: not determined, *B peptide tetramer / Control peptide tetramer
HLA-A24/R49.2 pepitde tetramer was used as control in the case 3 patient.
HLA-A24/HIV pepitde tetramer was used as control in the other patients.
Figure 3 Frequency of CTLs analyzed by HLA-A24/peptide tetramers in the case 4 patient. Frequencies of each analysis were described in Table 2.
Table 3 depicts the results of CTL induction by in vitro stimulations with SYT-SSX B peptide. Before vaccination, CTLs specific for SYT-SSX B peptide were successfully induced from one patient (case 2) who showed a high frequency of CTL precursors. After the first or third vaccination, CTLs were induced from four of five patients. Figure 4 represents the results of cytotoxicity assay. As shown, CTLs induced from the case 4 patient exhibited cytotoxic activities against T2-A*2402 cells pulsed with SYT-SSX B peptide, and synovial sarcoma cell lines expressing HLA-A24 and SYT-SSX (Fuji and HS-SY-II) in various effecter/target ratios examined. In contrast, the cytotoxity was less than 10% against T2-A*2402 cells without peptide pulsation, those pulsed with irrelevant HIV peptide, and tumor cells lacking HLA-A24 and SYT-SSX (SW982 and K562). These findings suggest that induction of peptide-specific immune responses in patients with synovial sarcoma, who received the SYT-SSX junction peptide vaccine.
Table 3 Induction of peptide specific CTLs
case Pre-vaccination After 1st vac. After 3rd vac. After 6th vac.
2 Failure Failure Success N.D
3 Success Success Success Failure
4 Failure Success Success N.D
5 Failure Success Failure Failure
6 Failure Failure Failure N.D
N.D: not determined
Figure 4 Cytotoxicity of CTLs induced from the case 4 patient. T2-P(-): T2-A*2402 cells without peptide pulsation, T2-B: T2-A*2402 cells pulsed with SYT-SSX B peptide, T2-HIV: T2-A*2402 cells pulsed with HIV peptide. Specific cytotoxicity was observed against T2-A*2402 cells pulsed with SYT-SSX B peptide, and synovial sarcoma cell lines expressing HLA-A24 and SYT-SSX (Fuji and HS-SY-II). E/T: Effecter cells/target cells ratio.
Discussion
The present study was designed to evaluate the in vivo immunological property of a 9-mer SYT-SSX junction peptide in patients with disseminated synovial sarcoma. A total of 16 vaccinations of the peptide in six patients revealed (i) no serious adverse effects in any case, (ii) suppression of tumor progression in one patient, (iii) increases in the frequency of peptide-specific CTLs in three patients and a decrease in one patient, and (iv) successful induction of peptide-specific CTLs from four patients. These findings suggest that the SYT-SSX junction peptide is safe to use as a vaccine and also has the property to evoke in vivo immunological responses.
With respect to the clinical efficacy, none of the enrolled patients showed signs of tumor remission. However, in the case 5 patient, tumors showed some dormancy during the vaccination period, in comparison to the other five patients in whom tumors grew rapidly from the early phase of the regimen. The case 5 patient had received one cycle of systemic chemotherapy four months before enrollment, making the possibility of residual chemotherapeutic efficacy unlikely. The other five patients, except in case 1, had received systemic chemotherapy one to two months before perticipation. Notably, frequency of circulating peptide-specific CTLs decreased in the case 5 patient, while CTL frequencies remained unchanged or increased significantly in the other four patients examined. Decrease in circulating CTLs in the case 5 patient may have resulted from accumulation of CTLs at the tumor sites, although biopsy of the tumors was not performed.
Vaccination trials of fusion gene-derived peptides have been reported with BCR-ABL in 12 patients with chronic myelogenous leukemia[16], EWS-FLI1 in 12 patients with Ewing's sarcoma[17], and PAX3-FKHR in four patients with alveolar rhabdomyosarcoma[17]. In addition, Matsuzaki et al.[18] reported a case of a synovial sarcoma patient who were treated with autologous dendritic cells pulsed with a mixture of SYT-SSX junction peptides (8–16 mer). In these studies, tumor remission was noted only in one patient with Ewing's sarcoma where IL-2 was concomitantly administered. These findings together with the results of our present trial have indicated the limited therapeutic efficacy of natural junction peptides. In this regard, we discovered improved in vitro immunogenicity of the SYT-SSX junction peptide by the substitution of an HLA-A24 anchor residue (position 9)[14]. Clinical study of this anchor-substituted peptide is currently underway. Besides modification of the peptide itself, concurrent use of adjuvants and cytokines, and adoptive T cell or/and dendritic transfer should further improve the therapeutic efficacy[8,19]. Also, it is important to determine the appropriate timing of vaccination and proper endpoints of clinical studies.
To monitor the immunological responses, we used HLA/peptide tetramer and in vitro CTL induction. Other monitoring procedures such as ELISPOT assay should provide further information. Nevertheless, the use of an internal control tetramer with HIV peptide added strength in this present comparative analysis. As shown in Table 2 and Fig. 4, reactivity of circulating T cells to HIV peptide tetramer remained under the background level throughout the vaccination period.
Due to the rarity and highly malignant nature of synovial sarcoma, three out of six patients failed to complete the six-time vaccination regimen. Such difficulty in continuation of vaccination has also been found in a trial of patients with Ewing's sarcoma and alveolar rhabdomyosarcoma[17]. Another limitation in the current study is lack of analysis on immunological significance of SYT-SSX variants (SYT-SSX1 and SYT-SSX2). This question should be addressed in a larger scale analysis.
Conclusions
This is the first clinical trial of SYT-SSX fusion gene-derived peptide in patients with synovial sarcoma. The present trial demonstrated the safety and immunogenic property of the peptide. Modification of both the peptide itself and the related protocol is required to further improve the therapeutic efficacy.
Abbreviations
CTL; cytotoxic T lymphocyte, HLA; human leukocyte antigen, MHC; major histocompatibility complex, PBMC; peripheral blood mononuclear cell
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
SK1 and YS carried out vaccinations and clinical evaluations. KI, TT, SK1,2, HA and KS carried out monitoring procedures. TW, SN, HS, HI, TT, HH, TI, ST, NS, and TY made substantial contributions to the design of the study.
Acknowledgements
We thank patients who participated in this trial, Drs. T. Goto and M. Egawa for referral of patients, and Drs. T. Nojima, H. Sonobe, and K. Kuzushima for kindly providing cell lines. We are also grateful to our colleagues and nurses on the 8th ward of Sapporo Medical University Hospital, who provided excellent clinical care to these patients.
Grant support: This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant No.15390462 to S. Kawaguchi, and Grant No. 12213116, 15659097 and 16209013 to N. Sato), Japanese Orthopaedic and Traumatology Foundation (Grant No. 0141 to S. Kawaguchi), and Japan Science and Technology Corporation (Grant No. H14-2 to N. Sato).
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| 15647119 | PMC545052 | CC BY | 2021-01-04 16:39:28 | no | J Transl Med. 2005 Jan 12; 3:1 | utf-8 | J Transl Med | 2,005 | 10.1186/1479-5876-3-1 | oa_comm |
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Microb Cell FactMicrobial Cell Factories1475-2859BioMed Central London 1475-2859-4-21563163410.1186/1475-2859-4-2ReviewProtein secretion in Lactococcus lactis : an efficient way to increase the overall heterologous protein production Le Loir Yves [email protected] Vasco [email protected] Sergio C [email protected] Daniela A [email protected] Anderson [email protected]údez-Humarán Luis G [email protected] Sébastien [email protected] Luciana A [email protected] Sophie [email protected] Jane E [email protected] Valeria D [email protected] Maricê N [email protected] Cathy [email protected] Michel [email protected] Philippe [email protected] Laboratoire de Microbiologie UMR1253 STLO, INRA-Agrocampus, 65, rue de Saint Brieuc CS84215, 35042 Rennes cedex, France2 Institute of Biological Sciences, Federal University of Minas Geiras (ICB-UFMG), Belo Horizonte-MG, Brazil3 Unité de Recherches Laitières et de Génétique Appliquée, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France2005 4 1 2005 4 2 2 13 10 2004 4 1 2005 Copyright © 2005 Le Loir et al; licensee BioMed Central Ltd.2005Le Loir 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.
Lactococcus lactis, the model lactic acid bacterium (LAB), is a food grade and well-characterized Gram positive bacterium. It is a good candidate for heterologous protein delivery in foodstuff or in the digestive tract. L. lactis can also be used as a protein producer in fermentor. Many heterologous proteins have already been produced in L. lactis but only few reports allow comparing production yields for a given protein either produced intracellularly or secreted in the medium. Here, we review several works evaluating the influence of the localization on the production yields of several heterologous proteins produced in L. lactis. The questions of size limits, conformation, and proteolysis are addressed and discussed with regard to protein yields. These data show that i) secretion is preferable to cytoplasmic production; ii) secretion enhancement (by signal peptide and propeptide optimization) results in increased production yield; iii) protein conformation rather than protein size can impair secretion and thus alter production yields; and iv) fusion of a stable protein can stabilize labile proteins. The role of intracellular proteolysis on heterologous cytoplasmic proteins and precursors is discussed. The new challenges now are the development of food grade systems and the identification and optimization of host factors affecting heterologous protein production not only in L. lactis, but also in other LAB species.
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Introduction
Lactic Acid Bacteria (LAB) are anaerobic Gram positive bacteria with a GRAS (Generally Regarded As Safe) status. They are also food grade bacteria, and therefore, they can be used for the delivery of proteins of interest in foodstuff or in the digestive tract. A last advantage compared to other well-known protein producers is that L. lactis does not produce LPS or any proteases as Escherichia coli or Bacillus subtilis do, respectively.
In the last two decades, genetic tools for the model LAB, Lactococcus lactis, were developed: transformation protocols, cloning- or screening-vectors [1,2], and mutagenesis systems [3] are now available. Moreover L. lactis genome is entirely sequenced [4]. Many protein expression- and targeting-systems have also been designed for L. lactis [5-7]. These systems have been used to engineer L. lactis for the intra- or extra-cellular production of numerous proteins of viral, bacterial or eukaryotic origins (Table 1). To produce a protein of interest in fermentors, secretion is generally preferred to cytoplasmic production because it allows continuous culture and simplifies purification. To use L. lactis as a protein delivery vehicle in the digestive tract of humans or animals, secretion is also preferable because it facilitates interaction between the protein (e.g. enzyme or antigen) and its target (substrate or immune system).
Table 1 Heterologous proteins produced in Lactococcus lactis.
Proteins Gene Origin Location References
Reporter
Nuc nuc Staphylococcus aureus Cytoplasmic / secreted / anchored [6, 16]
β-lactamase bla Escherichia coli secreted [44]
β-galactosidase β-gal Clostridium acetobutylicum cytoplasmic [45]
lactamase lacL, lacM Leuconostoc mesenteroides cytoplasmic [46]
α-amylase amyS Geobacillus (formerly Bacillus) stearothermophilus secreted [47] [18]
α-amylase amyL Bacillus licheniformis secreted [48]
Chloramphenicol Acetyl Transferase cat-86 Bacillus pumilus cytoplasmic [49]
M6 Streptococcus pyogenes anchored [12]
Green fluorescent protein gfp Aequoria victoria (jellyfish) cytoplasmic [50]
luciferase luxAB Vibrio harveyi cytoplasmic [51]
luciferase Vf lux Vibrio fischeri cytoplasmic [52]
Streptavidin SA Streptomyces avidinii anchored [11]
β-glucuronidase gus Escherichia coli cytoplasmic [53]
Bacterial antigens
L7/L12 L7/L12 Brucella abortus Cytoplasmic/secreted/anchored [19]
Urease subunit B Helicobacter pilori secreted [54]
TTFC ttfc Clostridium tetani secreted [55]
Eukaryotic antigen
GLURP-MSP3 fusion protein Plasmodium falciparum secreted [56]
Viral antigens
E7 E7 HPV type-16 cytoplasmic/secreted/anchored [20] [57]
NSP4 NSP4 Bovine coronavirus cytoplasmic [29]
BCV epitope BCV Bovine coronavirus secreted [58]
VP8 subunit of VP4 VP8* rotavirus secreted [59]
Interleukins
IL-2 IL-2 Mouse secreted [60]
IL-6 IL-6 Mouse secreted [61]
IL-10 IL-10 Mouse secreted [21]
IL-12 IL-12 Mouse Secreted [22]
IFN-ω IFN-ω Ovine secreted [5]
Allergens
BLG Blg Bovine cytoplasmic/secreted [13, 30, 36]
Epitope Blg41–60 Bovine secreted
Virulence factors
Fibronectin binding protein A fnbpA Staphylococcus aureus anchored [62]
Clumping factor A clfA Staphylococcus aureus anchored [63]
Clumping factor A and B clfB Staphylococcus aureus anchored [64]
serine-aspartate repeat protein sdrE Staphylococcus aureus anchored [64]
Protein A spA Staphylococcus aureus anchored [11]
Enterotoxin A sea Staphylococcus aureus secreted C. Charlier(a)
Unpublished results
Aggregation substance asc10 Enterococcus faecalis anchored [65]
Capsular polysaccharides cps genes Streptococcus pneumoniae CPS excreted [66]
Internalin inlA Listeria monocytogenes anchored V. Guimarães(b)
Unpublished results
Bacteriocins
ABP-118 abp118 Lactobacillus salivarius subsp. salivarius secreted [67]
Enterocin A ent genes Enterococcus faecium secreted [68]
Pediocin PA-1 ped genes Pediococcus acidilactici secreted [68]
colicin V Escherichia coli secreted [69]
Enzymes
heat-stable alpha-glucosidase malA Sulfolobus solfataricus cytoplasmic [70]
Bacteriophage lytic enzyme ply 118 Listeria monocytogenes bacteriophage secreted [71]
lysozyme hel Hen egg white cytoplasmic [72]
Neutral protease npr Bacillus subtilis secreted [73]
Aminopeptidase N pepN Lactobacillus helveticus secreted [74]
Cell Surface Protease prtB Lactobacillus delbrueckii subsp. bulgaricus anchored [13]
Dextrane sucrase dsrD Leuconostoc mesenteroides secreted [28]
Streptodornase sdc Streptococcus equisimilis secreted [75]
prochymosin PC Bovine secreted [76]
lipase lip Staphylococcus hyicus secreted [77]
plasmin Bovine secreted [78]
others
F18 fimbrial adhesin (receptor binding domain) fedF Escherichia coli Secreted / anchored [27]
S-layer protein slpH Lactobacillus helveticus cell wall associated [79]
(a) : Laboratoire de Microbiologie UMR1253 INRA Agrocampus, 65 rue de Saint Brieuc CS84215, 35042 Rennes cedex
(b) : Unité de Recherches Laitières et de Génétique Appliquée, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France
In LAB, like in other Gram positive bacteria, secreted proteins are synthesized as a precursor containing an N-terminal extension called the signal peptide (SP) and the mature moiety of the protein. Precursors are recognized by the host secretion machinery and translocated across the cytoplasmic membrane (early steps). The SP is then cleaved and degraded, and the mature protein is released in the culture supernatant (late steps). Sometimes, secreted proteins require subsequent folding and maturation steps to acquire their active conformation [8].
In most of the works describing heterologous protein production by recombinant lactococci, only one cellular-location (i.e. cytoplasm, external media or surface anchored) is described. Only a few works report the production of a given protein in different locations using the same backbone vector, the same induction level and or promoter strength, allowing thus a rigorous comparison of the production yields of cytoplasmic and secreted forms.
Here, six examples of different heterologous proteins produced in L. lactis in both secreted and cytoplasmic forms are reviewed and discussed. Our major conclusion is that the best production yields are observed in most of these cases with secretion (up to five-fold higher than with cytoplasmic production). Moreover, engineering the expression cassette to enhance the secretion efficiency (SE, proportion of the total protein detected as mature form in the supernatant) resulted in increased overall amounts of the protein. L. lactis is able to secrete proteins ranging from low-(< 10 kDa) to high-(> 160 kDa) molecular mass through a Sec-dependant pathway. Altogether, these observations suggest that i) heterologous proteins produced in L. lactis are prone to intracellular degradation whereas secretion allows the precursor to escape proteolysis, and ii) conformation rather than protein size is the predominant feature that can impair SE. New perspectives are now opened in the studies of heterologous protein production in L. lactis. Indeed, there is a need for food grade systems and for a better understanding of the host factors influencing heterologous protein secretion in L. lactis . For example, HtrA-mediated proteolysis (HtrA is the unique housekeeping protease at the cell surface) is now well-characterized in L. lactis [9] and can be overcome by use of a htrA L. lactis strain designed for stable heterologous protein secretion [10]. However, intracellular proteolysis (involving Clp complex -the major cytoplasmic housekeeping protease-, and probably other cellular components) remains poorly understood and is also discussed here.
Get out to get more
Genetic tools to target a given protein in different cellular compartments were developed using several reporter proteins [6,11-13] (Table 1). The staphylococcal nuclease (Nuc) is a well-characterized secreted protein whose activity is readily detectable by petri plate assay and it has been used as a reporter protein for secretion studies in several Gram positive hosts [14-16]. In L. lactis, Nuc was used to develop protein targeting- [6] and SP screening-systems [1,2]. Nuc was chosen to develop the pCYT and pSEC vectors for controlled production in L. lactis of cytoplasmic or secreted forms of a protein of interest, respectively (Fig. 1) [5]. The pCYT and pSEC plasmids, where expression is controlled by a nisin inducible promoter, should be used in L. lactis NZ9000 (hereafter referred to as NZ) strain bearing a nisR,K chromosomal cassette, required for the nisin signal transduction [17]. In each case described below, protein sample concentration was adjusted to the cell density of the producing culture (for details see [18]). At similar induction levels in lactococcal strains containing pCYT:Nuc and pSEC:Nuc vectors, the highest production yields were observed with the secreted Nuc form (Table 2). Similar results were obtained with constitutive nuc expression cassettes for cytoplasmic and secreted forms. Nuc was the first heterologous protein where highest protein yields were obtained with the secreted form.
Figure 1 Schematic representation of Nuc cassettes for controlled and targeted production in L. lactis. For details about plasmid constructions and contents see Bermúdez-Humarán et al. (2003) [5]. Plasmid backbone is a derivative of the rolling circle plasmid pWVO1, an E. coli-Gram positive shuttle vector. Arrows (1) indicate the presence of the nisin-inducible promoter (PnisA); solid vertical bars (2) indicate the Ribosome Binding Site of the usp45 gene; the striped bar indicate signal peptide of the usp45 gene (SPUsp); the white bar indicates the insertion of LEISSTCDA synthetic propeptide [18]; dark gray bars indicates Nuc mature coding sequence; stem-loop structures indicate trpA transcription terminators (not to scale). A NsiI restriction site comprises the ATG start codon (in pCYT) or the last two residues of SPUsp (pSEC) and allows a simple and one-step cloning of the cassettes corresponding to the mature proteins for cytoplasmic production (pCYT) or secretion (pSEC).
Table 2 Comparison of the protein yields in secreted vs cytoplasmic production.
Protein Quantification of the secreted form1 Quantification of the cytoplasmic form1 Ratio sec/cyto References
Nuc 20 mg/L 3 mg/L 6 [5]
L7/L12 3 mg/L 0.5 mg/L 6 [19]
E7 (expo)* nd nd 2 to 3 [20]
E7 (stat)* nd nd > 10 [20]
IFN-ω 309 mg/L 159 mg/L 2 [5]
1: protein samples were adjusted to the cell density and protein quantification was performed as described in the references either by western blot or by ELISA.
*: E7 was not quantified but ratio was calculated by scanning the western blot signals and comparing their intensity as described in the corresponding reference.
nd: not determined
Similar results were obtained for the production of a Brucella abortus ribosomal protein. B. abortus is a facultative intracellular Gram negative bacterial pathogen that infects human and animals by entry through the digestive tract. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate for the development of oral live vaccines against brucellosis using L. lactis as a delivery vector. L7/L12 was produced in L. lactis using pCYT and pSEC vectors [19]. Similarly to Nuc production, the production yield of secreted L7/L12 was reproducibly and significantly higher than that of the cytoplasmic form (Table 2).
Another example of higher protein yields in secreted vs cytoplasmic form is the production the human papillomavirus type 16 (HPV-16) E7 antigen, a good candidate for the development of therapeutic vaccines against HPV-16 induced cervical cancer. The E7 protein is constitutively produced in cervical carcinomas and interacts with several cell compounds. E7 was produced in a cytoplasmic and a secreted form in L. lactis [20]. Using similar induction level in exponential phase cultures, E7 production was higher for the secreted form than for the cytoplasmic form (Table 2). This difference was even higher when induction occurred in late-exponential phase, where intracellular E7 was detected at only trace amount whereas secreted E7 was accumulated in NZ(pSEC:E7) culture supernatant (see below). Thus, production of E7 clearly illustrates the fact that secretion results in higher yields in L. lactis.
Production of ovine interferon omega (IFN-ω) further illustrates this observation. In the case of poorly immunogenic antigens, co-delivery of an immuno-stimulator protein can enhance the immune response of the host. In order to optimize the use of lactococci as live vaccines, the production of cytokines was investigated in L. lactis [5,21,22]. IFN-ω is a cytokine able to confer resistance to enteric viruses in the digestive tract by reduction of viral penetration and by inhibition of intracellular multiplication of the viruses. Delivery of ovine IFN-ω in the digestive tract by recombinant L. lactis strains could therefore induce anti-viral resistance and could protect the enterocytes. Ovine IFN-ω cDNA was cloned into pCYT and pSEC plasmids for intracellular (pCYT:IFN) and secreted (pSEC:IFN) production respectively [5]. Induction of recombinant NZ(pCYT:IFN) and NZ(pSEC:IFN) strains were performed at equal level and IFN-ω production was measured. The levels of IFN-ω activity showed that i) an active form of IFN-ω was produced in both strains, and ii) the activity of IFN-ω found in the supernatant and cell fractions of NZ(pSEC:IFN) strain was about two-fold higher than that observed for the cytoplasmic form (Table 2). Similarly to what was observed for Nuc and E7, secretion leads to higher heterologous protein yields.
Better secretion for better yields
L. lactis has been engineered to secrete of a wide variety of heterologous proteins from bacterial, viral or eukaryotic origins (Table 1). There are reports about secretion bottlenecks and biotechnological tools for heterologous secretion in model bacteria such as Escherichia coli and Bacillus subtilis [23,24], but only few data are available concerning this aspect in L. lactis. Protein size, nature of the SP and presence of a propeptide are parameters that may interfere with protein secretion. Some data available about these features are compiled here.
To optimize secretion and thus production yields, the nature of the SP was the first parameter to modify on heterologous precursor as previously shown using Nuc as a reporter protein. The replacement of the native staphylococcal SPNuc by the homologous lactococcal SPUsp45 to direct the secretion of Nuc in L. lactis led to an increased SE [25] (Table 3). On the other hand, the replacement of SPNuc by SPUsp45 did not enhance the SE of NucT (a truncated mature moiety of Nuc devoid of N-terminal propeptide) suggesting the importance of the propeptide in the SE for Nuc [25] (Table 3). However, in several cases, the use of a homologous SP (and especially SPUsp45) allows a better SE compared to a heterologous one. Screening vectors were thus developed to search for new homologous secretion signals in L. lactis [1,2]. These screening works offer now a panel of SPs that are suitable for heterologous secretion. However, when compared to SPUsp45, the newly described SPs were less efficient to direct secretion of Nuc [1]. Even after a direct mutagenesis on SP310, one of these new SPs identified using a screening strategy [1], the enhanced SE was still lower than the one measured with SPUsp45 [26]. However, a recent study by Lindholm et al. showed that a Lactobacillus brevis SP (originated from a S-layer protein) drove the secretion of the E. coli FedF adhesin more efficiently than SPUsp45 [27]. High SE might thus result, at least in part, from good adequacy between the mature protein and the SP used to direct secretion.
Table 3 Effect of the signal peptide and of the insertion of the LEISSTCDA synthetic propeptide on the secretion efficiency.
Protein SEa with SPNuc SE with SPUsp45 Reference
Nuc 60 % >95 % [25]
NucT 30 % 30 % [25]
Protein SE without LEISS SE with LEISS Reference
Nuc 60 % 80 % [18]
NucT 30 % 90 % [25]
L7/L12 35 % 50 % [19]
AmySb + +++ [18]
a: SE, secretion efficiency is the proportion of total protein which is present in the mature secreted form.
b: SE was not determined by western blot and immuno revelation and thus could not be quantified but the activity plate assay demonstrated a clear secretion enhancement (+ to +++) with LEISS.
The fusion of a short synthetic propeptide between the SP and the mature moiety is another innovative biotechnological tool to enhance protein secretion. One such propeptide (composed of nine amino acid residues, LEISSTCDA) was developed and was shown to enhance the SE of several heterologous proteins in L. lactis: NucB, NucT, (Table 3) [18], the B. abortus L7/L12 antigen (Table 3) [19], and the α-amylase of Geobacillus stearothermophilus (Table 3) [18]. Directed mutagenesis experiments demonstrated that the positive effect of LEISSTCDA on protein secretion was due to the insertion of negatively charged residues in the N-terminus of the mature moiety [25]. Furthermore, the enhancement effect does not depend on the nature of the SP, since the secretion of NucB fused to either SPNuc or SPUsp45 was enhanced by LEISSTCDA insertion [25]. Strikingly, the enhancement of SE was reproducibly accompanied by an overall increase of protein yields as determined in Western blot experiments. This observation suggests that heterologous precursors are degraded by intracellular proteases when they are not efficiently secreted and that a higher secretion could be a way to escape proteolysis.
Protein conformation rather than protein size can impair the heterologous protein secretion in L. lactis
Proteins with molecular mass ranging from 165 kDa (size of DsrD, the Leuconostoc mesenteroides dextransucrase, [28]) to 9.8 kDa (size of Afp1, a Streptomyces tendae anti-fungal protein; Freitas et al., submitted) have been successfully secreted in L. lactis. This suggests that protein size is not a serious bottleneck for heterologous protein secretion in L. lactis. In contrast to protein size, conformation may be a major problem for heterologous secretion in L. lactis as illustrated by some recent examples. The first example is the production of the non-structural protein 4 (NSP4) of the bovine rotavirus, the major etiologic agent of severe diarrhea in young cattle. In order to develop live vaccines against this virus, the NSP4 antigen was successfully produced in L. lactis [29]. Derivatives of pCYT and pSEC plasmids were constructed to target NSP4 into cytoplasmic or extracellular location. The highest level of production was obtained with the secreted form. However, no secreted NSP4 was detected in the supernatant and both SPUsp45-NSP4 precursor and NSP4 mature protein were detected in the cell fraction. Two degradation products were detected in addition to the NSP4 precursor and mature protein. These results suggest that the cytoplasmic form of NSP4 was probably totally degraded inside the cell whereas fusion to the SPUsp45 protected NSP4 protein against intracellular proteolysis.
Similar results were obtained when pCYT and pSEC vectors were used to produce the B. abortus GroEL chaperone protein: only pSEC:GroEL plasmid was obtained and subsequently the fusion SPUsp45:GroEL was detected in Western blot experiments (V. Azevedo, unpublished data). In this case, B. abortus GroEL is likely to interact with lactococcal cytoplasmic proteins leading to severe cellular defects and thus to a lethal phenotype. On the other hand, fusion of SPUsp to GroEL might keep the chimeric protein in an unfolded and/or inactive state allowing thus its heterologous production.
Another example is the production of the bovine β-lactoglobulin (BLG) in L. lactis [30,31]. BLG, a 162 amino acid residues globular protein, is the dominant allergen in cow's milk and was produced in L. lactis to test the immunomodulation of the allergenic response in mice when BLG is delivered by a bacterial vector [30]. Western blot and ELISA showed that BLG production was significantly higher when BLG was fused to SPUsp45 although the SE was very low, with no detectable BLG in the supernatant of pSEC:BLG strains [30]. Further studies revealed that a fusion between the LEISS propeptide and BLG could not enhance the SE of BLG above ~5%, as determined by ELISA [31].
For rotavirus NSP4, B. abortus GroEL, and BLG (which are medium-sized compared to DsrD or Afp1), either very low secretion yields or absence of secretion was observed in L. lactis. In all cases, fusion to a SP stabilizes heterologous protein production even though they are not efficiently secreted. These results could be due either to the SP itself that reportedly acts as an intramolecular chaperone or to the protection of the chimeric precursor from intracellular proteolysis by the cytoplasmic chaperones of the Sec-machinery. GroEL (a cytoplasmic chaperone), NSP4 (a structural protein), and BLG (a globular protein) have dramatically different primary sequences. A higher affinity of intracellular housekeeping proteases for these particular sequences cannot be hypothesized since the fusion of a SP leads to the stabilization of the protein. Change of conformation is therefore the predominant criterion involved in the stabilization of the precursors and the higher yields observed. On the other hand, these proteins might undergo rapid folding right after their synthesis, which interferes with (or hampers) the secretion process. Such interferences between protein conformation and SE were previously shown in E. coli and B. subtilis [32,33]. Altogether, these results suggest that protein conformation rather than protein size is a major problem for heterologous protein secretion in L. lactis as well.
A labile protein can be stabilized by fusion to a stable protein
It was clearly demonstrated that the secreted form of E7, a reportedly labile protein, can be stabilized by fusion to Nuc [20,34]. Nuc is reportedly a stable protein and its use, as a fusion partner, does not affect its enzymatic activity. The production of the resulting chimerical protein is thus easy to follow. The cytoplasmic form of E7 was stabilized by the fusion to Nuc even when the production was induced in stationary phase (Fig. 2A), whereas cytoplasmic E7 alone was degraded (see below; Fig. 3). Thus, fusion to the stable Nuc could rescue E7 production in L. lactis and allowed higher protein yields compared to E7 alone [20]. Stabilization by fusion to Nuc was observed for several secreted proteins as well. First, a Nuc-E7 fusion on a pSEC backbone resulted in higher production yield although the SE was altered (Fig. 2B). Fusion to the synthetic propeptide LEISSTCDA in a pSEC:LEISS:Nuc:E7 construction restored an efficient secretion yield [34]. Second, in an attempt to increase the protein yield of the secreted L7/L12, a fusion to Nuc (pSEC:Nuc:L7/L12) resulted in a 2.5-fold increase in production yield (Fig. 2B) [19]. Recent results concerning the production of BLG provide a third example of yield enhancement by fusion to Nuc. A pSEC:Nuc:BLG construction allowed a 2-fold increase in BLG yields compared to pSEC:BLG [31]. These results show that Nuc is a stable carrier protein and has a protective effect on labile heterologous chimerical proteins by reducing its sensitivity to intracellular proteolysis. To our knowledge, Nuc is the fusion partner most commonly tested so far for stabilization in L. lactis. Bernasconi et al (2002) fused the Lactobacillus bulgaricus proteinase PrtB to BLG, which was subsequently stabilized by the PrtB carrier [13]. It is thus difficult to postulate any rule concerning the stabilization effect. Different results (i.e. no stabilization) could perhaps be observed with a different partner and thus could help to determine the mechanism of the stabilization effect. In biotechnological use of recombinant L. lactis strains for protein production, fusions can also facilitate purification (e.g. His-tag strategy). Protein fusion has also been successfully used to optimize the production of the two subunits of heterodimeric complexes as demonstrated with murine interleukin-12 in L. lactis [22] or with heterodimeric enzymes in E. coli [35]. In both cases, the resulting fusion had the expected properties. In other cases however, such fusions might dramatically interfere with the conformation of one or both of the proteins, which might be deleterious for the expected activity. Nevertheless, when L. lactis is used as an antigen delivery vector, fusions can be envisioned since it was demonstrated that both moieties of the chimerical protein are still recognized by the corresponding antiserum [10,20,34] and are immunogenic [36].
Figure 2 Fusion to Nuc rescue E7 in intracellular production and increase protein yields for the secreted forms of E7 and L7/L12. A. A DNA fragment encoding the mature moiety of Nuc was fused to the fragment encoding E7 (pCYT:Nuc:E7). Production of Nuc-E7 analyzed by Western blot using anti-E7 antibodies on protein samples prepared from induced cultures harvested either at exponential (exp) or stationary (stat) phase. Positions and sizes of molecular weight marker (M) are indicated at left. B. The mature Nuc fragment was inserted between SPUsp45 and the fragment encoding E7 (pSEC:Nuc:E7) or L7/L12 (pSEC:Nuc:L7/L12). Secretion of the fusion proteins was analyzed by Western blot using either anti-E7 or anti-L7/L12 antibodies. C, cell lysates; S, supernatant fraction. Positions of precursor (prec) or mature forms of E7, Nuc-E7, L7/L12, NucB-L7/L12, and NucA-L7/L12 are indicated by arrows.
Figure 3 Native E7 production in wt L. lactis depends on growth phase. E7 production and secretion were analyzed by Western blot from cultures induced at different times so that, 1 hour after nisin induction, the samples are harvested at exponential (OD600 = 0.5–0.6, upper panels) or stationary phase (OD600 = 1.5, lower panels). wt/pCYT-E7, NZ(pCYT-E7) strain (encoding native E7, cytoplasmic form). wt/pSEC-E7 NZ(pSEC-E7) strain (encoding the precursor preE7). Positions of E7 mature and precursor forms are given by arrows. C, cell lysates; S, supernatant fraction. ClpP is not involved in the intracellular degradation of E7 in L. lactis. Analysis by western blot shows that a strain of L. lactis deficient in the intracellular protease ClpP cannot rescue cytoplasmic E7 production. Induced cultures samples of wt L. lactis or L. lactis clpP mutant strain containing pCYT-E7 (clpP/pCYT-E7) or pSEC-E7 (clpP/pSEC-E7) taken at exponential- (upper panel) or stationary- (lower panel) phase.
Secretion avoids proteolysis?
Several of the results mentioned above suggest that secretion could be an efficient way to escape intracellular proteolysis. This hypothesis was particularly tested in E7 production [20]. E7 was indeed degraded when intracellular production was induced in late exponential or early stationary growth phase (Fig. 3). E7 production was then tested in a clpP deficient strain (ClpP is reportedly the major house keeping protease in L. lactis; [37]) and in a dnaK deficient strain (DnaK is an intracellular chaperone that may promote proteolysis by maintaining the protein in an unfolded state; [38]). In exponential or stationary phase cultures, no significant difference in E7 patterns was observed between wild type and clpP- (Fig. 3) or dnaK- (not shown) strains: E7 was equally degraded in the cytoplasm and remained unchanged in supernatants samples. Altogether, these results indicate that E7 intracellular proteolysis is ClpP- and DnaK- independent. Until recently, only two cytoplasmic proteases, ClpP and FtsH [39], have been identified in L. lactis. The existence of a third, as yet unidentified protease was postulated by studies of a clpP mutant suppressor [40]. E7 may thus be a useful screening target to identify a putative L. lactis protease that, as suggested by our data, is activated in stationary phase.
Besides the features of the precursor itself, these results also rise that host factors are involved in protein stability and SE (Fig. 4). Research efforts are now focusing on the analysis of host factors involved in protein production and secretion by either directed or random mutagenesis in L. lactis [41].
Figure 4 Schematic presentation of the molecular tools and the cellular events that can affect the production yields of heterologous protein in L. lactis. Thicknesses of the arrows are proportional to the final production yields. All the host factors involved in the cellular events are not identified and or characterized yet. SP, signal peptide (encoded in pSEC constructions), +Nuc, fusion between the protein of interest and the stable Nuc protein.
Although L. lactis possesses a wide range of enzymes (peptidases, housekeeping proteases) dedicated to intracellular proteolysis, it possesses only one extracellular housekeeping protease (HtrA) [9] and its major extracellular scavenger protease, PrtP, is plasmid encoded [42]. Thus, a plasmidless strain does not present any protease activity in the medium. Better production yields could then be expected when secretion is used versus cytoplasmic production. These results give clues and provide the research workers with target proteins to study intracellular proteolysis and protein stability inside and outside the host strain. Such studies already led to the development of htrA deficient L. lactis strains. Heterologous protein secretion and anchoring in a htrA deficient strain allowed higher protein stability at the cell surface for several heterologous proteins [10].
Perspectives
Current research works are now focusing on other host factors that affect protein production and secretion in L. lactis. L. lactis complete genome sequence analysis revealed indeed that the Sec machinery comprises fewer components than the well-characterized B. subtilis Sec machinery. Notably, L. lactis does not possess any SecDF equivalent and complementation of the lactococcal Sec machinery with B. subtilis SecDF results in better secretion yields as determined for Nuc reporter protein (Nouaille et al., submitted). Random mutagenesis approaches also revealed that features of some cell compartment, such as the cell wall, play an important role in the secretion process [41]. Similar approaches allowed the identification and characterization of genes of unknown functions specifically involved in production yields of the secreted proteins in L. lactis (Nouaille et al., in preparation).
Many molecular tools are now available to direct heterologous protein secretion in L. lactis and the list of heterologous proteins produced in this bacterium is regularly increased. The reports where cytoplasmic and secretion production can be compared mostly show that secretion allows better protein yields compared to intracellular production; and allow a better understanding of the protein production and secretion process in L. lactis.
Future works should investigate the L. lactis capacities for protein modifications. For example, we showed that proteins that require a disulfide bond (DSB) to acquire their native conformation can be efficiently produced and secreted in L. lactis [5,22,27]. However, no equivalent of E. coli dsb or B. subtilis bdb, the genes involved in DSB formation, was found by sequence comparison in L. lactis. Similarly, other folding elements (i.e. PPIases, so-called maturases...) are still to be identified and the L. lactis capacities for post-translational modifications are still to be investigated.
Altogether, these works will contribute to the development and the improvement of new food-grade systems for L. lactis [43] and should lead, in a near future, to the construction of lactococcal strains dedicated to high-level production of proteins of interest. The GRAS status of L. lactis and LAB in general, is a clear advantage for their use in production and secretion of therapeutic or vaccinal proteins.
Acknowledgements
Anderson MIYOSHI, Daniela FREITAS, Luciana RIBEIRO, Jane E. GABRIEL, Sophie LECLERCQ, Maricê N. OLIVEIRA, and Valeria D. GUIMARÃES were recipients of a CAPES fellowship (project CAPES-COFECUB #319-II). Luis BERMUDEZ and Sébastien NOUAILLE were recipients of a fellowship from the French Ministry of Education and Research. INRA and Région Ile-de-France also financed L. BERMUDEZ and V. GUIMARAES. Cathy CHARLIER is recipient of a fellowship from INRA and Région Bretagne.
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| 15631634 | PMC545053 | CC BY | 2021-01-04 16:38:15 | no | Microb Cell Fact. 2005 Jan 4; 4:2 | utf-8 | Microb Cell Fact | 2,005 | 10.1186/1475-2859-4-2 | oa_comm |
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Health Qual Life OutcomesHealth and Quality of Life Outcomes1477-7525BioMed Central London 1477-7525-3-11563435410.1186/1477-7525-3-1ResearchCan the concepts of depression and quality of life be integrated using a time perspective? Moore Margaret [email protected]öfer Stefan [email protected] Hannah [email protected] Lena [email protected] Royal College of Surgeons in Ireland, Department of Psychology, Mercer St Lwr, Dublin 2, Ireland2 Belfast City Hospital, Department of Health Clinical Psychology, Northern Ireland, UK2005 5 1 2005 3 1 1 5 10 2004 5 1 2005 Copyright © 2005 Moore et al; licensee BioMed Central Ltd.2005Moore 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 understood about the conceptual relationship of depression and quality of life (QoL). Judgments concerning both, implicitly or explicitly, involve a time perspective. The aim of this study was to test de Leval's theoretical model linking depression and QoL with a time perspective. The model predicts that changes in cognitions about one's past, present and future QoL, will be associated with changes in depressive symptomatology.
Methods
Eighteen psychiatric in-patients with a clinically confirmed diagnosis of depression were assessed on commencing treatment and 12 weeks later. QoL was assessed by the Schedule for Evaluation of Individual Quality of Life (SEIQoL), depression by the Beck Depression Inventory (BDI-II) and hopelessness by the Beck Hopelessness Scale (BHS). Time perspective was incorporated by asking QoL questions about the past, present and future.
Results
Depression and hopelessness were associated with a poorer present QoL. Depression lowered present QoL but did not alter future QoL, as these remained consistently high whether participants were depressed or recovering. However, depressed individuals had a larger gap between their actual present QoL and future (aspired to) QoL. Changes in QoL were influenced by depression and hopelessness. Contrary to the model, perception of "past" QoL was not affected by depression or hopelessness.
Conclusions
de Leval's model was largely confirmed. Thus depression and hopelessness influence a person's present and future QoL. The analysis of a temporal horizon was helpful in understanding the link between depression and QoL.
depressionquality of lifeSchedule for the Evaluation of Individual Quality of Lifetheorytime orientation
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Background
Assessment of quality of life (QoL) has become increasingly important in health care, particularly as an evaluative method to measure outcomes of the impact of disease and interventions. To date it is unclear how research on QoL relates to other psychological constructs such as depression and anxiety. Many clinical studies assess a number of related psychosocial dimensions but without a theoretical basis for the unique contribution of each. On an intuitive level, QoL and depression can appear as opposing phenomena – crudely representing all the positive and negative aspects of well-being. Poor QoL is sometimes seen as a consequence of depression [1-3]. On the other hand, poor QoL may also be a precursor to depression. In other formulations, depression can be seen as a component of QoL. Whatever the implicit models of their interrelationships, there has been little theoretical attention or research to understand the relationship between depression and QoL.
A theoretical approach developed by de Leval [4,5,8] tries to capture and highlight possible relationships between depression and QoL in a "three-time-dimension" theory. This theory links depression and QoL on a timeline of the past-present-future. Time can be perceived objectively and subjectively. It consists of three dimensions: past, present and future. The presence of psychopathology, e.g. depression has been found to influence time perception. For instance, individuals who are depressed have been reported as finding that time passes more slowly [11]. In comparison to others, depressed individuals also have a temporal focus, which is less future directed and more focused on the past.
The "three-time-dimension" theory describes the dislocated temporal horizon of the depressed patient. It situates both depression and quality of life as part of a continuum in time rather than as independent phenomena. De Leval proposes that, for the depressed individual, time passes slowly, the present is dissociated from the past and the potential for the future is lost or viewed with hopelessness [4]. During the course of their depression, individuals want to go back to their past when things were perceived as better. This searching for the past becomes the individual's future. According to de Leval, depressed individuals have two pasts: the actual past when they were well and the past as a position they wish to regain or aspire to, i.e. the 'therapeutic future', when things were better than they are in the present. In the proposed model by de Leval, in accordance with DSM-III-R criteria, depression is referred to as "ill-being". Ill-being is the current or present state of the patient experiencing depression. De Leval uses the term depression for "phenomenological-depression", i.e., depression as perceived by the individual in question and it is placed on the past-present timeline. She proposes that this "phenomenological-depression" is related to the perception of a gap between a healthy past and a present ill-being. The greater the gap between past and present, the greater the phenomenological-depression.
In de Leval's theory, QoL is perceived as the gap between actual experience and future aspirations. Whereas depression is placed on the past-present timeline, QoL is placed along the timeline using present and future. QoL according to this model is defined as being "the appropriateness of future aspirations to the present" or "the making present of the future". The larger the gap, the lower the QoL of the individual (Figure 1). Although not included explicitly in de Leval's theory, the concept of hopelessness as described by Beck [2] is worth considering given its timeline focus on negative evaluations of the future.
Figure 1 Illustration of de Leval's model
In a cross-sectional study, de Leval [5] examined the three-time-dimension theory in a group of 110 clinically depressed psychiatric patients. They completed a 30-item questionnaire – the Three-Time-dimension Synoptic Scale (3TSS – French version) developed by the author [6]. Questions were chosen to reflect the content of existing mental health scales and each question asked about their feelings now, their feelings in the past (before being depressed) and what they wanted to feel in the future. Findings indicated preliminary support for the theory. No other study has to our knowledge tested Leval's theory empirically.
The present study sought to advance the understanding about the conceptual relationship of depression and QoL by empirically testing de Leval's model in a longitudinal study. It also sought to assess the model using a previously developed individualised system for assessing QoL. If de Leval's proposals concerning the centrality of the temporal assessment are correct, then any QoL assessment instrument which measures the present can be adapted to measure the past and the future as aspired to. In this study, the model was operationalised as follows: scores on standardised depression measures were taken to demonstrate an individual's level of depression, i.e., 'present ill-being'. QoL was the gap between an individual's present status ('present ill-being' in the case of depression) and anticipated status (i.e 'therapeutic future') and was measured by the discrepancy between present and future actual QoL scores. The level of phenomenological depression – i.e. the gap between a person's perceived past and present "ill-being" was measured by the discrepancy between the past and present actual QoL scores. These gaps are referred to as time comparisons gaps. The full model is displayed in Figure 1. In addition to components of de Leval's model, aspirational QoL was measured and assessed for all three time periods: past, present and future. This was included to provide further information about the gap between where an individual is and where he/she would like to be. These gaps are referred to as preference comparisons gaps. It was hypothesized that the greater the gap between actual and aspirational scores at any time (past/present/future), the worse the QoL and vice versa. This reflects Calmans definition of QoL. He defines QoL as the gap between actual QoL and preferred QoL [3]. As an additional measure of an individual's perception of the future hopelessness was included . The model was tested at two time points: when individuals were clinically depressed (time 1) and approximately three months later when their progress towards recovery could be estimated (time 2).
The following hypotheses were tested in order to validate de Leval's model and additional components:
Hypothesis 1: The size of the gap between actual and past QoL scores is larger when patients are depressed, in comparison to when they are less/not depressed.
Hypothesis 1a: The therapeutic future is the view of a healthy past. Therefore scores for past and future QoL are equal.
Hypothesis 2: The change in depression scores (present "ill-being") from time 1 to time 2 predicts the change in the gap between actual and aspirational QoL scores.
Hypothesis 3: The change in hopelessness scores from time 1 to time 2 predicts the change in the gap between actual and aspirational QoL scores.
Hypothesis 4: A reduction in depression narrows the gap between the past and present, and enhances QoL by narrowing the gap between present and future QoL.
Methods
Consecutive psychiatric in-patients (N = 27) were approached to participate in a longitudinal study of depression. Study inclusion criteria were age ≥ 18 years, diagnosis of moderate to severe uni-polar depression (DSM-IV-criteria), a moderate or severe depression rating on the Beck Depression Inventory II [1], no cognitive disabilities (Mini Mental State Exam, [7]). Exclusion criteria were diagnosis of severe psychopathology or borderline personality disorder, more than two previous episodes of depression that were of more than three months duration and history of prolonged substance abuse or use of psychotropic medication, which would impede participation in a research interview. Patients were recruited as soon after admission as was deemed appropriate by medical staff.
Instruments
Quality of life (QoL)
QoL was assessed by the Schedule for the Evaluation of Individual Quality of Life (SEIQoL) [12], a well-established method of assessing QoL which incorporates the value system of the individual participant. To reduce interview demands for depressed participants, the shorter direct weighting (SEIQoL-DW) procedure was applied [9]. In the first step of the standard SEIQoL procedure, participants are asked to nominate the five areas of their life (cues) that are most important to them. In the second step, participants rate their current status or level of functioning on each cue. Ratings are against a vertical axis anchored from "worst possibly" to "best possibly".
The final assessment step in SEIQoL involves quantifying the relative importance (weight) of each cue to the judgment of participants' overall QoL. This is obtained by using a weighting disk, consisting of five interlocking coloured disks that can be rotated around a central point to form a pie chart. These disks are labelled with the five nominated cues and are adjusted by participants until they are satisfied that the proportion of the pie chart displayed by each life area accurately reflects the relative importance they attach to these life areas. The SEIQoL Index score is calculated by multiplying each cue weight with the relevant level of functioning. These five scores then are summed. The scale of the SEIQoL Index score ranges from worst possible (0) to best possible (100). This was done for each time anchor and point.
Time perspective
To measure the temporal nature of the gap in terms of perceptions of their actual and aspirational past, present and future QoL according to the Leval's theory and Calman's definition of QoL, participants were asked to rate the status of each nominated SEIQoL cue (i.e. how were you doing?) based on the following questions:
Past:
- Where were you before you got depressed? (actual past)
- Where would you have liked to have been? (aspirational past)
Present:
- Where are you now? (actual present)
- Where would you like to be? (aspirational present)
Future:
- Where do you expect to be in a year’s time? (actual future)
- Where would you like to be in a year’s time? (aspirational future)
Thus scores were taken for actual present, aspirational present, actual past, aspirational past and actual future, aspirational future.
Depression
The Beck Depression Inventory (BDI), a 21 item self-report instrument for measuring the severity of depression in adults, was developed for the assessment of symptoms corresponding to criteria for diagnosing depressive disorders based on the DSM-IV. In this study an updated version is used (BDI-II) [1]. The BDI-II requires about 10 minutes to complete and has a two week time-frame including today. Items are rated on a four point scale ranging from 0 to 3, with higher scores reflecting more severe depression. A cut-off score of 20 was used. The psychometric properties have been well established [1].
Hopelessness
The Hopelessness Scale [2] is a 20 item self-report instrument designed to assess pessimistic expectations. Items are rated true (1) or false (0), with a higher score indicating hopelessness. Internal consistency (split-half reliability) exceeds .90 in a range of samples and concurrent validity has been established [2].
Results
Of 27 patients approached, 24 gave written consent and took part in the study (89% response rate). Over the three month follow-up period, six patients dropped out (75% follow-up rate). Information on the complete sample of 18 patients was analysed. Sociodemographic and clinical details are presented in table 1.
Table 1 Sociodemographic and clinical profile of recruited sample (N = 24)
Mean ± SD % Min Max
Gender (% male) 50
Age (years) 36.7 ± 14.4 18 72
Marital status
Married/partner 42
Single 54
Separated 4
Occupation:
Employed 50
Unemployed/retired 16
Student 21
Homemaker 13
Residence (% urban) 71
Time since hospital admission (days) 5 ± 2.7 1 9
No. of previous episodes of depression 0.7 ± 0.7 0 2
Duration of previous episodes of depression (weeks) 2.6 ± 3.6 <1 12
Baseline scores of the depression measure (BDI-II) showed high levels of severity, which were reduced significantly after three months (p < .001, table 2). While at baseline all 18 patients scored above 20, only six patients remained above this threshold at three months follow-up. The mean hopelessness score (BHS) of 10 at baseline dropped significantly by 5 (p < .002).
Table 2 Profile of mental health at baseline and three month follow-up (N = 18)
Baseline three months
Mean SD Mean SD t p
Depression
BDI-II 34.0 9.5 15.0 11.4 4.8 <.001
Hopelessness
BHS 10.0 5.8 5.0 4.9 3.2 .002
The five most commonly mentioned cues for the SEIQoL at time 1 were: mental health, family of origin, work marriage/relationship, friends and leisure. There were no new cues introduced at time 2. This is consistent with previous research on cue profiles across varying populations. The actual present level of overall QoL was very low at baseline (time 1, see table 3). This increased significantly over three months. Aspirational level of present QoL (where one would like to be now) was significantly higher at both baseline and three months then the actual present level. QoL did not change significantly over time (p = .102).
Table 3 Quality of life (QoL) over time and actual and aspirational "gaps" at baseline and three months measured by SEIQoL Index Score
Actual t p Aspirational t p t p
Present Baseline 35.4 ± 19.1 88.2 ± 11.4 10.5 <.001
3.2 .002 1.3 .102
three months 61.1 ± 22.8 88.5 ± 8.5 5.5 <.001
Past Baseline 70.2 ± 13.7 87.0 ± 10.5 4.8 <.001
.2 .411 1.5 .07
three months 68.7 ± 18.8 83.7 ± 8.4 4.4 <.001
Future Baseline 66.8 ± 21.0 90.8 ± 10.5 5.1 <.001
.3 .362 .2 .396
three months 74.4 ± 23.6 91.5 ± 7.5 3.6 .001
Actual past level of overall QoL did not change from baseline to three months later. Aspirational past QoL (where one would have liked to be then) was significantly higher at baseline and three months than actual past and this did not change over time (table 3). Actual future QoL did not change significantly over the three months follow-up. Actual future QoL was significantly lower at baseline and three months than the aspirational future QoL. Aspirational future QoL was consistently high over time.
The gap between actual and aspirational QoL is presented in table 4. The biggest gap was perceived between baseline actual present level of QoL and aspirational present QoL. This large gap was significantly reduced after three months. This confirms hypothesis 1. Other gaps remained unchanged (figure 2). Correlations between the preference comparison QoL gaps for present, past and future assessments with the severity of depression (d) showed significant high correlations for the present (p) and future (f) gap at baseline (1) and three months (2) (rpd1 = .468, rfd1 = .475, both p < .05, rpd2 = .815, rfd2 = .730, both p < .01). In addition, correlations between the QoL preference comparison gaps for present, past and future assessment and hopelessness (h) were statistically significant at baseline for the present (p) gap and at three months (2) for present (p) and future (f) gaps (rph1 = .567, rph2 = .586, rfh2 = .422, all p < .05). There was no difference between actual past and actual future QoL scores at baseline and three months (p = .475, p = .528). This confirms hypothesis 1a.
Table 4 Differences between actual and aspirational QoL "gaps" at baseline and three months as measured by the SEIQoL Index Score
Time Baseline actual/aspirational gap (M ± SD) Three months actual/aspirational gap (M ± SD) F Time
Present 52.7 ± 21.0 27.6 ± 21.2 9.21 .007
Past 16.8 ± 14.7 14.9 ± 14.9 .154 .699
Future 23.9 ± 19.6 17.0 ± 17.9 2.86 .109
Figure 2 Illustration of the preference comparison gaps (actual/aspirational QoL) for the three time points (past, present and future) at baseline and three months. Δ represents the change from baseline to three months for the time anchor "present" ** p < .001
Multiple regression was conducted to investigate whether depression and hopelessness contribute to the change in the size of the gap of actual and aspirational QoL over time. Changes in depression and hopelessness scores over time were entered as independent variables in a multiple regression, while the change in gap scores was entered as the dependent variable. A significant amount of variance was explained by changes in the present and future gaps for actual and aspirational QoL (52.4%, 59,9% respectively both p < 0.01). Change in depression contributed to change (reduction) in the gap between present actual and aspirational Qol (β =.676). Change in hopelessness (β =.589) was the significant contributing factor for change (reduction) in the gap between actual and aspirational future QoL (table 5). This confirms hypothesis 2 and 3 in parts.
Table 5 Multiple regression analyses to explain the change in the actual/aspirational gap by change in Beck Depression Inventory (BDI) and Beck Hopelessness Scale (BHS) change scores from time 1 to time 2.
Δ in actual/aspirational Gap Variable name1 R2 p-value β p
Present .524 .004
Δ BDI12 .676 .020
Δ BHS12 .063 .813
Past .043 .720
Δ BDI12 .053 .888
Δ BHS12 .165 .661
Future .599 .001
Δ BDI12 .230 .352
Δ BHS12 .589 .026
1Change of Beck Depression Inventory (BDI) and change of Beck Hopelessness Scale (BHS) were entered as predicting variables in these analyses
In table 6, the differences of present and past (phenomenological-depression) and present and future QoL (quality of life) are assessed according to de Leval's model. As already reported, baseline actual present QoL scores were considerably lower than actual past scores (35.4 ± 19.1 vs. 70.2 ± 13.7, p <.001). This significant gap (34.8 ± 22.5) between present and past QoL was significantly reduced over three months to a gap of 7.6 ± 30.9 (p = .011). At the three month follow-up, there was no statistical difference in the gap between actual present and past QoL perception (figure 3). The gap between present and future QoL was large and significant at baseline (31.3 ± 27.2, p < .001). Differences remained significant at the 3 month follow-up (13.3 ± 13.5, p = .001) with higher QoL levels for the future. However, the reduction in the gap over time was also significant (p = .010).
Table 6 Testing de Leval's model: time gap comparison for actual QoL scores measured by SEIQoL Index Score
QoL at Baseline t p QoL at three months t p
Past 70.2 ± 13.7 68.7 ± 18.8
6.5 p < .001 1.0 .310
Present 35.4 ± 19.1 61.1 ± 22.8
Present 35.4 ± 19.1 61.1 ± 22.8
4.8 p < .001 4.2 .001
Future 66.8 ± 21.0 74.4 ± 23.6
Figure 3 Illustration of the time comparison gaps (past-present/present-future QoL; shaded areas) for the three time points (past, present and future) at baseline and three months. Δ represents the change from baseline to three months for the time anchor "past" and "future" ** p < .001; ns ... not significant
To investigate how depression and hopelessness may influence the change in the size of the gaps proposed by de Leval, multiple regression analysis was conducted. Change in depression and hopelessness scores over time were entered as independent variables in a multiple regression, while the change in the gap over time, was entered as the dependent variable. Changes in the size of the two time comparison gaps (past/present; present/future) were not influenced by change in depression or hopelessness scores (R2 = .281, p = .09; R2 = .162, p = .266). This partly disconfirms hypothesis 4
Discussion
In this study the relationship of QoL and depression was investigated in two ways. First the preference comparison gaps were assessed, i.e. where patients perceive themselves to be and where they wish to be. This was done for all three time dimensions (past, present and future). Secondly, the time comparison gap was assessed, i.e. where patients see themselves now in comparison to the past, and in comparison to the future. Both approaches were carried out on the basis of de Leval's model linking depression to all three time dimensions.
Our findings are consistent with previous results, showing that people use their current affective state as a basis for making judgments of how happy and satisfied they are with their lives. A depressed person will usually see his or her well-being, social functioning and living conditions as worse than they appear to an independent observer or to patients themselves after recovery – the so called "affective fallacy" [10]. Clinically depressed patients were assessed at two time points before and three months after admission to a psychiatric hospital. Clinical measures (depression and hopelessness) showed significant improvements for all 18 patients, indicating successful treatment. Alongside the reduction in depression, actual present QoL improved significantly over time. In contrast, actual past QoL did not change over time. Successful treatment of depression had no influence of the individual's perception of the past. In addition, there was no significant change in actual future QoL, i.e. how patients realistically estimated their QoL to be one year from now. Thus assessments of the past and of the expected future did not change when patients recovered from a depressive episode. Aspirational QoL was the same between the three time dimensions and remained constant over time. There was always a preference comparisons gap over time – independent of depression or recovering depression.
Our findings suggest the larger the present preference comparison gap, the greater the depression. While the preference comparison gap of the past did not change over time with the decrease of depression, there was a trend for the future preference comparison gap that might become statistically significant with a larger sample size. However, aspirations were not affected by depression, since they remained the same for past, present and future from baseline in hospital to three months later. Overall these findings may indicate that a reduction in the present preference gap should be the main goal for therapeutic actions for depressed patients. An achievable target may be to keep the same preference comparison gap between and over time points to maintain a healthy homeostasis and a good QoL (figure 2). Interestingly, the mean level of aspirational QoL was not at the top of the possible scale (100). This may reflect an individual "QoL set-point" as discussed by Seligman [14]. He proposes that an individual might be genetically predetermined, to a particular set-point, to which he/she might return after both ups and downs. In therapy then the goal might be to help an individual actively work to return to his/her set point as soon as possible.
Successful treatment of depression, indicated by a significant change of BDI-II and BHS scores, was accompanied by a significant reduction in the gap between present actual and aspirational QoL. A reduction in the gap between actual present QoL with the aspirational QoL ("where one would like to be right here and now") reflects a more positive view of the present and is accompanied by significant improvements in QoL. Depression was consistently correlated with the time comparison gap between present and future QoL assessments. However, depression was more highly associated with the present and future preference comparison gap at three months. This suggests, that depression may be more highly associated with the actual/aspirational gap when people are less depressed. In contrast, hopelessness was consistently correlated with the preference comparison future gaps and only at baseline with the gap of present actual/aspirational QoL. The gap between the actual past and aspirational past QoL was not explained by either depression or hopelessness. Change in depression or hopelessness was important in reducing the preference comparison gap for present and future QoL assessments. Change in depression was important in reducing the size of the present gap. Change in hopelessness significantly contributed to reducing the size of the future gap. Given that present "ill-being" is seen as depression, while hopelessness is future orientated and directed, this finding makes sense. This has implications for therapy: to improve the QoL of patients with depression, relief from depression must be provided. Hopelessness needs to be tackled in addition to achieving a realistic future QoL perspective.
To a large extend this study confirms de Leval's theory. The assessment of de Leval's model: present and future QoL (quality of life) and past and present (phenomenological-depression) showed interesting results. The theoretical time comparison gap between a "healthy past" and present "ill-being" was confirmed by the significant difference between patients' perceptions of actual present and future QoL, at baseline. Over time, following appropriate treatment, this time comparison gap should close and QoL should improve, if the treatment is considered to be successful. The significant reduction of the past-present gap over time, and the loss in significance in size of this gap three months later, further confirms de Leval's model. This reduction of the time comparison gap between the present "ill-being" and a "healthy past" resulted in an improvement in QoL. The reduction was achieved by a change in the actual view of the present, leaving the view of the past unchanged (figure 3).
According to de Leval's model, recovery from depression is achieved when the appraisal of present and past QoL are very close, i.e. a very narrow gap. This means that a reduction of the gap between present and future QoL should improve current QoL. Although this study showed a decrease in depression over time by standard measures of depression and the size of the time comparison gap was significantly reduced between the two time points, the size of the gap did remain significant between the present and future at three months follow up. This finding may either indicate that the patients were still depressed at three months, or this part of the model could not be confirmed (figure 3). de Leval argues that therapeutic future is the view of a healthy past. Therefore one would expect similar scores for past and future QoL. This was supported by our findings for both time points. The change in the size of the time comparison gaps was not predicted by the change in either hopelessness or depression scores over time. This suggests that the change in time comparison gap's may be influenced by other variables, not assessed in this study. Depression lowered a person's actual QoL, but not their aspirational QoL. This was consistent whether a person was depressed or improving and for each of the time dimensions (past-present-future). The recollection of the past, either actual or aspirational did not change for the depressed or recovering person, neither did their view of the future. Thus for this patient group, the perception of the past and the future was not altered by depression status, which might be linked to individual QoL set points.
Limitations
Since we tested the model in a small and specialised sample, the findings are not generalisable. However, our study did test de Leval's model in a clinically depressed sample which is the focus of the most likely benefit of understanding the interplay of the two phenomena of depression and quality of life. It is also a challenging group to recruit, engage with and maintain in a research project.
Conclusion
This study showed that depression influenced individual QoL by lowering the person's actual QoL. Thus depression was associated with a larger gap between current reality (actual perceptions) and patient aspirations and realistic expectations. Aspirations for past, present and future QoL remained the same over time. However, patients actual appraisal of their present QoL improved with successful treatment of depression, reflected by a closure of the present preference gap.
de Leval's model was largely confirmed. Thus depression and hopelessness influence a person's present and future QoL. The analysis of a temporal horizon was very helpful in understanding the link between depression and QoL. Therapeutic interventions can be considered as closing the gap between a person's present QoL and their present aspirations and realistic expectations for the future. This could be achieved, according to the data presented here, by changing the view of the present and not necessarily by changing the level of expected future QoL. Knowing the persons aspirational QoL level may be a helpful guide for the therapist in that it provides important information about a depressed person's position in the progress towards what he or she considers to be recovery.
Authors' contributions
Margaret Moore contributed to conception and design, acquisition, analysis and interpretation of the data and revised the article. Stefan Höfer drafted the article and contributed to the analysis and interpretation of the data. Hannah McGee contributed to the conception and design and interpretation of the data and revised the article. Lena Ring contributed to the interpretation and revised the article.
All authors have given final approval of the version to be published
Acknowledgements
The authors thank Dr. Patrick McKeon, Dr. Fionnuala O'Loughlin and staff at St. Patrick's Hospital Dublin for assistance with this study. The research was completed in part fulfilment of a doctoral programme in clinical psychology at University College Dublin by Margaret Moore. The paper was completed while Dr. Stefan Höfer and Dr. Lena Ring were EU Marie Curie Research Fellows at the Department of Psychology, Royal College of Surgeons in Ireland.
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| 15634354 | PMC545054 | CC BY | 2021-01-04 16:38:15 | no | Health Qual Life Outcomes. 2005 Jan 5; 3:1 | utf-8 | Health Qual Life Outcomes | 2,005 | 10.1186/1477-7525-3-1 | oa_comm |
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Epidemiol Perspect InnovEpidemiologic perspectives & innovations : EP+I1742-5573BioMed Central London 1742-5573-1-51560147710.1186/1742-5573-1-5Analytic PerspectiveYears of Life Lost due to exposure: Causal concepts and empirical shortcomings Morfeld P [email protected] Institute for Occupational and Social Medicine, Cologne University Medical School, 50931 Cologne, Joseph-Stelzmann-Str. 9, Germany2 Institute for Occupational Sciences of RAG Aktiengesellschaft, 44369 Dortmund, Hülshof 28, Germany2004 16 12 2004 1 5 5 10 9 2004 16 12 2004 Copyright © 2004 Morfeld; licensee BioMed Central Ltd.2004Morfeld; 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.
Excess Years of Life Lost due to exposure is an important measure of health impact complementary to rate or risk statistics. I show that the total excess Years of Life Lost due to exposure can be estimated unbiasedly by calculating the corresponding excess Years of Potential Life Lost given conditions that describe study validity (like exchangeability of exposed and unexposed) and assuming that exposure is never preventive. I further demonstrate that the excess Years of Life Lost conditional on age at death cannot be estimated unbiasedly by a calculation of conditional excess Years of Potential Life Lost without adopting speculative causal models that cannot be tested empirically. Furthermore, I point out by example that the excess Years of Life Lost for a specific cause of death, like lung cancer, cannot be identified from epidemiologic data without assuming non-testable assumptions about the causal mechanism as to how exposure produces death. Hence, excess Years of Life Lost estimated from life tables or regression models, as presented by some authors for lung cancer or after stratification for age, are potentially biased. These points were already made by Robins and Greenland 1991 reasoning on an abstract level. In addition, I demonstrate by adequate life table examples designed to critically discuss the Years of Potential Life Lost analysis published by Park et al. 2002 that the potential biases involved may be fairly extreme. Although statistics conveying information about the advancement of disease onset are helpful in exposure impact analysis and especially worthwhile in exposure impact communication, I believe that attention should be drawn to the difficulties involved and that epidemiologists should always be aware of these conceptual limits of the Years of Potential Life Lost method when applying it as a regular tool in cohort analysis.
years of life losteffect measurementcounterfactualsbias
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Introduction
The most common epidemiological exposure-disease effect measures are based on exposure or disease frequency statistics, like risks or odds. Such frequency statistics focus on the question whether an exposure or disease occurred in a population. This information is used to measure the effect of exposure on disease by comparisons of such statistics. Although these measures have been proven by practice and theory to be useful for this purpose, these frequency statistics are unable to reflect all causal effects of exposure in general (Greenland and Robins 1988 [3], Robins and Greenland 1989 [4]). One reason stems from the fundamental fact that exposure and disease are processes in time. In particular, if time plays a major role in the link between exposure and disease which is certainly true for long-term exposures and chronic diseases, the question when a disease occurs becomes of paramount relevance. It is important to note, although not widely recognised, that the temporal shift of the onset of disease caused by exposure falls beyond the grasp of conventional statistics based on risks or odds, at least in part. And this shortcoming is even true, albeit perhaps counter-intuitive at first glance, when time-dependent incidence rates are analysed by applying sophisticated time-related statistical procedures like Cox modelling with or without adjustment for time-dependent covariates (Rothman and Greenland 1998 [5], Greenland 1999 [6], Morfeld and Piekarski 2001 [7]). An illustrative example of a Cox analysis in which the true probability of causation can not be derived correctly from the hazard ratio estimate due to an incompletely reflected temporal shift of the disease onset is given as an endnote (see endnote 1).
Therefore, alternative measures that focus more directly on the time-shift of events or the time-shift of frequency statistics would be most welcome. One such approach aims at Years of Life Lost (YLL). Interestingly, even in the title of one of the very first articles about Years of Life Lost, Dempsey [8] expressed the opinion that important aspects are missed by frequency statistics that could be well covered by Years of Life Lost methodology. An overview of different explications of the concept of Years of Potential Life Lost (YPLL) was given by Gardner and Sanborn 1990 [9]. Moreover, the authors presented a unifying conceptual framework for all these explications of YPLL. In the past the method of Years of Potential Life Lost was mainly used to describe the impact of different causes of death on the survival of a population. This concept was developed further trying to estimate the health effects of specific exposures like smoking (Quellet et al. 1979 [10], Centers for Disease Control 1989 [11]). For this purpose, excess Years of Potential Life Lost due to exposure (e-YPLL) were calculated in two steps: first, for each age group the number of excess deaths among the exposed was multiplied by the expected remaining years of life at age at death, given no exposure, and second, these products were summed over all age categories.
Recently, this approach was extended by Park et al. 2002 [2] from SMR-based calculations to YPLL-estimates based on Poisson regression models. The authors illustrated the method with the Colorado Plateau Uranium Miners Cohort (Lundin et al. 1971 [12]) estimating Years of Life Lost per years worked as a uranium miner, in particular focusing on premature lung cancer deaths. A further application was published by Bailer et al. 2003 [13] in an analysis of occupational fatal injuries.
However, in a letter to the editor, Morfeld 2003 [14] claimed that the proposed method of estimating excess Years of Life Lost due to exposure is potentially biased in certain settings. The arguments presented were based on the fundamental but abstract article written by Robins and Greenland 1991 [1] who investigated the estimability of expected Years of Life Lost due to a hazardous substance. The critique raised in Morfeld 2003 [14] focused on the unavoidable non-identifiability of the true excess Years of Life Lost due to exposure after conditioning on age. However, the train of thoughts was presented in a rather condensed way, and obviously, the arguments were not easy to digest (compare the response by Park et al. 2003 [15])
Here I try to explain the reasoning in detail. I expand the critique demonstrating additionally that the e-YPLL method is unjustified when applied to specific causes of death like lung cancer – the main topic of Park et al. 2002 [2]. First, a general framework of causal thinking in epidemiology is developed. This provides a background to analyse the validity of estimation procedures. In the second Chapter the most elementary setting is used to introduce the concepts of excess Years of Life Lost (e-YLL), Years of Potential Life Lost (YPLL) and excess Years of Potential Life Lost (e-YPLL). Then I show that e-YPLL is an unbiased estimator of e-YLL when considering all causes of death. In the third Chapter I prove the potential bias of the e-YPLL estimator when conditioning on age. The proof is given in an elementary setting. Moreover, I illustrate this bias by a realistic life table example. The next Chapter deals with the potential bias of e-YPLL when applied to specific causes of death. This potential distortion is proved in a simple epidemiological setting and demonstrated by a realistic life table calculation. All life tables presented are designed to discuss the analysis published by Park et al. 2002 [2]: their potentially biased applications of the Years of Potential Life Lost method specific for lung cancer or conditioned on periods of age. These tables are provided in pdf-format as Additional files 1, 2 and 3. They are based on two spreadsheets that are available as Additional files 4 and 5. The final Chapter deals with generalized scenarios, alternative estimation procedures and a brief discussion of the counterfactual approach to causality, even including a hint at its important link to ontological concepts in quantum mechanics.
To summarize, the main focus of this paper is first, on a new and simple proof of the potential bias of the e-YPLL method, and second, on an illustration of the degree of potential bias involved by realistic life table examples. These examples are constructed to critically discuss the e-YPLL analysis of a cohort of US uranium miners presented by Park et al. 2002 [2]. The demonstrated limitation of the e-YPLL approach was first proven rigorously in Jamie Robins's and Sander Greenland's 1991 [1] breakthrough article on the estimability of expected years of life lost due to a hazardous exposure. My didactic intention is to prove and illustrate the problems involved on a much simpler level of argumentation.
Analysis
1. Causal concepts: setting out a cohort gold standard
The aim of this paper is to scrutinise the validity of an exposure effect statistic. Therefore, a gold standard is needed against which the statistic can be evaluated to identify and to measure potential biases. A comprehensive cohort investigation is the most natural and purposeful epidemiologic approach towards causality when leaving aside all practical obstacles linked to this study design (Rothman and Greenland 1998 [5]). To approximate the theoretical gold standard I suppose such a cohort study as being free of selection biases, information errors, losses to follow up, and inferential problems. However, to define an optimal study scenario, I need to assure additionally that contrasts between response statistics of differently exposed sub-cohorts measure correctly the true effect of exposure. This "no confounding" assumption can be visualised by exchangeable sub-cohorts, the most elementary one consisting of only one subject each.
These differently exposed but exchangeable subjects are assumed to be like ideal twins: if exposure status had been interchanged between both twins the health response of the subject exposed to exposure level A would have been exactly the response of his/her twin exposed to level B and vice versa. This scenario of totally exchangeable twins is at the bottom of the so called "counterfactual" approach to causality (Lewis 1973 [8], Rubin 1974 [18], Maldonado and Greenland 2002 [23]). It idealises the experimental approach often applied in science by carefully preparing test objects as similary as possible. The aim of this preparation is to measure the effect of an independent variable on a dependent variable in a series of experiments as unconfounded as possible. A mathematical outline of this approach was developed by Neyman 1923 [16] and even extended to non-experimental studies by Simon and Rescher 1966 [17], Rubin 1974 [18] and Holland 1986 [19]. Overviews are given by Rosenbaum 1995 [20] and Pearl 2000 [21]. In addition, Robins 1997 [22] applied the counterfactual approach to conceptualise and analyse scenarios with interdependent exposure variables, response variables and other covariates that develop in time. I will come back to a critical discussion of this fundamental approach to causality within the Discussion section. At the moment it is sufficient to understand that the gold standard for epidemiology is set out here as an ideal cohort study where exposure levels are allocated to n-tuples of exchangeable subjects.
Hence, a gold standard scenario with a binary point exposure assumes that each exposed subject has an ideal unexposed twin so that the exposed sub-cohort consists of all the exposed twins and the unexposed sub-cohort comprises all the unexposed twins. Each causal comparison between the sub-cohorts is based on the pairwise comparison of the differently exposed but perfectly exchangeable twins, at least indirectly (Maldonado and Greenland 2002 [23]). Obviously, even randomisation of exposure is unnecessary to gain an unbiased estimate of the exposure effect in such a gold standard study.
Note that the sub-cohort of unexposed twins constitutes an ideal reference population. It is ideal with respect to 1) being an optimal reference group to define the response of the exposed cohort had it not been exposed (comparison on the group level). Over and above this optimality criterion, the reference is ideal with respect to 2) being an optimal reference on the individual level: the response of each exposed subject can be compared to the response of his/her twin. Note that optimality criterion 2) entails optimality criterion 1). Whereas an external reference population as often chosen in concrete epidemiological studies (Rothman and Greenland 1998 [5], Breslow and Day 1987 [24]) can be supposed to fulfil criterion 1), at least approximately, the optimality criterion 2) will not be guaranteed. The reason is that the external reference population cannot provide an unexposed control partner on the individual level. Thus, statistics like SMRs tailored for analysis of such data do not rely on individual level comparison information. It is important to note that these statistics, due to their definition, could not make use of such information even if it were available. Chapter 2 demonstrates that the e-YPLL method can be applied unbiasedly given optimality criterion 2) when applied to all causes of deaths and without any stratification. Chapters 3 and 4 show that e-YPLL stratified on age or specified for certain endpoints, like lung cancer, may be potentially biased even if criterion 2) is assumed. This is so because e-YPLL ignores the information about individual matching. However, this information is crucial as I will demonstrate in Chapters 3 and 4. It follows that a stratified or specified version of e-YPLL is potentially biased if it is used to analyse cohort studies in comparison to an external reference population.
2. Years of Life Lost in an ideal study
2.1 Elementary situation with one pair of twins only
In this section I define excess Years of Life Lost (e-YLL) and excess Years of Potential Life Lost (e-YPLL). Then I analyse their relationship in the most simple setting of an ideal study: a pair-matched cohort study with binary point exposure and a death process as outcome. The cohort is assumed to consist of only two subjects, both being differently exposed but totally exchangeable twins. Figure 1 illustrates the scenario.
Figure 1 Two exchangeable twins 1 and 2. Birth at b(1) = b(2); allocation of exposure at t(1) = t(2): twin 1 exposed, twin 2 unexposed; death at d(1) ≤ d(2) ; excess years of life lost due to exposure e-YLL = d(2) - d(1); potential death time under no exposure pd(1) = d(2); pd(2) = d(2). Years of potential life lost YPLL(i) = pd(i) - d(i), i = 1,2; excess years of potential life lost e-YPLL = 1(pd(1) - d(1))-0(pd(2)-d(2)) = e-YLL.
Both twins (1 and 2) were born on the same day, b(1) = b(2). For simplicity I rescale the time axis and set b(1) = b(2) = 0. Thus, time is identical with age throughout this paper. The point exposure was allocated at an age of t(1) = t(2) > b(1) = b(2) = 0: twin 1 was exposed, twin 2 was never exposed. The twins died at ages d(1) > t(1) and d(2) > t(2), respectively, and deaths are understood as deterministic responses on the individual level. I assume the exposed twin dies earlier or at the same date as the unexposed control twin, d(1) ≤ d(2). Since both subjects are perfectly exchangeable exposure was neutral or detrimental but not preventive. Exposure caused an advancement of death by d(2) - d(1) years. Thus, the true excess Years of Life Lost due to exposure are e-YLL = d(2) - d(1).
Next, I introduce potential death times pd(i), i = 1, 2. If both subjects had been unexposed, I would have expected them to die at pd(i) when relying upon the sub-set of the reference population surviving at least to d(i). Since the ideal reference population consists of the unexposed subject only, it immediately follows pd(1) = d(2) and pd(2) = d(2). For this reason I set pd(1) and d(2) equal in Figure 1. In general, methods like those proposed by BEIR IV 1988 [25] may be applied to the life table of the reference population to estimate pd(i).
I now calculate life expectancy at age at death, YPLL(d(i)), based on the reference population. These Years of Potential Life Lost are defined as YPLL(d(i)) = pd(i) - d(i). Hence, YPLL(d(1)) equals pd(1) - d(1) = d(2) - d(1) = e-YLL in this elementary setting. Obviously I have YPLL(d(2)) = 0. Note that the identity of YPLL(d(1)) and e-YLL relies on the elementary scenario and on the exchangeability condition (together with other usual conditions necessary for study validity).
Next, the effect estimate based on YPLL is introduced. The effect estimate of exposure on health is defined as the excess Years of Potential Life Lost, e-YPLL, which is the sum of excess deaths among the exposed (i.e., the observed exposed deaths minus the expected exposed deaths) times Years of Potential Life Lost at age at death, where the summation runs over all death times t (Park et al. 2002 [2]):
e-YPLL = Σ (observed(t) - expected(t)) YPLL(t).
Since the number of events is discrete this can always be understood as
e-YPLL = Σ observed(d(i)) YPLL(d(i)) - Σ expected(d(j)) YPLL(d(j)),
where the first sum is running over all death times d(i) among the exposed twins and the second sum is running over all death times d(j) among the unexposed twins.
The number of expected deaths among the exposed at age t, expected(t), is calculated as the product of the risk among the unexposed at t and the number of exposed at t. This definition is analogous to the calculation of exposed cases in an SMR analysis (Breslow and Day 1987 [24]):
baseline risk at t = # unexposed events at t / # unexposed twins at t
expected(t) = (baseline risk at t) (# exposed twins at t).
If there are no unexposed twins at age t, I define expected(t) to be zero.
Two risk sets are given in the simple scenario discussed here: one exposed death occurs at d(1) and one unexposed death at d(2). At d(1) two subjects are under risk: one exposed, the other unexposed. Since the unexposed subject does not die at d(1) we get expected (d(1)) = (0/1) (1) cases among the one (1) exposed at d(1). At d(2) no exposed but one unexposed subject is under risk. Since the unexposed dies at d(2) this leads to expected(d(2)) = (1/1) (0) cases among the zero (0) exposed at d(2). Because one exposed death is observed at d(1) and zero exposed deaths at d(2), i.e., observed(d(1)) = 1 and observed(d(2)) = 0), it follows that
e-YPLL = (1 - (0/1)1) YPLL(d(1)) + (0 - (1/1)0) YPLL(d(2))
= YPLL(d(1)) = e-YLL.
Of course, using the second representation of e-YPLL given above one also gets
e-YPLL = 1(d(2) - d(1)) - 0(0) = d(2) - d(1) = e-YLL.
Hence, e-YPLL measures e-YLL unbiasedly, given the elementary scenario and the exchangeability condition (together with other usual conditions necessary for study validity).
2.2 Scenario with two pairs of twins
I have proven that e-YPLL is identical to e-YLL in an elementary setting of a cohort consisting of two exchangeable subjects given appropriate conditions. But does this hold if the cohort consists of two or more pairs of exchangeable twins? For didactic reasons I will study a cohort of two pairs of twins first. If we assume that exposure is never preventive, we are left with only two principal scenarios when investigating the situation with two pairs of twins. Indexing the pairs with a and b, the first principal scenario supposes the death times of the four subjects to be ordered as da(1) ≤ da(2) ≤ db(1) ≤ db(2). Obviously
e-YLL = da(2) - da(1) + db(2) - db(1).
Now I will calculate the Years of Potential Life Lost under this scenario 1. For the sake of clarity, I add a second argument to the YPLL function denoting the pair under consideration (a or b). To simplify the notation of YPLL, I drop the letter d that is superfluous since we analyse a scenario without censoring (all subjects die under observation) (i.e., YPLL(a,1) = YPLL(da(1))). The same simplified notation is used to denote observed and expected numbers of cases. Following Gardner and Sanborn 1990 [9] and using the ideal unexposed twins as the reference population the Years of Potential Life Lost at da(1) are given as
YPLL (a,1) = da(2) - da(1) + 0.5 (db(2) - da(2)).
This follows because all unexposed subjects survive at least to da(1) and half of the unexposed subjects survive at least to da(2). Analogous considerations yield
YPLL (a,2) = db(2) - da(2)
YPLL (b,1) = db(2) - db(1)
YPLL (b,2) = 0.
The observed cases among the exposed are
observed (a,1) = 1
observed (b,1) = 1
and the expected, based on the unexposed, are given as
expected(a,2) = (1/2)(1) = 0.5
expected(b,2) = (1/1)(0) = 0.
Finally, this leads to
e-YPLL = da(2) - da(1) + 0.5 (db(2) - da(2)) -
- 0.5 (db(2) - da(2)) +
+ db(2) - db(1)
= e-YLL.
Therefore, the equality of e-YPLL and e-YLL is proven for scenario 1. Next, I will investigate the other principal scenario. In contrast to scenario 1 the second principal scenario assumes the ordering da(1) ≤ db(1) ≤ da(2) ≤ db(2).
Given this scenario 2 we have
e-YLL = da(2) - da(1) + db(2) - db(1)
and
YPLL(a,1) = da(2) - da(1) + 0.5(db(2) - da(2))
YPLL(a,2) = db(2) - da(2)
YPLL(b,1) = da(2) - db(1) + 0.5(db(2) - da(2))
YPLL(b,2) = 0.
Moreover, it follows
expected(a,2) = (1/2)0 = 0
expected(b,2) = (1/1)0 = 0.
Therefore,
e-YPLL = (1)YPLL(a,1) + (1)YPLL(b,1) - (0)YPLL(a,2) - (0)YPLL(b,2)
= da(2) - da(1) + da(2) - db(1) + db(2) - da(2)
= da(2) - da(1) + db(2) - db(1) = e-YLL.
If we assume exposure to be never preventive all possible configurations of the death times of both twins can be mapped onto these two principal scenarios by renaming the subjects accordingly. Thus e-YPLL = e-YLL is always valid in such an ideal study with two pairs of twins, although e-YPLL does not use the individual matching information.
It is important to note the following: the true excess Years of Life Lost can be calculated for each single pair as "observed cases times potential years of life lost" minus "expected cases times potential years of life lost" only in scenario 1. This calculation is obviously invalid under scenario 2. Only the total e-YPLL equals the total e-YLL in both scenarios. Thus, scenario 2 indicates that we have to be careful when a specified version of e-YPLL is applied to estimate the excess Years of Life Lost given specific conditions. Such an analysis is possible without bias if first, optimality criterion 2 is fulfilled as introduced in the last paragraph of Chapter 1, and second, if this individual matching information is used in the analysis. However, this kind of application is not justified if only criterion 1 holds. I will deal with this important issue of potential biases in Chapters 3 and 4.
2.3 Scenario with n pairs of twins
A general proof of e-YPLL = e-YLL can be constructed using the principle of mathematical induction. Hence, this Chapter is a little bit technical. However, it may be skipped on first reading because it is not necessary to understand this proof to follow the arguments on potential biases developed in Chapters 3 and 4.
For the sake of clarity I introduce an additional functional argument denoting the number of pairs in the study: e-YLL(n) denotes the total excess Years of Life Lost due to exposure in a study with n pairs of twins. To start the inductive argument let us assume that e-YPLL = e-YLL has already been proven to be valid in all studies with n pairs of twins: e-YPLL(n) = e-YLL(n). This is the so called induction assumption. Now I add another pair of twins to the study and ask whether e-YPLL(n+1) = e-YLL(n+1). If I can prove this equality then it follows from the principle of mathematical induction that e-YPLL = e-YLL in all studies. This extension is true because I already have demonstrated that e-YPLL = e-YLL if n = 1 (start of induction).
To simplify the argument I now suppose the additional pair to have an unexposed twin with minimal survival among the unexposed n+1 subjects in the extended study. This simplication can always be assumed without any loss of generality for the following reason. If the added pair is not the one with the minimal death time among the unexposed I focus on a pair that fulfils that condition. Of course, such a pair always exists. Let us denote this pair with x and the death times of its twins with dx(1) and dx(2) accordingly. Regarding the other n pairs we know that e-YPLL(n) = e-YLL(n) due to the induction assumption. Hence it suffices to show that e-YPLL(n+1) = e-YLL(n+1) holds in the scenario with the added pair being pair x. This will prove the equation e-YPLL = e-YLL convincingly for all studies with n+1 pairs.
Since we assume throughout that exposure is never preventive we know that dx(1) ≤ dx(2). This means that the survival function of the exposed is the same in the extended study group with n+1 pairs as in the study group with n pairs, given we restrict the analysis to all exposed subjects with age at death greater than or equal to dx(2). Moreover, because dx(2) is minimal among the death times of all unexposed subjects the survival function of the unexposed is unchanged too. It follows that the Years of Potential Life Lost (i.e., conditional life expectancy) calculated from the unexposed reference population remains the same for all exposed subjects with age at death greater than or equal to dx(2). In addition, the expected number of cases among the exposed calculated from the unexposed remains unchanged. Therefore, the difference between e-YPLL(n+1) and e-YPLL(n) can only stem from events occurring before dx(2). In the next step I will calculate the change in e-YPLL caused by these events occurring before dx(2).
I assume that k of the n exposed in the study with n pairs may die before dx(2), 0 ≤ k ≤ n. Thus, there are n+1 - (k+1) = n - k exposed subjects of the extended study group under risk at dx(2), and it follows expected(x,2)=(n-k)/(n+1). The factor 1/(n+1) is to apply since the first event among the n+1 unexposed subjects occurs at dx(2). The amount of change in e-YPLL due to the additionally expected deaths at dx(2) is therefore given by -[(n-k)/(n+1)]YPLL(x,2). This is the first kind of contribution to the change in e-YPLL I have to consider. The second kind of contribution stems from the new exposed case dying at dx(1). This amount of change is simply the same as the Potential Years of Life Lost for this case: YPLL(x,1) = dx(2) - dx(1) + [n/(n+1)]YPLL(x,2) where n/(n+1) is the probability to survive dx(2) given the unexposed reference population. The third kind of contribution to the difference between e-YPLL(n+1) and e-YPLL(n) stems from the k exposed cases dying before dx(2). Their potential survival is shortened after introducing the new additional death at dx(2). Note that the reference-based probability to die at dx(2) is 1/(n+1) for each of these exposed cases. Therefore, the Years of Potential Life Lost is reduced for each of these cases by the amount [1/(n+1)]YPLL(x,2). This sums to [k/(n+1)]YPLL(x,2) for all k exposed cases dying before dx(2). Now I can calculate how e-YPLL changes when the pair x is added to the n pairs:
e-YPLL(n+1) = e-YPLL(n) + dx(2) - dx(1) +
+ [n/(n+1) - (n-k)/(n+1) - k/(n+1)] YPLL(x,2)
= e-YPLL(n) + dx(2) - dx(1)
= e-YLL(n) + dx(2) - dx(1)
= e-YLL(n+1).
The second to last equation follows from the induction assumption. The last equality is based on the obvious fact that the true excess Years of Life Lost increases by dx(2) - dx(1) if the pair x is added to the n pairs I started with. Hence, I have proven that e-YPLL equals e-YLL in all ideal studies consisting of pairs of exchangeable twins given that exposure is never preventive. Note that the measures are always calculated with respect to deaths from all causes. It is remarkable that the equality e-YPLL = e-YLL holds although e-YPLL does not make use of the individual matching information.
3. Non-identifiability of e-YLL conditional on age at death
I have shown in Chapter 2 that the total e-YLL of the whole study group can be measured accurately by the total e-YPLL provided the assumptions of an ideal study hold including optimality criterion 2 (cf. last paragraph of Chapter 1) and provided the analysed response is death from all causes. However, this identity of e-YLL and e-YPLL no longer holds if I am interested in e-YLL conditional on age at death. To see why consider the scenarios illustrated in Figure 2.
Figure 2 Two scenarios of different exposure effects in two pairs of twins. The pairs are indexed by a and b. As before, da(1), db(1) are death times of the exposed and da(2), db(2) are death times of the unexposed twins. Scenario 1 assumes causal effects of the same degree in both pairs, da(2) - da(1) = db(2) - db(1) = n years. Scenario 2 supposes no effect in pair b, db(1) - db(2) = 0 years, and a large one in pair a, da(1) - da(2) = 2n years.
In both scenarios (Fig. 2) the total e-YLL's are identical and thus, are also the total e-YPLL's. However, the e-YLL conditional on age at death varies with the scenarios: the Years of Life Lost due to exposure at da(1) are smaller in scenario 1 than in scenario 2 whereas it is vice versa at db(1). Irrespective of the scenario, the e-YPLL are always calculated in the same way. Therefore, we get in both scenarios the same e-YPLL at da(1) as well as the same e-YPLL at db(1). Note that the calculation of e-YPLL does not make any use of the individual matching information.
Hence, the true excess Years of Life Lost conditional on age at death are not identifiable without having access to a perfect control twin or without supposing a specific mechanism for how exposure causes death. Note that criterion 1 alone – as defined in the last paragraph of Chapter 1 and hopefully fulfilled in usual cohort data – does not render the excess Years of Life Lost stratified on age at death identifiable. Consequently, estimates based on e-YPLL conditional on age at death are potentially biased in all usual settings.
Next, I analyse a theoretical birth cohort of 100,000 men to demonstrate this potential bias by a realistic life table example (Table 1, see Additional file 1). I assume the death rates of the male US population as tabulated in BEIR IV 1988 [25] as reference rates. The example is restricted to the unexposed men surviving at least to an age of 30 years.
Life expectation at death is calculated from the size of the unexposed population and the number of unexposed deaths, presuming that all deaths occur at the midpoint of age categories. Numbers of exposed deaths include a certain advancement of age at death without changing the totals. Excess deaths are calculated as the difference between the number of exposed deaths and the expected deaths among the exposed. The latter is determined by multiplying the size of the exposed population by the fraction of deaths among the unexposed population conditional on age. Finally, e-YPLL is derived according to Park et al. 2002 [2] by multiplying the number of excess deaths by the life expectation at death in each age category (see Chapter 2.1 for details). The total number of excess Years of Life Lost due to exposure sums up to 255,094 years.
Table 2 (see Additional file 2) describes the impact of two different causal exposure-response mechanisms, both compatible with the data presented in Table 1 (see Additional file 1). Mechanism 1 assumes two different response types of subjects. One type is immune and does not react under exposure in comparison to no exposure (no advancement of age at death). The other type shows an adverse effect of exposure because his death is advanced by five years. In contrast, mechanism 2 supposes three different reaction types: an immune one and two types adversely affected by exposure. The affected types differ in their degree of response (advancement by five years or ten years). Tables 1 and 2 are based on a spreadsheet that is supplied as an additional data file (see Additional file 4).
Note that the total numbers of deaths, unexposed and exposed, as well as the total excess Years of Life Lost are identical in both sub-tables of Table 2 (see Additional file 2) although the sub-tables reveal a different causal impact of the varied mechanisms. Note further that the distribution of unexposed deaths as well as the distribution of exposed deaths are constant across both sub-tables and Table 1 (see Additional file 1). Moreover, the total e-YLL calculated in the sub-tables of Table 2 (see Additional file 2) agrees with the total e-YPLL derived in Table 1 (see Additional file 1). But obviously, the excess Years of Life Lost conditional on age differ between mechanism 1 and 2, and moreover, diverge from the conditional excess Years of Potential Life Lost calculated in Table 1 (see Additional file 1). Figure 3 contrasts the distributions.
Figure 3 Excess Years of Potential Life Lost e-YPLL and true excess Years of Life Lost e-YLL1 and e-YLL2, assuming two different causal mechanisms, conditional on age at death. Basic data, exposure impact, and mechanism producing e-YPLL, e-YLL1, and e-YLL2 are given in Table 1 and Table 2. Lost Years, true and estimated, sum up to 255,094 years always.
The excess Years of Potential Life Lost differ remarkably from the true excess Years of Life Lost. The difference depends on the chosen mechanism. In this example e-YPLL overestimates the true excess Years of Life Lost up to an age of about 65 years and underestimates the impact at higher ages. The zigzag increase of e-YPLL may reflect a real phenomenon since mechanism 2 produces a similar structure. However, the somewhat surprising zigzag increase of e-YPLL may be totally artificial because mechanism 1 causes a nearly smooth incline. Thus, it is not justified to interpret the form of the increase of e-YPLL across age as a true phenomenon without assuming untestable mechanistic assumptions about how exposure causes death. Note that such naive interpretations can be grossly misleading.
Hence, as illustrated by this example, the true excess Years of Life Lost conditional on age at death cannot be identified from cohort data like that presented in Table 1 alone (survival curves, see Additional file 1). The estimator e-YPLL conditional on age is shown to be potentially biased even if optimality criterion 2 (as defined in the last paragraph of Chapter 1) is fulfilled. The reason is that e-YPLL conditional on age does not make use of the relevant individual matching information.
4. Non-identifiability of Years of Life Lost due to a specific cause of death
The equality of total e-YPLL and total e-YLL was proven for deaths from all causes given the assumptions mentioned in Chapter 2. However, such an equality does not hold for a specific cause of death like lung cancer. To see why, consider the simple scenario presented in Figure 4.
Figure 4 Two exchangeable twins 1 and 2. The exposed twin died from lung cancer at age d(1), the unexposed twin 2 died from heart attack at d(2) > d(1).
Figure 4 illustrates an elementary gold standard cohort study performed with two twins, one exposed and the other unexposed. Since the subjects are assumed to be exchangeable, exposure caused a premature death with an overall e-YLL = d(2) - d(1). However, if we are interested in the exposure impact on age at death for lung cancer we are left with an identification problem. Obviously, if twin 2 had died from lung cancer also the causal effect on age at death from lung cancer would have been d(2) - d(1). But in case of the scenario illustrated in Figure 4, one can only conclude that the effect on age at death from lung cancer must be at least d(2) - d(1). Therefore, as a simple but important consequence, the causal effect on age at death from lung cancer is not identifiable even in an optimal study comprising exchangeable twins as long as competing causes of death exist. Since this causal effect cannot be identified even in an optimal study (i.e., fulfilling condition 2 defined in the last paragraph of Chapter 1) it does not make sense to estimate it without assuming untestable mechanistic assumptions about how exposure causes death from lung cancer.
Note, however, that this causal effect is not the e-YLL due to exposure and manifested by lung cancer, e-YLL(lung cancer) for short. In contrast to the causal effect, e-YLL(lung cancer) comprises exactly that part of e-YLL from all causes of death that is due to the occurrence of lung cancer deaths among the exposed. In the example (Figure 4) these e-YLL(lung cancer) are d(2) - d(1) which is the exposure effect on the age at death from all causes, e-YLL(overall) for short. The reason ist that there are no other causes of death among the exposed besides lung cancer. Whereas the causal effect of exposure on age at death from lung cancer cannot be identified in an optimal study with pairs of twins fulfilling optimality criterion 2 (defined in the last paragraph of Chapter 1) without adopting further untestable assumptions we may ask whether at least e-YLL(lung cancer) can be estimated unbiasedly assuming criterion 2. To give an answer we must investigate the situation in more detail.
The concept presented supposes that each subject has a set of hypothetical (deterministic) death times for each combination of level of exposure and cause of death. Just one of these death times is effective, the others are latent. For the exposed twin 1 lung cancer is effective as a cause of death and heart attack is latent. It is vice versa for the unexposed twin 2. Both causes of death compete within each twin for the effective position. Of course, the result of this competition depends on the exposure conditions applied. This result motivates to introduce a second (competing) exposure into the scenario, in which I deal with two competing responses (lung cancer and heart attack). Figure 5 expands on this concept accordingly.
Figure 5 Effective and latent causes of death in a quadruplet of exchangeable subjects. Each subject is supposed to have been allocated to a different combination of two binary exposures, illustrated here by asbestos and smoking. Without any exposure and as well as after asbestos exposure the subjects are assumed to have died from lung cancer, LC (effective cause). Lung cancer was latent in the smoker not exposed to asbestos while heart attack, HA, became the effective cause in this situation. Asbestos exposure had no causal effect on age at death from heart attack but from lung cancer. Smoking affected age at death from heart attack and lung cancer, the latter to the same amount as asbestos did. Under joint exposure a synergistic effect on age at death from lung cancer is assumed, but not for heart attack.
In Figure 5 both exposures are assumed to be never preventive for all endpoints of interest (LC = lung cancer, HA = heart attack). However, when contrasting the two situations without asbestos exposure smoking obviously leads to a decrease in the number of lung cancer deaths, and correspondingly, asbestos exposure causes a decrease in the number of deaths from heart attack given smoking. This spuriously preventive effects of both exposures are an outcome of the competing causes of death structure. In the following, I prove that this structure leads to a potential bias of e-YPLL(lung cancer). Note that the misleading impression of a preventive effect is produced by relying on a risk or rate statistic only. As I emphasized in the Introduction section, these statistics count accurately how many responses of what kind occurred ("whether"), but unfortunately, mainly ignore the specific causal structure in time ("when").
From Figure 6 I conclude
Figure 6 The effective data of Figure 5 analysed in an ideal cohort study consisting of two pairs of twins which are supposed to form a quadruplet of exchangeable subjects, cmp Figures 1 and 2. The pairs are indexed by a and b: the twins in pair a are assumed to have been smokers, those in pair b non-smokers; da(1), db(1) are death times of the asbestos exposed and da(2), db(2) are death times of the twins not exposed to asbestos. The effective cause of death, lung cancer LC or heart attack HA, are denoted at age at death, accordingly.
e-YLL(overall) = e-YLL(lung cancer) = e-YPLL(overall) =
da(2) - da(1) + db(2) - db(1).
The first equation holds because the only cause of death among all asbestos exposed is lung cancer and the second because I have shown that e-YLL(overall) = e-YPLL(overall) is true in an ideal study given the aforementioned assumptions. The third equation follows from a simple evaluation of e-YLL(overall).
Next, I calculate e-YPLL(lung cancer) according to Park et al. 2002 [2]:
e-YPLL(lung cancer) = Σ (observedlungcancer(t) - expectedlungcancer(t)) YPLL(t).
The only change in comparison to the formula of e-YPLL introduced in Chapter 2.1 is the specification of the observed and expected cases. The important point here is that the procedure, as defined by Park et al. 2002, only uses information at age at death from lung cancer. For the YPLL-terms involved I get
YPLL (a,1) = da(2) - da(1) + 0.5 (db(2) - da(2))
YPLL (b,1) = db(2) - db(1),
from which I derive
e-YPLL(lung cancer) = da(2) - da(1) + 0.5 (db(2) - da(2)) +
+ db(2) - db(1)
= e-YLL(lung cancer) + 0.5 (db(2) - da(2)),
since no expected lung cancer deaths are to be subtracted.
Hence, e-YPLL(lung cancer) is biased upward by 0.5 (db(2) - da(2)) which is half of the causal effect of smoking on age at death from all causes (overall) according to Figure 5. This overestimate is due to the fact that no lung cancer deaths are expected to occur among the exposed during follow-up. This empirical shortcoming of e-YPLL(lung cancer) could only be overcome in general if we were able to identify the matched partner of the lung cancer case not dying from lung cancer and if we use this information in the analysis. Since this is impossible when relying on usual cohort data (survival curves) I have proven the non-identifiability of the excess Years of Lost Life due to exposure for a specific cause of death, given the natural situation of competing causes for death and usual cohort data. I demonstrate the potential bias of e-YPLL(lung cancer) by a life table example additionally (Table 3, see Additional file 3). This table relies on a spreadsheet which is also supplied (see Additional file 5).
Table 3 (see Additional file 3) uses the same basic data as Table 2 (see Additional file 2) to describe the unexposed population but is extended by an additional column presenting the number of lung cancer deaths by age. The impact of exposure on lung cancer death is defined by an advancement of age at death by five years in all situations. Thus, factual (effective cause) age at death as well as hypothetical (latent cause) age at death is assumed to occur five years earlier under exposure. Furthermore, it is supposed that the advancement of latent lung cancer deaths leads to an excess of 50% among unexposed lung cancer deaths in each age category, if exposed. This advancement means that 3367 (= 10102 - 6735) subjects died from lung cancer after exposure who are assumed to have died from other causes if unexposed. A mixture of advancements greater or equal than 0 years, differing in amount between other causes of death than lung cancer, is assumed to produce the distribution of the number of deaths from all causes among the exposed. Hence, the overall excess Years of Life Lost for lung cancer are less than the number of exposed lung cancer deaths times five years (i.e., 50,509 years). This number is an upper bound because the additional lung cancer deaths occurring under exposure are related to a loss of life in the individual of at most five years while the cause of death changes from, say, heart attack to lung cancer.
Applying the procedure proposed by Park et al. 2002 [2] I calculate a total e-YPLL(lung cancer) of 86,373 years. Thus, the estimate is biased upward by more than 35,000 years which corresponds to a relative bias of at least 70%. In addition, the life table analysis demonstrates that the e-YPLL method is also biased when used to estimate the causal effect of exposure on age at death from lung cancer among the exposed lung cancer cases, which exactly amounts to 50,509 years. Moreover, the causal effect of exposure on the lung cancer death times among all subjects was defined to be 5(92,219) years = 476,095 years, again clearly different from e-YPLL(lung cancer) = 86,373 years.
5. Discussion
I have shown that the total excess Years of Life Lost (e-YLL) for death from all causes can be accurately determined by calculating the corresponding excess Years of Potential Life Lost (e-YPLL) provided conditions hold that are also often cited for general study validity. However, the equality of e-YLL and e-YPLL does not hold in general under certain conditions of interest because under these certain conditions we need to assure criterion 2: each exposed subject has to have an ideal unexposed control partner so that the effect of exposure can be measured on the individual level (see also the last paragraph of Chapter 1). The equality also requires that this information about individual matching is used in the analysis. I pointed out that the e-YPLL conditional on age at death are potentially biased as they were published in some analyses (see e. g., Figure 1 in Park et al. 2002 [2]). Furthermore, the excess Years of Potential Life Lost calculated for a specific cause of death are potentially biased also. Hence, estimates of Years of Life Lost presented for chronic diseases like lung cancer (see for example the detailed lung cancer Poisson model analysis by Park et al. 2002 [2]) are unjustified without referring to untestable assumptions about the causal mechanism involved.
I should add, just for the sake of clarity, that in all my examples presented that cast doubt on the validity of e-YPLL, exposure was always assumed to be never preventive. Therefore, preventive effects of exposure are not the reason for these problems. The deeper reason of these validity problems of e-YPLL is a non-identifiability of excess Years of Life Lost due to exposure in an ideal but unmatched cohort study. Note further that the calculation of e-YPLL will ignore this important matching information even if such data are available. This structural problem of non-identifiability was already clearly described, analysed and discussed on an abstract level by Robins and Greenland 1991 [1], even under the generalized scenario of stochastic responses on the individual level. For further theoretical discussions of the problems involved and for an investigation into untestable assumptions that render identification possible, I therefore like to refer to this important publication. I simply remark here that mechanisms 1 and 2 (Table 2, see Additional file 2) do not preserve ranks.
It is important to note that the validity problems of e-YPLL cannot be resolved by applying the rare disease assumption or by restricting the discussion to a situation with a constant relative risk across age. Rather, the problems are structural ones. For example, if the additional lung cancer cases under exposure are calculated in Table 3 (see Additional file 3) by applying an increasing factor with age at death instead of using the constant value of 1.5, the SMR's for lung cancer can be kept almost constant across age. However, e-YPLL still overestimates the true excess Years of Life Lost due to lung cancer by a considerable amount. A substantial upward bias, nearly independent of the increasing factor, can be demonstrated by simulations that assume an isolated and homogeneous detrimental effect of exposure on lung cancer. This describes a scenario where other endpoints are only changed due to exposure because they are competing with lung cancer. Again using the data from BEIR IV 1988 [25] (Table 3, see Additional file 3) I find an upward relative bias of at least 30% if the additional lung cancer deaths (that take over the effective position from competing causes if exposed) amount to about half of the lung cancer deaths shifted to a younger age at death (under exposure) without changing the endpoint. The examples I studied show that the relative bias is at least 70% if both groups are of the same size.
In agreement with the direction of bias indicated by the abstract quadruplet analyses of total excess Years of Life Lost for lung cancer (Figures 5 and 6) I show an upward distortion of e-YPLL(lung cancer) in the life table example (Table 3, see Additional file 3). This upward bias is due to an inappropriate mixture of life expectancy at age at death from lung cancer, based on data from all later deaths, with lung cancer excess deaths only, while ignoring the corresponding excess deaths from competing causes among the non-exposed that are produced as a side-effect of the causative exposure (against the interpretation of Park et al. 2003 [15]). However, total e-YPLL for a specific cause of death can be used to assess total e-YLL for this cause unbiasedly if despite potentially competing causes no change of the cause of death occurred within the whole cohort after a different exposure level of interest had been allocated counterfactually. Unfortunately, this assumption is not provable. When excess cases of the cause of death of interest are observed among the exposed – a situation quite natural in occupational cohort studies – this assumption definitely does not hold.
My analysis reported so far on problems with ideal studies with complete follow-up. Obviously, if a constant end of follow-up is introduced into the scenarios of Figure 5 and 6 so that the number of lung cancer deaths are affected, the calculated e-YPLL can change whereas the true e-YLL always remain constant. Hence, the degree of bias depends additionally on the censoring structure even if left censoring is totally uninformative. This dependence means that censoring is not taken into account adequately by the e-YPLL method when applied to specific endpoints.
One important point to discuss is whether the constructed life table examples are realistic ones in comparison to the data and results published in Park et al. 2002 [2]. All tables presented here are based on the mortality experience of the male US population as published by BEIR IV 1988 [25], Table 2A-10, p. 139. Since the paper of Park et al. 2002 [2] analysed the mortality of white US uranium miners, hired during 1950 - 1963 and followed until 1990, the choice of this reference population appears to be appropriate.
Park et al. 2002 [2] described the total excess Years of Life Lost per cohort member as 3.1 years (Table IV, page 6) and an effect of similar magnitude was chosen here: 2.7 years per cohort member (n = 95,219) according to Tables 1 or 2 (see Additional file 1 and 2), and 3.3 years per cohort member according to Table 3 (see Additional file 3). In Park et al. 2002 [2] the lung cancer SMR dropped from 7.6, age period 30–34, to 5.4, age band 45–49, and even further to SMR = 2.6, age period 65–69 (Table III, page 5). The example given in Table 3 (see Additional file 3) tries to mimic this downward trend starting with 7.0 in age category 30–34, then decreasing to 3.0 in age band 45–49 and to 1.7, age period 65–69. Thus, the overall downward trend is somewhat more pronounced here than in Park et al. 2002 [2]. However, I try to simulate a trend of SMR's across age that is quite similar to the relative decrease observed within the main body of data in Park et al. 2002 [2] (age bands 65–69/45–49): 2.6 / 5.4 = 48% (Table III, Park et al. 2002 [2]) and 1.7 / 3.0 = 57% (Table 3 of this paper, see Additional file 3).
The overall lung cancer SMR can be calculated from Table 3 (see Additional file 3) as 1.74, less than the value of 3.8 observed by Park et al. 2002 [2] (Table III, page 5). A lower SMR was chosen here to avoid an exaggeration of potential biases due to overstating the effect of exposure in the constructed example. Accordingly, the e-YPLL for lung cancer was presented as 1.47 years per cohort member by Park et al. 2002 [2] (Table IV, p. 6) but only 0.91 years per cohort member (n = 95,219) can be calculated from Table 3 of this paper (see Additional file 3).
Hence, the life table examples presented in Tables 1, 2 and 3 (see Additional file 1, 2 and 3) are realistic ones and the magnitude of potential biases identified appears to be defensible. The potential biases derived are fairly extreme: the true total Years of Life Lost are overstated by 100% (mechanism 1) or 33% (mechanism 2) in age band 50–54 but understated by more than 40% in age period 75–79 (Figure 3 and Tables 1 and 2, see Additional file 1 and 2). Moreover, a relative upward bias of more than 70% is demonstrated for the e-YPLL related to lung cancer (Table 3, see Additional file 3). These findings render the central results published by Park et al. 2002 [2] scientifically unreliable (i.e., the age-specific analysis of excess Years of Life Lost due to exposure as well as all results published on excess Years of Life Lost in relation to lung cancer). Note that the latter was the main topic of Park et al. 2002 [2].
Due to its definition (Breslow and Day 1987 [24]) the average lung cancer SMR is calculated as observed / expected = exposed / (exposed - excess) which leads to 10,101.74 / (10,101.74 - 4,286.88) = 1.74 (Table 3, see Additional file 3). For the sake of clarity I should note that this value does not coincide with the average causal case ratio (exposed / unexposed = 10,101.74 / 6,734.49 = 1.5). The discrepancy is due to the fact that the construction of all life tables in this paper strictly follows counterfactual principles: the elevated death rates in the exposed population affects the person-years yielding an expected number of lung cancer cases smaller than the true observed number of lung cancer cases in the unexposed population (cp. Keiding and Vaeth 1986 [26], Greenland 1996 [27]).
Statistical measures that focus on the advancement of disease onset are assumed to be helpful in communicating risk factor impact on disease (e.g., Peto 1980 [28]). Fischhoff et al. 1993 [29], Jardine and Hrudey 1997 [30] as well as Yamagishi 1997 [31] pinpointed problems in risk perception and communication when frequency statistics are solely presented as descriptors of health effect. Weinstein et al. 1996 [32] demonstrated additionally a considerable effect of framing risk levels on the perception of risk.
Statistics conveying information about the advancement of disease onset are therefore helpful in exposure impact analysis and especially worthwhile in exposure impact communication. However, attention should be drawn to the difficulties involved and that epidemiologists should always be aware of the conceptual limits of the Years of Potential Life Lost method when applying it as a regular tool in cohort analysis.
Aside from Years of Life Lost, other approaches are available to convey information on the impact of a risk factor on the onset of a disease and may thus facilitate communication of epidemiological findings. One such concept is the risk and rate advancement period (RAP) introduced by Brenner et al. 1993 [33] which could easily be calculated from standard software output. Risk and rate advancement periods are the time periods by which the risk or rate of disease is advanced among exposed subjects given a disease-free survival to some baseline age. An application of RAPs in assessing the impact of risk factors on myocardial infarction was published by Liese et al. 2000 [34]. However, the RAP statistic cannot be applied if disease rates do not increase strictly with age. Another approach describing the exposure impact on disease onset, complementary to RAP, was developed by Boshuizen and Greenland 1997 [35]. The authors estimated the time shift in average age at first occurrence of disease due to exposure as a measure of disease advancement. Although especially valuable when the background incidence of the disease is high, a technical drawback of this procedure stems from the necessity to correct regression model likelihoods for left truncation, an option that is not offered in standard statistical packages. Of course, none of these procedures can be applied sensibly if, according to study design, cases and controls or exposed and unexposed were matched on age. A rigorous causal approach in estimating the shrinkage or extension of time to an event, like lung cancer death, due to exposure was developed by Jamie Robins and is called G-estimation (for a brief overview see Rothman and Greenland 1998 [5], p. 424, 425). Since this method has a profound logical basis and can be applied even in complicated longitudinal scenarios with interrelated time-dependent covariates, and in addition, is programmable in standard software like SAS or STATA it should become a regular tool in cohort analysis, in particular when Years of Life Lost are to be estimated.
The main conclusions about non-identifiability of e-YLL are derived as an application of a causal theory based on counterfactuals (for a review see Greenland 2000 [36]). Some authors seem to believe that this approach has no clear logical and philosophical backing, judging Greenland's reasoning as purely academic (Armstrong and Thieriault 1996 [37]) or Robin's counterfactual based methods like G-estimation as not applicable in occupational epidemiology (Steenland et al. 1996 [38]). The arguments and examples given in this article prove the applicability and practical relevance of the counterfactual approach in occupational epidemiology. However, this does not help to eliminate a more fundamental philosophical resistance I experienced in a number of controversial discussions (e. g., with epidemiologist from NIOSH, Cincinnati). Thus, some explanation of the philosophical background of counterfactual reasoning should be given here.
The ideas of counterfactual reasoning can at least be traced back to philosophers in the eighteenth and nineteenth centuries, like Hume and Mill. Even the oldest clearly structured theory of causality, developed by Aristotle, has some similarities to counterfactual reasoning due to its manipulative four-causes concept (Vorländer 1979 [39]). In its most simple form, the counterfactual approach assumes the existence of potentially different hypothetical response variables for the same subject depending on differently assumed exposure conditions. At most one of these conditions and responses can become true, the others remain hypothetical, so counterfactual. A causal comparison is understood as a comparison of the responses due to different exposure conditions within the same subject. Indeed, this is the rigorous version of ideal twins I have used throughout to derive statements about causality (see Figures 1, 2, 4, 5, 6). In their famous statistical textbook, Cox and Oakes 1985 [40], p. 64 implicitly used this approach to define the stronger version of the accelerated failure time model: "any individual having survival time t under z = 0 would have survival time t/Ψ under z = 1". The counterfactual nature of this statement is reflected by the tense used (conditional II). Note that modern physics also uses counterfactual reasoning, expressed in conditional II, to define causes in the framework of the general theory of relativity: "We say that an event, let us call A, is in part the cause of another event, B, if A was necessary for B to occur. If A had not occurred, B could not have. In this case I can say that A was a contributing cause of the event B" (Smolin 2001 [41]). And note further that also everyday causality is often expressed by statements in conditional II (i.e., by statements that describe situations that contrary to fact did not occur – "If the train had not stopped for such a long time in front of the railway station I would have reached the connecting train"). A detailed philosophical discussion of the counterfactual causal model is given in Lewis 1973 [42].
Some authors resist a counterfactual approach and argue against the speculative and metaphysical background of counterfactual worlds (Dawid 2000 [43]): How can one object have two different contradictory properties like being exposed (factually) and simultaneously not being exposed (counterfactually)? In addition, the units of observation are assumed to exist in different states simultaneously in the counterfactual world if the properties have more than two distinct values. This duality appears to be even more puzzling. However, exactly such weird statements are made by quantum mechanics about objects like electrons or Fulleren molecules which are part of the real world we live in (Mittelstaedt 1972 [44], Feynman 1985 [45], Zeilinger 2003 [46]). It is important to understand that this superposition principle ("superposing" contradicting properties simultaneously on the same object) is no marginal phenomenon but lies at the center of modern physics (Dirac 1967 [47]).
Note further that the mathematically consistent analytical treatment of causal questions by counterfactual theory is obviously related to the so called multiverse approach (Everett 1957 [48]). The latter is a suggestive ontological interpretation of abstract Hilbert space theory that was introduced into modern physics to represent quantum states (Weberruβ 1998 [49], Penrose 1994 [50], Deutsch 1997 [51]). A new mathematical approach was necessary because – due to the superposition principle – quantum states are much richer than classical states and therefore fall beyond the grasp of Newtonion and Einsteinian terminology and theory (Hughes 1999 [52]). Critics of counterfactual logic, like Dawid 2000 [43], overlook this deep connection to quantum mechanics that renders the approach empirically plausible and mathematically as consistent as modern physics. In other words, a fundamental philosophical attack of counterfactual reasoning leads inevitably into an attack of quantum physics, which has survived successfully all criticism raised, empirically and theoretically, during the last century (Feynman 1985 [45], Hawking 1996 [53], 2001 [54]). In a discussion of the so called null measurements in quantum mechanics Roger Penrose described the link of quantum mechanics to counterfactuals as follows: "It is quite extraordinary that quantum mechanics enables you to test whether something might have happened but didn't happen. It tests what philosophers call counterfactuals. It is remarkable that quantum mechanics allows real effects to result from counterfactuals!" (Penrose 2000 [55], p. 67).
In addition, this link to quantum mechanics disproves the repeatedly made statement by Dawid (cp. the discussion following Maldonado and Greenland 2002 [23]) that counterfactual reasoning were subject to a deterministic or 'fatal' world view. On the contrary, quantum mechanics is the realm of pure chance and statistics: counterfactuals are therefore clearly consistent with indeterminism and stochastic principles. Finally, critics of counterfactual logic, like Dawid 2000 [43], have not presented an appealing and logically consistent alternate mathematical theory of causality.
Whereas the counterfactual approach can help to clarify terminology and substance of causal relations, it points simultaneously at some ambiguities when discussing competing causes of death. In this scenario, it is unclear what kind of action should be taken to cause a suppression of competing risks (Greenland 2002 [56]). Hence, the logical and causal background of the example presented with competing causes of death (Figure 4, 5, 6 and Table 3, see Additional file 3) appears to need further elaboration.
Conclusion
In conclusion, the excess Years of Potential Life Lost estimates the excess Years of Life Lost due to exposure unbiasedly if we are interested in a) death from all causes and b) total excess Years of Life Lost summed up across the whole cohort. However, the method of calculating excess Years of Potential Life Lost due to exposure is potentially biased if it is applied 1) to estimate the impact of exposure on specific causes of death, like lung cancer, in the presence of competing causes or 2) to estimate the impact of exposure (overall or cause specific) conditional on age at death. These potential biases can be rather severe in published analyses (e.g., Park et al. 2002 [2]). Rigorous causal thinking (Greenland et al. 1999a [57], Greenland et al. 1999b [58]) can help to identify and avoid such empirical shortcomings. Fortunately, as an important outcome of these causal considerations, methods are available (Robins 1997 [22]) and readily applicable in occupational epidemiology (Witteman et al. 1998 [59]) to estimate the cause-specific reduction in life span due to occupational exposures unbiasedly given a specified failure time model and the assumption of no unmeasured confounders. These methods are valid even under the complicated but often realistic conditions of dependent censoring, survivor biases and intermediate confounding (Keiding et al. 1999 [60], Morfeld et al. 2002 [61]).
Competing Interests
The author(s) declares that he has no competing interests.
Endnote 1
The following example is based on the counterfactual framework presented in Chapter 1. It demonstrates that neither relative risks nor relative rates can be used in general to estimate the probability of causation unbiasedly. In particular, I show that an estimate of the attributable hazard derived from a Cox model (Breslow and Day 1987 [24]) fails to describe the causally attributable fraction among the exposed cases. The example demonstrates that this shortcoming is due to the fact that an advanced onset of disease is not reflected completely by risk or rate statistics. As in Chapter 1, a binary exposure indicator is assumed for simplicity in the following.
The cohort is supposed to comprise four subjects: A, A's twin, B, and B's twin. In addition, it is assumed that the cohort is followed up in mortality until the fixed censoring date tend>0. The time scale can be chosen arbitrarily as age, calendar time or time since start of follow-up without affecting the following arguments. I assume that the exposed subject A may experience the event (death) during the follow-up period at t1 years (t1 > 0), if unexposed counterfactually (A's twin) at t2 > t1, t2 < tend. No event may occur in subject B and his/her twin during follow-up (i.e., neither under exposure nor when unexposed). Thus, among the two exposed subjects, A and B, only one case occurs and this case is causally affected by exposure since t1 < t2. It follows that the probability of causation (i.e., the percentage of exposed cases occurring during follow-up which are causally affected by exposure) is exactly 100%.
In contrast, the incidence proportion (Rothman and Greenland 1998 [5]) is 0.5 among both, the exposed and unexposed, leading to a relative risk of 1 and an attributable risk among the exposed of 0. Hence, the attributable risk calculated from incidence proportions fails to reflect the causal impact of exposure on subject A's event time (true probability of causation among the exposed = 100% >> attributable risk among the exposed calculated from incidence proportions = 0%). I'd like to emphasize that the reason for this shortcoming is the fact that relative risks reflect excess cases only but no advanced cases.
Note that a change from risks to rates does not overcome the problem. The rate among the exposed is 1/(t1 + tend) and among the unexposed is 1/(t2 + tend). Consequently, the rate ratio is (t2 + tend)/(t1 + tend) > 1 because t2 > t1. The attributable rate among the exposed can be determined as (rate ratio -1) / rate ratio = 1 - 1/rate ratio. Thus, I conclude 0 < 1 - (t1 + tend)/(t2 + tend) < 1, again proving a systematic underestimate of the true probability of causation among the exposed by the attributable rate ratio. Note that the rare disease assumption is of no help. Assuming n exposed subjects (n>>1) that neither react nor do their n unexposed twins, we get an attributable rate among the exposed of 1 - (t1 + (n)(tend)) / (t2 + (n)(tend)) < 1. If n approaches infinity the attributable rate among the exposed decreases to zero whereas the true probability of causation remains always constant at 100%. Hence, the discrepancy is even sharpened under the rare disease assumption.
Last, I try to escape these problems by performing a Cox analysis. Due to its construction, the whole cohort (exposed and unexposed) comprises 4 subjects and 2 risk sets. One set is generated by the exposed case (A), the other set by the unexposed (A's twin). Therefore, a Cox analysis of the cohort yields the following.
Assuming a relative hazard (rate) in the Cox model of
λ / λ0 = exp (β exposure), exposure = binary exposure indicator
we get the partial likelihood
which is maximized at b = ln 2/2.
Therefore, the estimated hazard ratio is
exp (b) = √ 2
yielding an estimated attributable hazard among the exposed of
1 - (√2)-1 < 1 = probability of causation.
Note that the rare disease assumption is again of no help to overcome this discrepancy because the estimated attributable hazard among the exposed approaches 0 when the number of controls is rising indefinitely.
Greenland 1999 [6] emphasizes correctly that this methodological error (i.e., using attributable risks or rates among the exposed to measure the probability of causation) has already become a social problem. Compensation of workers who developed a disease after occupational exposure is often decided by impact measures based on this erroneous identification of probability of causation and attributable risk or rate among the exposed. Usually a threshold value of 50% is chosen in Germany, which corresponds to the so called doubling of risks (i.e., a relative risk or rate of 2). Thus, compensation is usually withheld if the attributable risk or rate is below 50% (Morfeld and Piekarski 2001 [7]). Similar procedures are applied in other countries (Greenland 1999 [6], Armstrong and Theriault 1996 [37]). However, as demonstrated in this endnote, such an argument based on the attributable risk or rate is not justified. Discussions have started about how the discrepancy between the calculated attributable risk among the exposed and the intended probability of causation can be accounted for in amended compensation schemes in Germany (Morfeld und Piekarski 2001 [7]). These amended compensation schemes should reflect the advancement of disease onset due to exposure more accurately than the conventional ones.
Supplementary Material
Additional File 1
Life table analysis with calculation of excess Years of Potential Life Lost e-YPLL according to Park et al. 2002. Basic data (unexposed) from BEIR IV (1988), Table 2A-10, p. 133: death rates of the male US population, surviving at least 30 years, applied to a birth cohort of 100,000. Exposure impact: advancement of certain fractions of deaths. For details of assumed mechanism see Table 2 (Additional file 2).
Click here for file
Additional File 2
Two different exposure-response mechanisms compatible with the life table analysis in Table 1. Fractions of the unexposed deaths are advanced by 0 yr or 5 yr according to mechanism 1 and by 0 yr, 5 yr or 10 yr according to mechanism 2. The age distribution of all exposed deaths is identical under both mechanisms. The distribution of the true excess Years of Life Lost e-YLL differs between mechanisms and both diverge from e-YPLL (Table 1, see Additional file 1), whereas the totals agree. (advcm = advancement)
Click here for file
Additional File 3
Life table analysis with calculation of excess Years of Potential Life Lost e-YPLL and true excess Years of Life Lost e-YLL for overall and lung cancer mortality (ICD9-162). Basic data (unexposed) from BEIR IV (1988), Table 2A-10, p. 133: overall and lung cancer death rates of the male US population, surviving at least 30 years applied to a birth cohort of 100,000. Exposure impact: advancement of factual and hypothetical lung cancer deaths by 5 years, mixture of advancements among deaths from all causes. It is assumed that the advancement of hypothetical lung cancer deaths leads to an excess of 50% of lung cancer deaths among exposed in each age category. The overall e-YLL for lung cancer are less than the number of exposed lung cancer deaths times 5 years. The e-YPLL for overall death and lung cancer death are determined according to Park et al. 2002. For all deaths e-YPLL must equal e-YLL, but e-YPLL is obviously biased for lung cancer death.
Click here for file
Additional File 4
Excel sheet explaining the calculations in Additional files 1 and 2.
Click here for file
Additional File 5
Excel sheet explaining the calculations in Additional file 3.
Click here for file
Acknowledgment
I would like to thank Sander Greenland, University of California, Los Angeles, for an outstanding series of e-mail exchanges on counterfactual reasoning, subjective probability and quantum mechanics, Stephen C. Newman, University of Alberta, Canada and David P. Smith, University of Texas, for reviewing the manuscript, two unknown editors for their help on copy-editing the paper, and Carl V. Phillips, Editor of Epidemiologic Perspectives and Innovation, for his support and very helpful comments on earlier versions of this article.
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| 15601477 | PMC545055 | CC BY | 2021-01-04 16:36:33 | no | Epidemiol Perspect Innov. 2004 Dec 16; 1:5 | utf-8 | Epidemiol Perspect Innov | 2,004 | 10.1186/1742-5573-1-5 | oa_comm |
==== Front
BMC GastroenterolBMC Gastroenterology1471-230XBioMed Central London 1471-230X-5-11563163610.1186/1471-230X-5-1Case ReportA rare case of ascending colon actinomycosis mimicking cancer Filippou Dimitrios [email protected] Ioannis [email protected] Diamanto [email protected] Spiros [email protected] Department of Surgery, GP Hospital of Piraeus "Tzanion", Tzani & Afentouli str, Pireaus-Athens, Greece2 Department of Pathology, GP Hospital of Piraeus "Tzanion", Tzani & Afentouli str, Pireaus-Athens, Greece2005 4 1 2005 5 1 1 19 7 2004 4 1 2005 Copyright © 2005 Filippou 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
Actinomycosis is a rare inflammatory disease caused by an anaerobic bacterium that can rarely affect the large intestine.
Case presentation
We present a rare case of a cecum and ascending colon actinomycosis in a 72 years old woman, mimicking clinically a malignant inflammatory tumor of the right colon. The patient complained of right lower quadrant pain. Although our first thought was a peri-appendiceal abscess, CT scan suggested a right colon tumor. The patient underwent a right colectomy and the histological examination of the specimen revealed colon actinomycosis.
Conclusions
Preoperative diagnosis in colon actinomycosis is difficult to achieve. Treatment of choice is antibiotics administration. A review of the possible pathogenesis and therapeutic modalities is also presented.
==== Body
Background
Actinomycosis is an uncommon inflammatory entity caused by the universally distributed anaerobic bacterium, Actinomyces Israeli. This Actinomyces is a gram-positive, rather microaerophilic bacterium, which consists a component of the normal human flora. Actinomyces requires the presence of many other bacteria, which destroy the over-vascularized regions and convert aerobic microenvironment to an anaerobic one. Then it's easy for Actinomyces to migrate, infect and proliferate in already injured tissue. Primary bowel involvement is rare, although it has been increased in frequency over the last years. The most common sites of the disease are the transverse colon and the cecum with the appendix [1,2].
Actinomycosis can mimic other abdominal diseases as diverticulitis, abscesses, inflammatory bowel disease and malignant tumors, presenting a diagnostic challenge, and identified post-operatively in most of the cases [3].
The treatment of choice is antibiotic administration, whenever it is possible due to diagnostic difficulties, although in most cases surgical intervention is performed Diagnosis can be achieved with endoscopy and imaging techniques, as computed tomography (CT scan) and magnetic resonance imaging (MRI). We present a rare case of right colon actinomycosis mimicking malignant tumor, causing bowel obstruction. The diagnosis was achieved only postoperatively.
Case presentation
A 72 years old woman proceeded to the Emergency Department complaining of acute lower right abdominal pain, mild fever, mild weight loss and constipation. The past medical history of the patient was free.
The patient was presented with severe lower right abdominal pain, with signs of local peritonitis and a palpable mass in the same region. The body temperature was 37.2–37.4°C, while arterial blood pressure and cardiac rate 150/90 mmHg and 90/min respectively. The signs of local peritonitis combined with the palpable mass of the lower right abdominal area suggested perforation of the appendix and abscess formation. The laboratory examinations of the patient showed leucocytosis with white blood cell count (WBC) 19000 (with macrophage prevalence: 89%). Colonoscopy revealed obstruction of the right colon. Biopsies were not acquired because the colon lumen was obstructed and the endoscope could not approach the lesion. Computerized tomography (CT) scans of the abdomen (Figure 1) revealed a soft lobular mass, measuring 5 × 5 cm, attached to the ascending colon and cecum, compatible with a tumor. The most possible diagnosis was that of a perforated colonic tumor. The patient underwent explorative laparotomy and right hemicolectomy with an end-to-end ileocolonic anastomosis. Thorough exploration of the abdominal cavity revealed no other pathologic findings.
The surgical specimen consisted of a 10 cm length of the terminal ileum and the whole of the right colon. The serosa was very heavily covered by suppurative exudates and fibrotic tissue.
Microscopic examination of the surgical specimen revealed thickening of the ascending colon wall with neutrophilic infiltration. Numerous polymorphonuclear leukocytes within the muscularis and a fibro-purulent reaction over the serosa with actinomycotic "sulfur granules" in it, were found in high power magnification (Figure 2).
Upon receiving the pathology report, systemic intravenous penicillin treatment was initiated. Therapy continued for 10 days and then followed by oral penicillin for 6 months. No postoperative complications were observed and the patient was discharged the 14th day.
Discussion
Actinomyces Israeli, a filamentous, gram-positive bacillus, is a constant part of the micro flora in the human oral cavity [4].
Actinomycosis presents a worldwide distribution and no sex predilection is obvious although most of the reported cases refer to males. Abdominal involvement occurs in only 20 percent of all cases of actinomycosis and can mimic malignancy, tuberculosis and inflammatory bowel disease [5].
Actinomyces is not always pathogenic, and normally exists in stagnated cecum or sigmoid colon. Predisposing factors include previous abdominal surgical operations, intestinal necrosis, foreign bodies, appendicitis and perforation. Some authors suggest that inflammatory or neoplastic processes may contribute to actinomycosis development [6,7].
Bowel obstruction and perforation due to actinomycosis without predisposing factors is very rare and only few cases have been described in the literature. Most commonly actinomycosis occurs in terminal ileus and appendix and rarely in the ascending colon, which is difficult to get obstructed. In our case actinomycosis affected cecum and ascending colon, with a dramatic clinical presentation. We searched the literature in Medline from 1997 to 2004 and we found that only a few cases with clinical presentation similar to our case have been reported.
Preoperative diagnosis is difficult although in some cases colonoscopy and histological examination of endoscopically acquired specimen can set the diagnosis. In our case the colon lumen was obstructed and no biopsies were taken. The CT findings suggested perforated colon tumor and an oncologic right hemicolectomy was performed. Actimomycosis usually mimics subacute infections or malignant tumors and the radiologic diagnosis of this entity may be difficult. Some authors suggest that abdominal CT scan with contrast enhancement may reveal a solid mass (intraluminal or extraluminal) with focal areas of attenuation invading the adjacent tissues and suggesting the diagnosis [8,9].
Most common findings in CT scan and/or barium study include mural invasion with stricture formation, mass effect with tapered narrowing of the lumen, and thickened mucosal folds. In many cases the radiologic findings are similar to those of Crohn's disease, intestinal tuberculosis, and excavated malignant tumors [10,11]. The most important CT feature for the correct diagnosis is a large mass adjacent to the involved bowel, which is also a very common finding in patients with colon actinomycosis. In rectosigmoid, colon cystic masses are more common, whereas in transverse or ascending colon purely solid masses are the predominant finding [12,13].
Goldwag et al suggest that CT guided fine needle aspiration can be both diagnostic and therapeutic. Microbiological analysis of material acquired by FNA may reveal sulfur granules, which are suggesting actinomycosis and nocardiosis. In most of the cases the sample receive is difficult especially when intestinal and colon are involved. We believe that in cases where the CT findings are non-specific, surgical exploration is necessary not only for diagnostic but also for therapeutic reasons [14].
High dose intravenous penicillin injection followed by orally administered penicillin for at least 6–12 months is the treatment of choice. Penicillin administration decreases morbidity and the patient may avoid an unnecessary operation [15,16].
Correct diagnosis is difficult and can be achieved preoperatively in only 10% of the cases, but it is of great importance because the appropriate treatment includes primarily penicillin administration. Surgical intervention is indicated only in cases with obscure diagnosis and for necrotic debridement removal. Although diagnosis only with imaging techniques and laboratory tests is difficult, abdominal actinomycosis should always be included in the differential diagnosis in patients with abdominal masses [1].
Conclusions
In conclusion, colon actinomycosis should always be included in the differential diagnosis of abdominal masses with tumor of inflammatory characteristics. It should be especially suspected when the appearance on CT scan is of a solid mass with focal areas of attenuation or a cystic mass with a thickened wall showing inhomogeneous contrast enhancement that tends to invade adjacent tissues or structures. Immediate and accurate diagnosis, usually by FNA and cytology examination can prevent unnecessary surgical treatment.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
All authors contributed equally to this work. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgments
Written consent was obtained from the patient or their relative for publication of the patient's details. We thank the patient for giving us written consent for publishing this study.
Figures and Tables
Figure 1 In abdominal CT scan a large inflammatory tumor like mass is identified. The characteristics of the mass do not indicate colon actinomycosis. The characters of the lesion and the coexisting edema and the severe and diffuse inflammation of the mesentery in the lower right quadrant probably suggest perforated ascending colon tumor.
Figure 2 The histological examination of the removed specimen (right hemicolectomy specimen) revealed actinomyces colonies in the upper part of the cecum and the ascending colon wall. The large spherical clusters, densely packed and branching, and PAS positive "sulfur granules" are specific of actinomycosis.
==== Refs
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| 15631636 | PMC545056 | CC BY | 2021-01-04 16:03:27 | no | BMC Gastroenterol. 2005 Jan 4; 5:1 | utf-8 | BMC Gastroenterol | 2,005 | 10.1186/1471-230X-5-1 | oa_comm |
==== Front
BMC GastroenterolBMC Gastroenterology1471-230XBioMed Central London 1471-230X-5-11563163610.1186/1471-230X-5-1Case ReportA rare case of ascending colon actinomycosis mimicking cancer Filippou Dimitrios [email protected] Ioannis [email protected] Diamanto [email protected] Spiros [email protected] Department of Surgery, GP Hospital of Piraeus "Tzanion", Tzani & Afentouli str, Pireaus-Athens, Greece2 Department of Pathology, GP Hospital of Piraeus "Tzanion", Tzani & Afentouli str, Pireaus-Athens, Greece2005 4 1 2005 5 1 1 19 7 2004 4 1 2005 Copyright © 2005 Filippou 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
Actinomycosis is a rare inflammatory disease caused by an anaerobic bacterium that can rarely affect the large intestine.
Case presentation
We present a rare case of a cecum and ascending colon actinomycosis in a 72 years old woman, mimicking clinically a malignant inflammatory tumor of the right colon. The patient complained of right lower quadrant pain. Although our first thought was a peri-appendiceal abscess, CT scan suggested a right colon tumor. The patient underwent a right colectomy and the histological examination of the specimen revealed colon actinomycosis.
Conclusions
Preoperative diagnosis in colon actinomycosis is difficult to achieve. Treatment of choice is antibiotics administration. A review of the possible pathogenesis and therapeutic modalities is also presented.
==== Body
Background
Actinomycosis is an uncommon inflammatory entity caused by the universally distributed anaerobic bacterium, Actinomyces Israeli. This Actinomyces is a gram-positive, rather microaerophilic bacterium, which consists a component of the normal human flora. Actinomyces requires the presence of many other bacteria, which destroy the over-vascularized regions and convert aerobic microenvironment to an anaerobic one. Then it's easy for Actinomyces to migrate, infect and proliferate in already injured tissue. Primary bowel involvement is rare, although it has been increased in frequency over the last years. The most common sites of the disease are the transverse colon and the cecum with the appendix [1,2].
Actinomycosis can mimic other abdominal diseases as diverticulitis, abscesses, inflammatory bowel disease and malignant tumors, presenting a diagnostic challenge, and identified post-operatively in most of the cases [3].
The treatment of choice is antibiotic administration, whenever it is possible due to diagnostic difficulties, although in most cases surgical intervention is performed Diagnosis can be achieved with endoscopy and imaging techniques, as computed tomography (CT scan) and magnetic resonance imaging (MRI). We present a rare case of right colon actinomycosis mimicking malignant tumor, causing bowel obstruction. The diagnosis was achieved only postoperatively.
Case presentation
A 72 years old woman proceeded to the Emergency Department complaining of acute lower right abdominal pain, mild fever, mild weight loss and constipation. The past medical history of the patient was free.
The patient was presented with severe lower right abdominal pain, with signs of local peritonitis and a palpable mass in the same region. The body temperature was 37.2–37.4°C, while arterial blood pressure and cardiac rate 150/90 mmHg and 90/min respectively. The signs of local peritonitis combined with the palpable mass of the lower right abdominal area suggested perforation of the appendix and abscess formation. The laboratory examinations of the patient showed leucocytosis with white blood cell count (WBC) 19000 (with macrophage prevalence: 89%). Colonoscopy revealed obstruction of the right colon. Biopsies were not acquired because the colon lumen was obstructed and the endoscope could not approach the lesion. Computerized tomography (CT) scans of the abdomen (Figure 1) revealed a soft lobular mass, measuring 5 × 5 cm, attached to the ascending colon and cecum, compatible with a tumor. The most possible diagnosis was that of a perforated colonic tumor. The patient underwent explorative laparotomy and right hemicolectomy with an end-to-end ileocolonic anastomosis. Thorough exploration of the abdominal cavity revealed no other pathologic findings.
The surgical specimen consisted of a 10 cm length of the terminal ileum and the whole of the right colon. The serosa was very heavily covered by suppurative exudates and fibrotic tissue.
Microscopic examination of the surgical specimen revealed thickening of the ascending colon wall with neutrophilic infiltration. Numerous polymorphonuclear leukocytes within the muscularis and a fibro-purulent reaction over the serosa with actinomycotic "sulfur granules" in it, were found in high power magnification (Figure 2).
Upon receiving the pathology report, systemic intravenous penicillin treatment was initiated. Therapy continued for 10 days and then followed by oral penicillin for 6 months. No postoperative complications were observed and the patient was discharged the 14th day.
Discussion
Actinomyces Israeli, a filamentous, gram-positive bacillus, is a constant part of the micro flora in the human oral cavity [4].
Actinomycosis presents a worldwide distribution and no sex predilection is obvious although most of the reported cases refer to males. Abdominal involvement occurs in only 20 percent of all cases of actinomycosis and can mimic malignancy, tuberculosis and inflammatory bowel disease [5].
Actinomyces is not always pathogenic, and normally exists in stagnated cecum or sigmoid colon. Predisposing factors include previous abdominal surgical operations, intestinal necrosis, foreign bodies, appendicitis and perforation. Some authors suggest that inflammatory or neoplastic processes may contribute to actinomycosis development [6,7].
Bowel obstruction and perforation due to actinomycosis without predisposing factors is very rare and only few cases have been described in the literature. Most commonly actinomycosis occurs in terminal ileus and appendix and rarely in the ascending colon, which is difficult to get obstructed. In our case actinomycosis affected cecum and ascending colon, with a dramatic clinical presentation. We searched the literature in Medline from 1997 to 2004 and we found that only a few cases with clinical presentation similar to our case have been reported.
Preoperative diagnosis is difficult although in some cases colonoscopy and histological examination of endoscopically acquired specimen can set the diagnosis. In our case the colon lumen was obstructed and no biopsies were taken. The CT findings suggested perforated colon tumor and an oncologic right hemicolectomy was performed. Actimomycosis usually mimics subacute infections or malignant tumors and the radiologic diagnosis of this entity may be difficult. Some authors suggest that abdominal CT scan with contrast enhancement may reveal a solid mass (intraluminal or extraluminal) with focal areas of attenuation invading the adjacent tissues and suggesting the diagnosis [8,9].
Most common findings in CT scan and/or barium study include mural invasion with stricture formation, mass effect with tapered narrowing of the lumen, and thickened mucosal folds. In many cases the radiologic findings are similar to those of Crohn's disease, intestinal tuberculosis, and excavated malignant tumors [10,11]. The most important CT feature for the correct diagnosis is a large mass adjacent to the involved bowel, which is also a very common finding in patients with colon actinomycosis. In rectosigmoid, colon cystic masses are more common, whereas in transverse or ascending colon purely solid masses are the predominant finding [12,13].
Goldwag et al suggest that CT guided fine needle aspiration can be both diagnostic and therapeutic. Microbiological analysis of material acquired by FNA may reveal sulfur granules, which are suggesting actinomycosis and nocardiosis. In most of the cases the sample receive is difficult especially when intestinal and colon are involved. We believe that in cases where the CT findings are non-specific, surgical exploration is necessary not only for diagnostic but also for therapeutic reasons [14].
High dose intravenous penicillin injection followed by orally administered penicillin for at least 6–12 months is the treatment of choice. Penicillin administration decreases morbidity and the patient may avoid an unnecessary operation [15,16].
Correct diagnosis is difficult and can be achieved preoperatively in only 10% of the cases, but it is of great importance because the appropriate treatment includes primarily penicillin administration. Surgical intervention is indicated only in cases with obscure diagnosis and for necrotic debridement removal. Although diagnosis only with imaging techniques and laboratory tests is difficult, abdominal actinomycosis should always be included in the differential diagnosis in patients with abdominal masses [1].
Conclusions
In conclusion, colon actinomycosis should always be included in the differential diagnosis of abdominal masses with tumor of inflammatory characteristics. It should be especially suspected when the appearance on CT scan is of a solid mass with focal areas of attenuation or a cystic mass with a thickened wall showing inhomogeneous contrast enhancement that tends to invade adjacent tissues or structures. Immediate and accurate diagnosis, usually by FNA and cytology examination can prevent unnecessary surgical treatment.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
All authors contributed equally to this work. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgments
Written consent was obtained from the patient or their relative for publication of the patient's details. We thank the patient for giving us written consent for publishing this study.
Figures and Tables
Figure 1 In abdominal CT scan a large inflammatory tumor like mass is identified. The characteristics of the mass do not indicate colon actinomycosis. The characters of the lesion and the coexisting edema and the severe and diffuse inflammation of the mesentery in the lower right quadrant probably suggest perforated ascending colon tumor.
Figure 2 The histological examination of the removed specimen (right hemicolectomy specimen) revealed actinomyces colonies in the upper part of the cecum and the ascending colon wall. The large spherical clusters, densely packed and branching, and PAS positive "sulfur granules" are specific of actinomycosis.
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| 15615593 | PMC545057 | CC BY | 2021-01-04 16:03:30 | no | BMC Infect Dis. 2004 Dec 22; 4:62 | latin-1 | BMC Infect Dis | 2,004 | 10.1186/1471-2334-4-62 | oa_comm |
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BMC BiolBMC Biology1741-7007BioMed Central London 1741-7007-3-11563894510.1186/1741-7007-3-1Research ArticleDiscs-large (DLG) is clustered by presynaptic innervation and regulates postsynaptic glutamate receptor subunit composition in Drosophila Chen Kaiyun [email protected] David E [email protected] Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60607, USA2005 8 1 2005 3 1 1 21 10 2004 8 1 2005 Copyright © 2005 Chen and Featherstone; licensee BioMed Central Ltd.2005Chen and Featherstone; 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
Drosophila discs-large (DLG) is the sole representative of a large class of mammalian MAGUKs, including human DLG, SAP 97, SAP102, and PSD-95. MAGUKs are thought to be critical for postsynaptic assembly at glutamatergic synapses. However, glutamate receptor cluster formation has never been examined in Drosophila DLG mutants. The fly neuromuscular junction (NMJ) is a genetically-malleable model glutamatergic synapse widely used to address questions regarding the molecular mechanisms of synapse formation and growth. Here, we use immunohistochemistry, confocal microscopy, and electrophysiology to examine whether fly NMJ glutamate receptor clusters form normally in DLG mutants. We also address the question of how DLG itself is localized to the synapse by testing whether presynaptic innervation is required for postsynaptic DLG clustering, and whether DLG localization requires the presence of postsynaptic glutamate receptors.
Results
There are thought to be two classes of glutamate receptors in the Drosophila NMJ: 1) receptors that contain the subunit GluRIIA, and 2) receptors that contain the subunit GluRIIB. In DLG mutants, antibody staining for the glutamate receptor subunit GluRIIA is normal, but antibody staining for the glutamate receptor subunit GluRIIB is significantly reduced. Electrophysiological analysis shows an overall loss of functional postsynaptic glutamate receptors, along with changes in receptor biophysical properties that are consistent with a selective loss of GluRIIB from the synapse. In uninnervated postsynaptic muscles, neither glutamate receptors nor DLG cluster at synapses. DLG clusters normally in the complete absence of glutamate receptors.
Conclusions
Our results suggest that DLG controls glutamate receptor subunit composition by selectively stabilizing GluRIIB-containing receptors at the synapse. We also show that DLG, like glutamate receptors, is localized only after the presynaptic neuron contacts the postsynaptic cell. We hypothesize that glutamate receptors and DLG cluster in response to parallel signals from the presynaptic neuron, after which DLG regulates subunit composition by stabilizing (probably indirectly) receptors that contain the GluRIIB subunit. The mechanism(s) stabilizing GluRIIA-containing receptors remains unknown.
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Background
The molecular mechanisms that target postsynaptic glutamate receptors to the postsynaptic membrane, and keep receptors clustered there, remain unknown. Membrane-associated guanylate kinase proteins (MAGUKs) are cell-cell junction proteins with multiple protein-interaction domains (PDZ, SH3, 4.1/Hook, and a catalytically inactive guanylate kinase/GUK domain) [1-3]. Synaptic MAGUKs are widely believed to be required for recruitment and/or stabilization of a variety of synaptic proteins, including glutamate receptors in the postsynaptic density (PSD) [2,4-6]. Although genetic evidence for MAGUK-dependent clustering of NMDA receptors is strongest, and consistent with a model wherein MAGUKs traffick NMDARs to the membrane [7,8], the evidence for scaffolding or trafficking of non-NMDA ionotropic glutamate receptors by MAGUKs is largely based on biochemical interactions and overexpression [9-12]. There is little evidence showing that glutamate receptors fail to cluster appropriately in the absence of MAGUKs – a critical prediction of the 'MAGUK scaffold' model.
Drosophila DLG is a prototypical MAGUK, containing three PDZ domains, an SH3 domain, a hook/4.1-binding domain, and a GUK domain [3,13]. DLG is the sole fly representative of a large group of mammalian MAGUKs, including SAP-90/PSD-95, SAP-102/NE-dlg, Chapsyn-110/PSD-93, and SAP97/human DLG [3]. DLG was originally isolated as a tumor suppressor due to loss of apicobasal polarity in dlg mutants and consequent tumorous overgrowth in imaginal disc epithelia [14,15]. Since then, DLG has been shown to be present at several types of cell junction, including the glutamatergic larval neuromuscular junction (NMJ) [16-19].
The Drosophila NMJ is a widely-used model glutamatergic synapse that is molecularly and developmentally similar to glutamatergic synapses in the mammalian CNS. Drosophila NMJs in DLG mutants show a variety of changes, including disrupted organization of synaptic shaker potassium channels and fasciclin II, plus subtle alterations in larval synaptic growth [17,20-22]. It is clear from previous studies that DLG is not absolutely required for glutamate receptor expression and localization in the NMJ. In fact, DLG mutant larvae display larger excitatory postsynaptic potential amplitudes [17]. However, this phenotype depends specifically on presynaptic, but not postsynaptic loss of DLG [17]; presynaptic loss of DLG has subsequently been shown to increase synaptic vesicle diameter and quantal size [23]. Thus, based on measures of NMJ transmission, it is difficult to determine,, whether subtle changes in glutamate receptor cluster formation have occurred. Another complication is that DLG mutant larvae show dramatic underdevelopment of the subsynaptic reticulum (SSR), a dense infolding of postsynaptic membrane that appears during larval NMJ growth [16,17,19,24]. This loss of postsynaptic membrane in DLG mutant larvae makes it difficult to evaluate changes in postsynaptic transmembrane proteins, such as receptors.
Thus, there has so far been no answer to the question of whether DLG is involved in the formation of postsynaptic glutamate receptor clusters in Drosophila. However, the aforementioned phenotypic and technical obstructions can be completely avoided in two ways. First, we can examine glutamate receptors in DLG mutant embryos rather than larvae. In embryos, the SSR has not yet formed [24]; therefore there are not yet any differences in postsynaptic membrane abundance between DLG mutant and control NMJs. Second, we can assay postsynaptic glutamate receptors directly, by immunohistochemistry and pressure ejection of glutamate onto voltage-clamped postsynaptic muscle cells [25]. This circumvents any presynaptic alterations. Immunocytochemical techniques are particularly valuable, because antibodies that recognize different receptor subunits can show whether DLG differentially regulates subpopulations of receptors that differ in subunit composition. Mammalian studies have made it increasingly apparent that many aspects of receptor assembly and trafficking depend on the presence of specific subunits. Evidence for molecularly distinct subpopulations of glutamate receptors in Drosophila NMJs has only recently been presented [26,27]. Differential regulation of these receptors has never before been demonstrated.
The Drosophila NMJ contains five different ionotropic glutamate receptor subunits, each encoded by a different gene: GluRIIA, GluRIIB, GluRIIC (also referred to as 'GluRIII'), GluRIID, and GluRIIE [26-30]. By sequence, fly NMJ subunits are most similar to mammalian kainate receptors. Mutations in GluRIIC, GluRIID, or GluRIIE are lethal, show loss of functional NMJ glutamate receptors, and eliminate immunoreactivity for other subunits [26,27,30]. Thus, GluRIIC, GluRIID, and GluRIIE are thought to be essential subunits contained by each glutamate receptor at the NMJ. In contrast, null mutations in either GluRIIA or GluRIIB individually are viable, but deletion of both GluRIIA and GluRIIB simultaneously is lethal [29,31]. Evidence from ligand binding studies and partial crystal structures strongly suggests that ionotropic glutamate receptors are tetramers [32-34]. Thus, it is thought that Drosophila NMJ glutamate receptors are heterotetramers composed of one GluRIIC subunit, one GluRIID subunit, and one GluRIIE subunit, plus either one subunit of GluRIIA or one subunit of GluRIIB [26,27]. This model is consistent with immunocytochemical results: immunoreactivity for GluRIIA only partially overlaps that of GluRIIB [30], suggesting that at least some receptors contain either GluRIIA or GluRIIB, but not both. In other words, the Drosophila NMJ contains two subclasses of ionotropic glutamate receptor: 1) GluRIIA-containing receptors and 2) GluRIIB-containing receptors.
Here, we use electrophysiology and immunocytochemistry to demonstrate selective loss of GluRIIB, but not GluRIIA, in DLG mutant Drosophila embryos. This is the first demonstration that DLG regulates synaptic glutamate receptor abundance in Drosophila, and the first evidence that fly NMJ receptors can be differentially regulated, based on subunit composition. We also explored the mechanisms by which DLG itself is localized at the NMJ. Neither GluRIIA nor GluRIIB are localized unless a presynaptic neuron first contacts the postsynaptic cell. DLG is also not clustered in the absence of presynaptic innervation. This neuronal contact-dependent clustering of DLG does not depend on the clustering or expression of glutamate receptors, because DLG is clustered properly in the absence of all postsynaptic glutamate receptors. Our results are consistent with a model in which an unknown signal from the presynaptic neuron triggers parallel clustering of both DLG and glutamate receptors, after which DLG promotes the synaptic stability of receptors containing GluRIIB, but not GluRIIA.
Results
DLG, GluRIIA, and GluRIIB are localized postsynaptically at the Drosophila NMJ
As previously demonstrated [16-19], DLG is abundantly expressed throughout the postsynaptic membrane surrounding presynaptic motor axon terminals (aka 'boutons') (Fig. 1A–B). DLG appears distributed throughout the postsynaptic membrane; there are no discernible DLG-positive domains smaller than the size of a bouton. In contrast, immunoreactivity for NMJ glutamate receptors has been shown to be restricted to specific postsynaptic domains directly opposite presynaptic active zones [26,35]. This restricted immunoreactivity is visible as distinct clusters within the bouton-wide area delimited by DLG staining (Fig. 1C–D). Thus, not all postsynaptic DLG appears associated with glutamate receptors. We cannot determine by light microscopy whether all glutamate receptors colocalize with DLG, although our staining is consistent with that conclusion.
Figure 1 DLG, GluRIIA, and GluRIIB are localized postsynaptically at the Drosophila NMJ A: Confocal projection of two boutons in a Drosophila third instar neuromuscular junction, visualized using the neuronal membrane marker anti-HRP (red) and anti-DLG antibodies (green). Scale bar = 2 μm. B: Isosurface projection generated from the confocal stack shown projected in A. At this stage of development, larval boutons are partially embedded in postsynaptic muscle membrane. DLG immunoreactivity surrounds the boutons, consistent with postsynaptic localization. C, D: Confocal projections of larval NMJs visualized using antibodies that recognize DLG (green) and the glutamate receptor subunits GluRIIA or GluRIIB (magenta, in panels C and D, respectively). Note the incomplete overlap of DLG and glutamate receptors; glutamate receptor immunoreactivity falls within the area stained by DLG, but not all DLG immunoreactivity overlaps with glutamate receptor immunoreactivity. Scale bar = 10 μm.
GluRIIB, but not GluRIIA, is lost from synapses in DLG mutants
In Drosophila embryos and larvae, the intersegmental nerve branch b (ISNb) innervates the ventral longitudinal muscles of each abdominal hemisegment [36]. In the confocal images shown in Fig. 2, ISNb is visualized using anti-HRP antibodies, which stain all neuronal membranes (green). Three NMJs on four muscles (not stained) are shown in each image (Fig. 2A–B). Ventral longitudinal muscles 7 and 6 are innervated via a NMJ that lies in the cleft between the two adjacent muscles. Muscles 13 and 12 are innervated by arborizations distal to the 7/6 NMJ. Each of these body wall NMJs contains multiple clusters of postsynaptic glutamate receptors that can be visualized using antibodies specific to either GluRIIA (Fig. 2A) or GluRIIB (Fig. 2B).
Figure 2 GluRIIB, but not GluRIIA, is lost from synapses in DLG mutants A: Confocal projections of late stage 17 embryonic NMJs visualized using antibodies to the neuronal membrane marker anti-HRP (green) and anti-GluRIIA subunit antibodies (magenta). Each panel shows NMJs on interior-most ventral longitudinal muscles in one hemisegment. Major anatomical landmarks are labelled: Intersegmental nerve branch b (ISNb) enters from the left (medial) and branches to form NMJs on muscles 7 & 6, 13, and 12. The top row of panels shows images from control embryos; the lower row of panels shows images from dlg mutant embryos. B: As in A, except anti-GluRIIB subunit antibodies (magenta) were used. Scale bar = 5 μm. C: Cumulative frequency plot of glutamate receptor cluster sizes, measured from images such as those shown in A &; B. GluRIIA cluster sizes (black squares) do not differ between control (filled squares) and dlg mutant (open squares) embryos. GluRIIB cluster sizes (magenta circles), however, are significantly smaller in dlg mutant embryos (open circles), compared to controls (filled circles).
To determine whether DLG is required for clustering of postsynaptic glutamate receptors, we visualized NMJ glutamate receptors in control and DLG mutant embryonic NMJs using GluRIIA and GluRIIB specific antibodies [30]. To manipulate DLG levels, we used embryos homozygous for the mutation dlgX1–2. In dlgX1–2 mutants, the S97N isoform of DLG, which is predominantly expressed in neurons and muscle, is reduced to undetectable levels [37]. Other isoforms of dlg (Drosophila expresses at least five) are expressed only at extremely low levels (approximately 5% normal) [37]. In addition, all isoforms (including S97) are truncated such that the C-terminus and GUK domains are completely removed [20].
Both control and dlgX1–2 mutant NMJs contain highly visible clusters of GluRIIA-containing receptors (Fig 2A; magenta). In Drosophila embryonic NMJs, the cluster area is directly proportional to the number of functional postsynaptic receptors measured using patch-clamp electrophysiology [25]. GluRIIA cluster area does not differ significantly between control and dlgX1–2 mutants (control cluster area = 0.68 ± 0.03 μm2, N = 103 clusters from 10 embryos; dlg = 0.75 ± 0.03 μm2, N = 99 clusters from 16 embryos; P = 0.15). Immunoreactivity for GluRIIB, on the other hand, appears dramatically decreased in dlgX1–2 mutants compared to controls (Fig. 2B). Indeed, GluRIIB cluster size is significantly decreased in dlgX1–2 mutants compared to controls (control cluster area = 0.45 ± 0.03 μm2, N = 88 clusters from 6 embryos; dlg = 0.31 ± 0.02 μm2, N = 57 clusters from 6 embryos; P < 0.001). Cumulative frequency histograms of GluRIIA and GluRIIB cluster sizes (Fig. 2C) represent the entire distribution of cluster sizes measured in control and mutant embryos. Fig. 2C shows that the distribution of GluRIIA cluster sizes is almost identical in control and dlg mutants. The GluRIIB cluster size curve in dlg mutants, however, is shifted toward smaller values. The largest shift occurs in the section of the curve where cluster sizes are largest, suggesting that the largest GluRIIB clusters are preferentially lost in dlg mutants. However, the smallest clusters approach the resolution limit of light microscopy, where reductions in object size are no longer detectable. Thus, the reduction in small cluster size is probably underestimated, and the average decrease of GluRIIB in dlg mutants may be larger than is measurable by immunocytochemistry.
Postsynaptic glutamate receptor current properties change in DLG mutants
The immunocytochemistry in Fig. 2 suggests that dlgX1–2 mutants specifically lose receptors that contain GluRIIB, but do not lose receptors containing GluRIIA. GluRIIA null mutants are viable, but mEJP amplitudes are smaller, glutamate receptor channel open times are reduced, and receptors show decreased sensitivity to the GluR antagonist argiotoxin 636 [31]. GluRIIB null mutants are also viable, but show no significant change in receptor function, suggesting that either the GluRIIB subunit plays a lesser role in channel function, or that the majority of native receptors lack GluRIIB.
To confirm our immunocytochemical results, and explore the functional changes resulting from loss of GluRIIB-containing receptors, we used electrophysiology. First, we compared single glutamate receptor channel properties in control and dlg mutant embryonic muscle 6 (Fig. 3A–B). Because Drosophila glutamate receptor conductance is relatively large and embryonic muscle input resistance is relatively high, single glutamate receptor channel currents are visible during the falling phase of some spontaneous synaptic events (Fig. 3B). DiAntonio et al. [31] showed that extrasynaptic larval muscle glutamate receptors in GluRIIB null mutants have slightly larger single channel currents (8.8 pA and 9.2 pA at -60 mV, for wild-type larvae and GluRIIB null mutants, respectively). We saw a similar, but larger increase in synaptic receptor single channel amplitudes in dlg mutant embryos (Fig. 3A; control = 9.3 ± 0.7 pA at -60 mV, N = 17; dlg = 14.1 ± 0.06 pA at -60 mV, N = 42; P < 0.001).
Figure 3 Postsynaptic glutamate receptor current properties change in DLG mutants A: Single glutamate receptor channel current amplitudes from synaptic glutamate receptors are significantly larger in dlg mutants, compared to controls. Single channel amplitudes were measured from channels displaying delayed closing during the falling phase of spontaneous synaptic currents; examples of sEJCs showing single channel currents are shown in B. C: Glutamate-gated currents, evoked using pressure ejection of 1 mM glutamate onto embryonic NMJs, are smaller in dlg mutants, compared to controls. Sample glutamate-gated currents are shown in D. E: The frequency of spontaneous excitatory junction currents (sEJCs) is reduced in dlg mutants, compared to controls. F: sEJC amplitudes are not significantly different in dlg mutants, compared to controls.
DiAntonio et al. [31] also examined single channel kinetics in the absence of GluRIIA or GluRIIB. Although loss of GluRIIA resulted in a dramatic decrease in average open channel times, loss of GluRIIB did not result in any significant change in open channel duration, compared to wildtype. If dlg mutants selectively lose GluRIIB, but not GluRIIA, then there should correspondingly be no change in single glutamate receptor channel kinetics in dlg mutants. Consistent with this, we observed no change in average duration of single channel currents visible during the falling phase of spontaneous synaptic currents (control channel open time = 14.3 ± 1.5 ms, N = 17; dlg mutant channel open time= 12.1 ± 0.7 ms, N = 42; P = 0.13).
All evidence strongly suggests that there are two subtypes of ionotropic glutamate receptors at the Drosophila NMJ: 1) receptors that are made up of the subunits GluRIIA+IIC+IID+IIE, and 2) receptors that consist of GluRIIB+IIC+IID+IIE. Our immunocytochemical results (Fig. 2) suggest that in dlg mutants, GluRIIB-containing receptors are selectively lost without any compensatory increase in GluRIIA-containing receptors. If this is true, the total number of glutamate receptors measurable electrophysiologically should decrease. To test this prediction, we measured the amplitude of glutamate-gated currents triggered by pressure ejection of 1 mM glutamate onto postsynaptic muscles. Figure 3C–D shows that, glutamate-gated currents were significantly smaller in dlg mutants, compared to controls (control = 1842 ± 255 pA at -60 mV, N = 10; dlg = 1044 ± 173 pA at -60 mV, N = 10; P = 0.018). Dividing by the single channel current amplitudes allows us to calculate the number of individual receptors opened. Control currents represent the opening of approximately 198 (1842/9.3) receptors. Currents in dlg mutants represent the opening of approximately 74 (1044/14.1) receptors. Since pressure ejection of glutamate onto embryonic muscles activates extrasynaptic as well as synaptic receptors, this decrease in electrophysiologically detectable glutamate receptors also demonstrates that the loss of immunocytochemically visible receptors shown in Fig. 2 is not due to dispersal of GluRIIB-containing receptors away from postsynaptic sites.
Recent studies suggest that Drosophila NMJ glutamate receptors are specifically localized opposite active zones, and that GluRIIA-containing receptors and GluRIIB-containing receptors are segregated from each other [26,30,35]. In other words, it is thought that individual postsynaptic densities (PSDs) contain either GluRIIA or GluRIIB, but not both. If this is true, then loss of one receptor subtype should cause some active zones to be without apposing receptor fields, while other active zones have relatively normal receptor fields. Our electrophysiological results (Fig. 3C–D) show that 63% (±12%) of all receptors are missing in dlg mutants. If GluRIIA and GluRIIB are segregated into different PSDs, and GluRIIB-containing receptors are selectively lost in dlg mutants, then 63% (±12%) of the individual synapses (active zone-PSD pairs) should be silent in dlg mutants. This should show up as a decrease in spontaneous excitatory synaptic current (sEJC) frequency, without a corresponding decrease in sEJC amplitude. sEJC frequency drops to 54% (±14%) of normal in dlg mutants (Figure 3E; control = 9.3 ± 1.6 Hz, N = 13; dlg = 5.0 ± 1.0 pA, N = 13; P = 0.03). sEJC amplitude in dlg mutants, however, is not significantly different compared to controls (Fig. 3F; control = 79 ± 7 pA, N = 13; dlg = 69 ± 9 pA, N = 13; P = 0.41). These results are consistent with selective loss of GluRIIB-containing receptors in NMJs where individual postsynaptic densities are composed exclusively of receptors containing GluRIIA or GluRIIB.
DLG mutants have fewer postsynaptic glutamate receptor clusters per presynaptic active zone
If postsynaptic glutamate receptor clusters are composed of receptors containing either GluRIIA or GluRIIB, and GluRIIB-containing receptors are selectively lost in DLG mutants, then there should be fewer postsynaptic glutamate receptor clusters per presynaptic active zone in dlg mutants. We tested this by triple staining the first instar NMJs with anti-HRP antibodies to visualize the presynaptic nerve, NC82, an antibody that marks presynaptic active zones [38], and anti-GluRIID antibodies [26], which label all postsynaptic glutamate receptors. The results are shown in Figure 4. NC82 and GluRIID immunoreactivity appear as distinct puncta where motor neurons contact postsynaptic muscle (Fig. 4A). In control larvae, each NC82 punctum is associated with a GluRIID punctum. Not every GluR cluster is associated with NC82 or HRP immunoreactivity, however, consistent with the previously-described presence of extrasynaptic glutamate receptors [39-41]. Thus, the ratio of postsynaptic glutamate receptor clusters to presynaptic active zones is greater than one (Fig. 4B). Specifically, control larvae show an average glutamate receptor cluster to active zone ratio of 1.33. In dlg mutants, however, this ratio is reduced to approximately one-half normal (Fig. 4B; control = 1.326 ± 0.15 GluR clusters/active zone, N = 312 GluR clusters from 5 animals; dlg = 0.64 ± 0.11 GluR clusters/active zone, N = 231 GluR clusters from 5 animals; P = 0.006). These results are consistent with a model in which: 1) GluRIIB-containing receptors are clustered independently of GluRIIA-containing receptors, 2) GluRIIB-containing receptor clusters are selectively lost in dlg mutants, and 3) selective loss of GluRIIB-containing receptors causes some presynaptic active zones to no longer be associated with postsynaptic glutamate receptor clusters.
Figure 4 DLG mutants have fewer postsynaptic glutamate receptor clusters per presynaptic active zone. A: Confocal projections of first instar NMJs visualized using three different antibodies: 1) the neuronal membrane marker anti-HRP (blue), 2) anti-GluRIID subunit antibodies which label all postsynaptic glutamate receptors (red), and 3) NC82 antibodies that label presynaptic active zones (green). Each panel shows NMJs on interior-most ventral longitudinal muscles in one hemisegment. Scale bar = 10 um. B: Number of postsynaptic glutamate receptor clusters relative to number of presynaptic active zones, showing that dlg mutants have fewer postsynaptic receptor clusters. C: High magnification images from dlg mutant NMJs showing glutamate receptor dispersion (arrows).
Interestingly, some receptor clusters opposite active zones in dlg mutants were visibly less distinct (Fig. 4C, arrows), suggesting that GluRIIB-containing receptors are not only lost, but some receptors are slightly mislocalized.
Postsynaptic localization of GluRIIA, GluRIIB, and DLG requires contact by the presynaptic neuron
Broadie & Bate [42] showed electrophysiologically that innervation triggers clustering and expression of functional glutamate receptors at the site of neuron-muscle contact. However, it has never been determined whether neuronal contact triggers localization of receptor protein, or local conversion of non-functional receptors to functional receptors. To answer this question, we repeated the critical experiments of Broadie & Bate but detected glutamate receptors immunocytochemically instead of electrophysiologically (Figures 5A–B).
Figure 5 Postsynaptic localization of GluRIIA, GluRIIB, and DLG requires contact by the presynaptic neuron, but localization of DLG does not depend on the presence of glutamate receptors A: Confocal projections of late stage 17 embryonic NMJs visualized using antibodies to the neuronal membrane marker anti-HRP (green) and anti-GluRIIA subunit antibodies (magenta). This image shows NMJs on interior-most ventral longitudinal muscles in two neighbouring hemisegments in a prospero null mutant. The muscles in the upper hemisegment are normally innervated; the muscles in the lower hemisegment are not innervated. Major anatomical landmarks are labelled: In the upper hemisegment, intersegmental nerve branch b (ISNb) enters from the right (medial) and branches to form NMJs on muscles 7 & 6, 13, and 12. ISNb is absent in the uninnervated hemisegment. Note the lack of GluRIIA clusters in this hemisegment. B: Uninnervated muscles 6 & 7 in a prospero mutant embryo, stained using antibodies against GluRIIB, DLG, and the neuronal membrane marker HRP. Note the lack of GluRIIB clusters, and dispersal of DLG throughout the muscle membrane. C: Innervated muscles 6 & 7 in a GluR-less Df(2L)SP22 mutant embryo, stained using antibodies against the synaptic vesicle protein cysteine string protein (CSP, green)) and DLG (magenta). Note that DLG clusters properly at the synapse. Scale bars (A, B, C) = 15 μm.
In prospero null mutants, motor axon outgrowth is delayed and impaired, such that embryonic body wall muscles are variably innervated [42]. Fig. 5A shows two neighboring hemisegments in a late stage (24 h AEL) prospero17 mutant embryo stained with anti-HRP to visualize motor axon terminals (green), and anti-GluRIIA antibodies (magenta). Ventral longitudinal muscles 12, 13, 6, and 7 (all unstained) are labelled in each hemisegment. The locations where the intersegmental nerve normally enter the ventral longitudinal muscle field in each hemisegment is marked with arrowheads. The muscles in the top hemisegment are innervated; ISNb (green) forms appropriate branches to each of the muscles, and GluRIIA clusters (magenta) are visible at the sites of innervation. As shown in Fig. 5A (top hemisegment, muscle 12), GluR clusters were typically visible even under growth cones, suggesting that receptors cluster at synaptic sites within minutes of nerve-muscle contact. In the neighboring uninnervated hemisegment (bottom of image), however, no GluRIIA clusters are visible. Thus, GluRIIA protein does not cluster in the absence of innervation. Similar results were obtained for GluRIIB (Fig. 5B). These results support the conclusion that postsynaptic glutamate receptors (containing either GluRIIA or GluRIIB) are clustered and upregulated only after contact by the presynaptic neuron. The signal between presynaptic neuron and postsynaptic muscle that triggers receptor clustering remains unknown.
Fig. 1 and previous studies [16-19,43] show that DLG (like GluRs) is largely restricted to the postsynaptic region. We used prospero mutants to determine whether DLG localization also depends on contact by the presynaptic neuron. Figure 5B shows muscles 6 and 7 in a non-innervated prospero17 mutant hemisegment triple-stained for GluRIIB (green), DLG (magenta), and HRP (blue). As previously noted, glutamate receptor clusters are not visible in uninnervated muscles. Without innervation, DLG is also not localized; DLG remains dispersed throughout the muscle membrane in a pattern reminiscent of that seen when DLG is missing PDZ domains 1 and 2 [13]. In innervated muscles, DLG clusters appropriately at sites of muscle-nerve contact (Fig. 5C).
Postsynaptic localization of DLG does not depend on the presence of glutamate receptors
Because glutamate receptor clustering requires neuron-muscle contact, and DLG clustering requires neuron-muscle contact, it is possible that DLG clustering requires glutamate receptors. We tested this by visualizing DLG in homozygous Df(2L)SP22 mutant embryos. Df(2L)SP22 mutants contain a deletion that removes the genes encoding GluRIIA and GluRIIB, resulting in complete loss of NMJ glutamate receptors [26,27,31]. Figure 5C shows an innervated hemisegment from a homozygous Df(2L)SP22 embryo, stained with antibodies to the presynaptic vesicle protein CSP (green) and DLG (magenta). The innervation-dependent clustering of DLG does not depend on glutamate receptors; DLG clusters opposite presynaptic boutons even in homozygous Df(2L)SP22 mutants (Fig. 5C). Note, however, that some extrasynaptic DLG remains. Extrasynaptic DLG remains prominent until approximately 48–60 h after hatching (mid second instar stage; data not shown).
Discussion
We have tested for the first time whether DLG is required for formation and/or stability of postsynaptic glutamate receptors in Drosophila. Our results show that DLG is indeed required, but only for a subset of receptors, those that contain the subunit GluRIIB. The molecules required for similar assembly and/or stabilization of GluRIIA-containing receptors remain unidentified. The molecular mechanism by which DLG regulates GluRIIB stability remains unclear. There is currently no evidence for a direct interaction between DLG and any Drosophila glutamate receptor subunit. Genome-wide yeast two-hybrid assays failed to identify any interactions between DLG and any Drosophila glutamate receptor subunit [44]. Similar results were obtained in two other independent and otherwise successful yeast two-hybrid screens: One, using the C-termini of GluRIIA, GluRIIB, and GluRIIC as baits failed to identify any interaction with DLG (S Sigrist, personal communication). Another screen independently used the SAP97-like N-terminus, the PDZ1-2 domains and the GUK domain of DLG as baits, but failed to identify any glutamate receptor subunits (U Thomas, personal communication).
Despite the fact that DLG clearly regulates the number of GluRIIB-containing receptors in the Drosophila NMJ, we do not believe that DLG specifies the location of GluRIIB-containing glutamate receptors. First, glutamate receptors are clearly not localized based on DLG alone, because DLG is present extrasynaptically in uninnervated muscle (cf. Fig. 5), and abundant throughout the postsynaptic membrane (c.f. Fig. 1), but glutamate receptors are tightly localized to discrete puncta that are mostly (but not exclusively) found opposite presynaptic active zones [26,35]. Thus, DLG is not sufficient for glutamate receptor clustering or stability. Second, as described above, there is no evidence for a direct interaction between DLG and glutamate receptors. Our data are most compatible with a model wherein DLG participates in the stability of receptors (possibly by regulating the assembly of a 'stability-promoting complex'), but does not 'scaffold' receptors. This conclusion derives from the observations that immunoreactive GluRIIB-containing receptor clusters are largely absent in dlg mutants, and glutamate-gated currents are smaller in dlg mutants. If receptors were dispersed, clusters would disappear but glutamate-gated currents should remain normal. However, some declustering of receptors was observed in dlg mutants (Fig. 4C), and it is possible that unclustered receptors are endocytosed and/or rendered nonfunctional.
If DLG does not determine where receptors go, then something else must. We do not know the identity of this protein. We show that localization of postsynaptic DLG, like localization of postsynaptic glutamate receptors, depends on contact by the presynaptic neuron. We do not know the mechanism by which presynaptic contact triggers localization of either glutamate receptors or DLG. However, the identification of DLG as a target for this process should help identify the molecules involved in this critical initial trans-synaptic signal.
Our results are the first evidence that glutamate receptors in Drosophila can be differentially regulated based on subunit composition. Mammalian ionotropic glutamate receptors also undergo subunit-dependent assembly and trafficking, suggesting that receptor subunit-dependent interactions are a conserved method for 'tuning' postsynaptic properties. In the Drosophila NMJ, the most critical role for DLG may therefore be as part of the machinery for regulating subunit composition. One possible mechanism for this process could be as follows. In the Drosophila NMJ, active CamKII phosphorylates DLG [19]. Constitutively active CamKII increases extrasynaptic DLG and phenocopies dlg mutants [19]. Our results therefore predict that synaptic activity, via activation of CamKII, would decrease the number of GluRIIB-containing receptors and silence some synapses (active zone-PSD pairs). Sigrist et al. [45] showed that NMJ activity leads to enhanced translation and insertion of GluRIIA-containing receptors (they did not assay GluRIIB). Thus, the overall result of increased NMJ activity is probably replacement of GluRIIB-containing receptors with GluRIIA-containing receptors – a 'switch' in postsynaptic receptor subunit composition.
DiAntonio et al. [31] studied the effects of selectively expressing GluRIIA or GluRIIB transgenes in Df(2L)SP22 mutant Drosophila, where endogenous GluRIIA and GluRIIB were eliminated. The most dramatic changes in receptor properties resulted from overexpression of GluRIIA in the absence of GluRIIB: mEJP amplitudes increased several-fold, receptor channel open times increased, and sensitivity to the antagonist argiotoxin decreased. Thus, their results show that switching from 'B-type' receptors (e.g. those containing GluRIIB) to 'A-type' receptors (e.g. those containing GluRIIA) at the NMJ leads to changes in postsynaptic properties. Overexpression of GluRIIA increases presynaptic growth in larval NMJs [46], suggesting that postsynaptic subunit switching might also play a role in presynaptic development.
Conclusions
Our results demonstrate that mutation of DLG causes loss of glutamate receptors containing GluRIIB, but not GluRIIA. We also show that, like glutamate receptors, DLG localization requires contact between pre and postsynaptic cells. DLG localization does not depend on the presence of glutamate receptors, since DLG is localized normally in the complete absence of postsynaptic glutamate receptors. Since glutamate receptor localization does not entirely depend on DLG, and DLG localization does not depend on glutamate receptors, we hypothesize that presynaptic nerve contact triggers localization of receptors and DLG in parallel, after which DLG promotes the stability of GluRIIB-containing receptors.
Methods
Genetics
'Control' genotypes were either Oregon R (OR) or non-homozygous mutant siblings of the appropriate genotype. No statistically significant difference in any measurement was observed between OR and any other control genotype used in this study. Homozygous mutant embryos were identified through the use of an appropriate balancer chromosome expressing GFP. prospero[17] mutants are nulls that were a gift from Dr Chris Doe, University of Oregon. Df(2L)SP22 mutants remove both GluRIIA and GluRIIB, as previously described [31]. dlg[X1–2] mutants [20] were gifts from Dr Vivian Budnik (University of Massachusetts Medical School).
Immunocytochemistry
Embryos and larvae were dissected and stained for immunocytochemistry and electrophysiology as previously described [43]. When antibodies against any of the glutamate receptor subunits were used, preparations were fixed 30 min in Bouin's fixative. Otherwise, fixations were 30 min in 4% paraformaldehyde. Antibodies against GluRIIA (8B4D2, used at 1:100) [25] were produced from hybridoma cells and obtained from the University of Iowa Developmental Studies Hybridoma Bank (DSHB). Mouse NC82 antibodies were a gift from Erich Buchner and used at 1:100. Rabbit polyclonal GluRIIB antibodies [30] were used at 1:1000. Rabbit polyclonal anti-DLG antibodies [16] were used at 1:1000. Rabbit polyclonal GluRIID antibodies [26] were a gift from Stephan Sigrist and used at 1:500. All primary antibodies were visualized using fluorescently-conjugated (fluorescein, rhodamine, or CY-5) secondary antibodies (Jackson Immuno Labs, West Grove, PA) generated against the appropriate species (mouse or rabbit) and viewed using an Olympus FV-500 laser-scanning confocal microscope. Presynaptic terminals were visualized using fluorescently conjugated anti-HRP antibodies (Jackson Immuno Labs) directly conjugated to FITC, TRITC, or CY-5.
Receptor cluster sizes (Fig. 2C) were measured using an automated edge-finding/threshold-based macro run within NIH ImageJ software (v. 10.2 for OS X). Results using the automated procedure avoid experimenter bias and agree quantitatively with careful manual measurements [25]. The 3D reconstruction of the portion of a larval NMJ shown in Fig. 1B was generated using Amira 3.1 (Mercury Computer Systems, Chelmsford, MA).
The number of glutamate receptor clusters per active zone (Fig. 4) was quantified as follows: first instar larvae of the appropriate genotype were dissected and triple-stained with antibodies against NC82, which marks presynaptic active zones, GluRIID, which marks all postsynaptic glutamate receptor clusters, and HRP to visualize the NMJ. These preparations were subsequently imaged using confocal microscopy. Z-projections from each image (which contained several NMJs and dozens of clusters) were split into the separate color channels using ImageJ, and the number of clusters in each channel (NC82 or GluRIID) was counted using ImageJ's particle analysis function. The number of GluRIID clusters in each image was then divided by the number of NC82 clusters in each image to calculate ratios that were then compared using a Student's T-test (Fig. 4B).
Electrophysiology
All electrophysiology (Fig. 3) was performed on ventral longitudinal muscle 6. whole-cell patch clamp measurements from embryonic muscles were performed as previously described [41,43]. Briefly, temporally and morphologically staged embryos were dechorionated in bleach, manually devitellinated and dissected, then treated with 1 mg/ml collagenase type IV (Sigma-Aldrich) for 60–90 s. Muscle 6 was whole-cell voltage clamped (-60 mV) in standard Drosophila embryonic saline using standard patch-clamp techniques. Data were acquired and subsequently analyzed using an Axopatch 1D amplifier and PClamp 9 (Axon Instruments, Union City CA).
Statistics
Statistical significance in figures is represented as follows: *** = p < 0.001; ** = p < 0.01; * = p < 0.05. Unless otherwise specified (e.g. Fig. 2C, 3F), all statistical comparisons were made using unpaired T-tests, or (in the case of distributions) Kolmogorov-Smirnov tests. All error bars represent S.E.M.
Authors' contributions
All immunocytochemistry and microscopy was performed and analyzed by DEF. All electrophysiology was performed and analyzed by KC. The manuscript was written by DEF with input from KC; both authors reviewed and approved the final manuscript.
Acknowledgements
This work was supported by a grant from the National Institutes of Health. We thank Stephan Sigrist, Ulrich Thomas, and Aaron DiAntonio for useful discussions, Erich Buchner for NC82 antibodies, and Aaron DiAntonio for extreme generosity with his excellent anti-GluRIIB antibodies.
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| 15638945 | PMC545058 | CC BY | 2021-01-04 16:39:16 | no | BMC Biol. 2005 Jan 8; 3:1 | utf-8 | BMC Biol | 2,005 | 10.1186/1741-7007-3-1 | oa_comm |
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BMC MedBMC Medicine1741-7015BioMed Central London 1741-7015-3-11562740210.1186/1741-7015-3-1Research ArticleRecurrent cardiac events in patients with idiopathic ventricular fibrillation, excluding patients with the Brugada syndrome Champagne Jean [email protected] Peter [email protected] François [email protected] Pedro [email protected] Cardiovascular Center, OLV Ziekenhuis, Moorselbaan 164, 9300 Aalst, Belgium2 Quebec Heart Institute, Laval Hospital, 2725, Chemin Ste-Foy, Quebec City, Quebec, Canada2005 1 1 2005 3 1 1 18 8 2004 1 1 2005 Copyright © 2005 Champagne et al; licensee BioMed Central Ltd.2005Champagne 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 recurrence of cardiac events in patients with idiopathic ventricular fibrillation (VF) excluding patients with the Brugada syndrome is unclear since this entity remains present in previous studies.
Methods
Since 1992, 18 patients (72% male) with idiopathic VF out of 455 ICD implants were treated with an implantable cardioverter defibrillator (ICD). The mean age at first ICD implantation was 42 ± 14 years. Brugada syndrome, as well as other primary electrical diseases (e.g. long QT), were systematically excluded in all patients by the absence of the typical electrocardiogram (ST elevation in the right precordial leads) at rest and/or after pharmacological tests (ajmaline, flecainide, or procainamide). Recurrence of cardiac events was prospectively assessed.
Results
During a mean follow-up period of 41 ± 27 months, VF recurrence with appropriate shock occurred in 7 patients (39%) covering a total of 27 shocks. The median time to first appropriate shock was 12 ± 9 months. There were no deaths. In the electrophysiological study, 39% of patients were inducible, but inducibility failed to predict subsequent arrhythmic events. Forty-four percent of patients suffered 21 inappropriate shocks, which were caused by sinus tachycardia, atrial arrhythmias or lead malfunction.
Conclusion
Idiopathic ventricular fibrillation patients have a high recurrence rate of potentially fatal ventricular arrhythmias, excluding patients with the Brugada syndrome or other known causes. ICD prevents sudden cardiac death but inappropriate shocks remained a major issue in this young and active population.
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Background
Idiopathic ventricular fibrillation (VF) is defined as spontaneous ventricular fibrillation in the absence of any structural heart disease, including coronary artery disease, valvular heart disease, myocarditis, cardiomyopathy or electrophysiological diseases, with a well-defined cause, such as the long QT syndrome, the Brugada syndrome, ventricular pre-excitation (WPW), and drug intoxication [1]. The consensus statement of the joint steering committees of the UCARE registry of Europe and of the idiopathic VF registry of US reported in 1997 that patients with the Brugada syndrome should be considered a variant of idiopathic VF [1].
Brugada syndrome is characterized by a unique electrocardiographic (ECG) pattern of right bundle branch block with ST elevation in the right precordial leads. Transient forms of the disease in which the ECG normalizes for a period of time have been described. Administration of sodium channel blockers will unmask the abnormal ECG in patients with transient normalized ECGs [2,3]. The American Heart Association has recently proposed diagnostic criteria for the Brugada syndrome [4]. Moreover, the incidence of Brugada syndrome among patients with idiopathic VF remains unclear. The prevalence of a Brugada type ECG pattern was reported in 21% [5] and 24% [6] of idiopathic VF populations. However, on the basis of pharmacological tests, it has been speculated that up to 40% – 60% of patients diagnosed with idiopathic VF might actually suffer from the Brugada syndrome [7]. Since this is a new clinical entity, older publications probably classified some patients with Brugada syndrome as idiopathic VF, since intravenous administration of a sodium channel blocker to unmask a concealed form of the disease was not systematically performed. Our report describes the clinical and electrophysiological characteristics of consecutive sudden cardiac arrest survivors from idiopathic VF who received an ICD, and in whom electrical and structural heart diseases including Brugada syndrome, long QT, arrhythmogenic right ventricular dysplasia, and hypertrophic cardiomyopathy characterized by a high recurrence rate, were systematically excluded.
Methods
Patient population
The total population under investigation included 455 consecutive patients who received a third generation ICD, with stored electrograms capabilities for hemodynamically poorly tolerated ventricular tachyarrhythmias, from 1992 to 2000. Of these, 29 were initially diagnosed as idiopathic VF associated with a structurally normal heart, normal left and right ventricular ejection fraction and normal coronary arteries. During the follow-up, 11 patients developed a new diagnosis potentially explaining VF, and were excluded from the final analysis. Four had right ventricular dysplasia, 5 had Brugada syndrome with the typical ECG manifestations after a drug challenge, and 2 patients had long QT syndrome. A final diagnosis of idiopathic VF was made in the remaining 18 patients. All patients had survived either an episode of cardiac arrest due to VF (12 patients) or a syncopal episode associated with documented self-terminating polymorphic ventricular tachycardia or fibrillation (6 patients). In 15 patients, the documented VF was seen during daily activity (one episode during sustained effort) and in 3 patients, during night-time. Out of the 18 patients, 10 had a previous history of unexplained syncope.
Definitions and investigation
Idiopathic VF was defined as VF in the absence of demonstrable cardiac abnormalities as previously reported [1]. Thus, we excluded other known causes of VT/VF such as WPW syndrome, congenital long QT syndrome, short-coupled torsade de pointes, catecholamine-induced polymorphic VT, and Brugada syndrome. No patient had a past history of ischemic heart disease, congestive heart failure or family history of unexpected sudden cardiac death. All patients had a normal physical examination, blood testing and exercise test. Structural heart disease was excluded in all by echocardiography, cardiac catheterization including right and left ventricular angiography and coronary angiography. Patients on anti-arhythmic therapy before their cardiac arrest were excluded from the study, as well as those with electrolyte disturbances, history of significant alcohol or drug abuse or prolonged QTc. Of the 18 patients, 10 patients had normal right ventricular endomyocardial biopsy and 8 patients had a normal cardiac magnetic resonance imaging. Moreover, in 5 patients, an ergonovine provocation test was performed to exclude coronary artery spasm. Brugada syndrome was systematically excluded in all patients by negative serial ECG recordings with the typical coved or saddle shaped-type ST-segment elevation in the right precordial leads. All patients received iv administration of a sodium channel blocker to unmask a concealed form of the disease and all tests were negative. Flecainide (2 mg/kg body weight) was used in 9 patients, procainamide (10 mg/kg body weight) in 4 patients, and ajmaline (0.7 mg/kg body weight) in 7 patients.
Electrophysiological study
Programmed electrical stimulation was performed in all patients using up to 3 extra-stimuli at 3 different drive cycle lengths (600, 500, and 430 ms) delivered to the right ventricular apex. The coupling interval of the first 2 extra-stimuli was not shorter than 180 ms and not shorter than 200 ms for the third extra-stimuli. In case of non-inducibility, programmed stimulation was also performed from the right ventricular outflow tract. Sustained VF was defined as VF at a cycle length ≤ 200 ms and lasting ≥ 30 seconds or requiring immediate defibrillation. Sustained ventricular tachycardia (VT) was defined as VT lasting ≥ 30 seconds or requiring termination secondary to hemodynamic instability. Non-sustained VT was defined as >6 consecutive ventricular complexes or VT lasting <30 seconds.
ICD implantation
All patients received a transvenous ICD with stored electrograms. Recorded episodes were reviewed and adjudicated as appropriate or inappropriate therapies. VF was defined at follow-up as consecutive ventricular beats recorded from the device at a cycle length of 240 msec or less. In all patients, the ICD was programmed with only 1 ventricular fibrillation detection zone at 180 bpm (333 msec). Only shock therapy was programmed. All patients were seen at follow-up in our centre.
Statistical analysis
All data are expressed as mean ± standard deviation. Kaplan-Meier analysis was used to analyze time intervals until the first appropriate shock.
Results
The study cohort consisted of 13 men and 5 women (Table 1), with a mean age at the first ICD implantation of 42 ± 14 years (range 20 to 70 years). Baseline electrocardiograms were normal in 16 patients. One patient had a left bundle branch block and 1 had a right bundle branch block. The mean QTc for the entire population was 410 ± 20 msec. All patients had a normal left ventricular ejection fraction (≥ 50%) without regional wall motion abnormalities. Mean left ventricular ejection fraction (EF) was 69 ± 8% (range 52 to 81%). No patient had significant coronary artery disease (defined as ≥ 50% stenosis). Microscopic examination of right ventricular biopsy specimens in 10 patients showed no evidence of a viral, infiltrative or dysplasic process in the myocardium. Baseline electrophysiological studies were performed in all patients and showed normal sinus node, atrioventricular node and His-Purkinje function in all. Inducible sustained ventricular arrhythmias occurred in 7 patients (39%). From these, 4 patients had sustained VF and 3 patients had sustained inducible monomorphic VT or ventricular flutter (cycle length 230 ± 20 ms). Two extra-stimuli were required in 5 patients and 3 extra-stimuli in 2 patients. Non-sustained VT-VF was induced in 3 patients. No arrhythmia could be induced in 8 patients.
Table 1 Clinical characteristics and outcome of 18 patients with idiopathic VF
Baseline Characteristics ICD Therapy
Patient Age (years) sex CE Drug test to exclude Brugada PES AS NSVT IS Ind S Time to 1st AS (months) Follow-up (months) AAD during follow-up
1 47,M Sy-VF F SMVT 6 7 - - 4 17 sotalol
2 51,F CA F NI 7 21 - - 1 19 sotalol
3 66,M CA AJ VF - - - - - 5 -
4 70,F CA P NI - - 6(af) - - 19 amiodarone
5 46,M CA F NI - 1 1(af) - - 23 sotalol
6 20,M Sy-VF A VF - - 3(st-lp) - - 92 b-blocker
7 46,F CA P VF - 120 - - - 27 sotalol
8 40,M CA A NSVT 1 - - - 13 75 -
9 33,M Sy-VF F NSVT - 1 - - - 19 -
10 26,M CA F NI 1 - 3(st-lp) - 7 9 sotalol
11 33,F CA F NI - - - - - 18 -
12 50,F CA F SMVT 6 - 1(st) - 13 86 flecainide
13 25,M CA AJ + P NSVT - 1 - - - 62 -
14 36,M CA AJ NI 2 - 2(af) 4 27 55 sotalol
15 58,M Sy-VF AJ NI - - 3(af) 2 - 57 sotalol
16 44,M CA F + P NI - - - - - 48 -
17 28,M Sy-VF F VF 4 - 2(st) - 18 55 sotalol
18 35,M Sy-VF A SMVT - - - - - 59 -
CE: clinical events; CA: cardiac arrest; Sy-VF: syncope with documented self-terminating polymorphic VT or VF
PES: programmed electrical stimulation; M: male; F: female
SM: sustained monomorphic; VT: ventricular tachycardia; VF: ventricular fibrillation
NS: non sustained; NI: non inducibility; af: atrial fibrillation; st: sinus tachycardia; lp: lead problem
AS: appropriate shock; IS: inappropriate shock; Ind S: indeterminate cause for shocks;
AAD: antiarrhythmic drug; F: flécaïnide; AJ: ajmaline; P: procainamide
Long-term outcome
After a mean follow-up of 41 ± 27 months (ranging from 5 to 92 months), 7 of the 18 patients (39%) with idiopathic VF had VF or sustained polymorphic VT recurrence and received appropriate shocks (Table 1). These patients experienced a total of 27 shocks; 5 of them had more than 1 episode (2 to 7). Mean time to first appropriate shock was 12 ± 9 months (ranging from 0.4 to 27 months). No arrhythmic storm was observed at follow-up (defined as ≥ 3 separate VT/VF episodes within 24 h). With the use of stored electrograms, 6 patients had documented non-sustained polymorphic VT or VF for a total of 149 episodes (range 1 to 120). Four of them have not received any appropriate shock. These episodes were detected by the ICD but shock delivery was appropriately aborted by the non-committed function of the device. There was no relationship between the initial clinical and arrhythmic presentation and subsequent arrhythmic events recorded from the ICD at follow-up (Table 1). From the intracardiac electrograms, the arrhythmia initiation was always associated with PVCs with a mean coupling interval of 300 ± 35 ms. A long-short initiating sequence of VF was never observed. In 5 patients, multiple VF episodes were recorded by the ICD, all of which for a single patient were associated with the same PVC coupling interval. Since the stored electrograms in the present study were obtained from endocardial sites and were single-channel recordings, we could not assess the origin of the PVCs. The mean QTc at the time of VF recurrence was normal in all patients (mean 418 ± 22 msec). The ICD effectively recognized and promptly treated all the polymorphic VT or VF recurrences and prevented the possible occurrence of sudden cardiac death. No death was reported during follow-up. Non-invasive follow-up examinations failed to detect any new structural heart disease or primary electrical disease such as long QT or Brugada syndrome.
Value of electrophysiological Testing
Programmed electrical stimulation failed to predict subsequent cardiac events. The sensitivity and specificity were 43 and 64%, respectively. The positive and negative predictive values were also 43 and 64%, respectively, that is not considered clinically useful.
Causes and incidence of inappropriate shocks
In this population, we observed a high incidence of inappropriate shocks. Eight of the 18 patients (44%) received an inappropriate shock for a total of 21 discharges. Atrial fibrillation was responsible for 57% of them (12 episodes in 4 patients). These were older patients with a mean age of 53 ± 14 years. Six inappropriate shocks (4 patients) were triggered by sinus tachycardia. These patients were younger with a mean age of 31 ± 13 years. Lead malfunction caused 3 inappropriate shocks in 2 patients. Six shocks in 2 patients were classified as of unknown cause, even after careful examination of the intracardiac electrograms. These shocks were probably inappropriate since no clinical symptoms occurred during these episodes and these 2 patients already experienced inappropriate shocks for atrial fibrillation.
Device follow-up
During the follow-up period, first-time generator replacement was performed in 9 patients after a mean of 43 ± 11 months after initial implantation (range 21 to 58). End of life battery was the indication for replacement in 8 patients, and 1 had an ICD component failure. A second replacement was done in 2 patients. Lead replacement was also indicated in 2 patients for lead insulation fracture. Adjunctive anti-arrhythmic drugs were required in 11 patients after ICD implantation in order to control for the occurrence of appropriate or inappropriate shocks (Table 1).
Discussion
To our knowledge, this report is the first study to describe the clinical outcome of ICD patients with a diagnosis of idiopathic VF in whom the Brugada syndrome, characterized by a high recurrence rate, was systematically excluded. Since there is a wide variability in the ECG expression in individual patients with the Brugada syndrome, we performed iv administration of sodium channel blockers to unmask the ECG features of the syndrome in all our idiopathic VF patients. Two studies reported the long-term outcome of patients with idiopathic VF without the Brugada syndrome [5,6]. Viskin et al. [5] performed iv administration of a sodium channel blocker to unmask a concealed form of Brugada syndrome in only 15 % of their idiopathic VF population, whereas Remme et al. [6] tested the effect of an iv sodium channel blocker on ECG morphology in only 30% of the patients. The unique finding of the present report is the high recurrence rate of sustained ventricular arrhythmias in this well-characterized idiopathic VF group, even after a careful systematic evaluation to exclude the presence of Brugada syndrome. After a mean follow-up of 41 ± 27 months, 39% of the patients received an appropriate ICD discharge for VF or polymorphic VT. In accordance with references [8,9], ICD therapy offered good protection against fatal outcome due to recurrent ventricular arrhythmias, with no mortality during the follow-up. The high recurrence rate is consistent with the relatively high recurrence rate of arrhythmic events or sudden death from the UCARE registry [10], as well as the high frequency of electrical discharges from ICDs reported in a large series of patients with idiopathic VF [6,8,9]. In comparison, appropriate ICD discharges have been reported in 48% of arrhythmogenic right ventricular dysplasia population [11], 40 to 56% of inducible population receiving an ICD [12,13] and 23% (7%/year) in the hypertrophic cardiomyopathy population [14]. However, some authors have reported a more benign clinical course of their idiopathic VF population with a lower recurrence rate [15-17]. Our high ICD discharge rate might also be explained by the rapid ICD intervention for fast ventricular arrhythmia that could have been self-terminated. The definition of idiopathic VF remains problematic, since numerous studies including idiopathic VF population are heterogeneous. Indeed, 61% (11/18 patients) of our cohort remained free of sustained arrhythmia recurrence during follow-up despite the same initial VF or syncopal presentation. Of note, idiopathic VF is always a diagnosis of exclusion. The patients in our cohort, as well as in other reported series, had normal hearts, as defined by clinical, non-invasive and invasive testing. Even after a careful investigation, a transient phenomenon such as a reversible localized myocardial disease, silent myocardial ischemia due to coronary artery spasm or sudden manifestation of an unknown primary electrophysiological disease could easily be missed by the clinician, and be responsible for the VF episode. Idiopathic VF is in fact an amalgam of different diseases in which the first clinical manifestation is VF. The ultimate answer probably lies in a more comprehensive approach and a more precise understanding of the molecular genetics and associated electrophysiological abnormalities in finding a specific treatment avoiding ICD implantation with its associated complications.
Anti-arrhythmic drugs have also been used in this clinical condition. Belhassen et al. [18] reported excellent results in their idiopathic VF population with EP-guided therapy using Class 1A drugs, primarily quinidine; no death occurred during a mean follow-up of >9 years. Recent evidence also suggests that frequent premature ventricular contractions arising from the Purkinje system are responsible for initiation of ventricular fibrillation, and can be mapped and ablated in selected patients [19]. Until another proven therapy has been assessed prospectively, the possibility of VF recurrence mandates ICD implantation as currently proposed in ICD guidelines [20].
Electrophysiological findings
The inducibility rate (39%) of sustained VT/VF observed in our study is also in accordance with the results of the UCARE registry [10]. Of importance, programmed electrical stimulation was of limited value with a poor sensitivity and specificity. Moreover, inducibility failed to predict subsequent arrhythmic events. Our findings differ from the observed 79% average inducibility rate reported by Belhassen et al. [17,18]. This may be due to different patient characteristics and stimulation protocol. Our study excluded patients with Brugada syndrome known to have a high inducibility rate [21]. Compared to our stimulation protocol, Belhassen et al. [22] used up to 3 extra-stimuli, 2 basic cycle lengths, 2 RV sites (first RVA, then RVOT), and repetition of extra-stimulation (n = 10 for double, and n = 5 for triple) at the shortest coupling intervals that resulted in ventricular capture. Three patients had a fast monomorphic VT inducible even if the clinical presentation was aborted sudden cardiac death. This may suggest an underlying structural heart disease undetected by the current investigation. Although general cardiac function can be normal, patients might have discrete abnormalities, which are currently unidentifiable. Better risk stratification is required to identify patients who will experience recurrent VF over time. Therefore, defibrillator implantation could be seen as the primary therapy in patients with idiopathic VF, since no stratification has yet identified patients at risk of arrhythmia recurrence.
ICD limitations
The impact of multiple battery replacements over time in this young population with idiopathic VF needs to be emphasized. It is noteworthy that half the population (9/18 patients) had an ICD replacement and 2 other patients had their transvenous lead replaced during follow-up. We also observed a high incidence of inappropriate shocks (44%), mainly caused by atrial fibrillation and sinus tachycardia. This is well in accordance with other studies where up to 40% inappropriate discharge rates were observed even with fourth generation ICDs [23]. The addition of an anti-arrhythmic drug to decrease the incidence of inappropriate shocks was required in this group. Four patients experienced atrial fibrillation, which might indicate the presence of an associated primary electrophysiological disease also affecting the atrium. In patients with idiopathic VF, the characteristics, is the recurrence of VF and not monomorphic VT compared to other ventricular arrhythmias. To decrease the number of inappropriate shocks, one should program a higher VF zone around 200–220 bpm and a lower monitoring zone to detect atrial arrhythmia. Third-generation ICD still have limitations and complications over time, with a significant proportion of patients having hardware-related complications or inappropriate shocks [24]. Future developments in ICD technology is needed, which will hopefully address these issues.
Conclusions
Idiopathic VF patients have a high recurrence rate of ventricular arrhythmias, even after the systematic exclusion of patients with the Brugada syndrome or other known electrical diseases from the analysis. ICD prevents sudden cardiac death in this population, but treat only the final manifestation of an unknown disease. Inappropriate shocks remain a major concern in this young population.
Competing interests
The author(s) declare that they have no competing interests.
Author's contributions
JC participated in the hypothesis generation and drafted the manuscript. PG and PB were project managers. FP participated in the final writing of the manuscript. All authors have read and approved the final version of the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
Dr Champagne was supported by a grant from the Quebec Heart Institute, Quebec City, Canada.
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| 15627402 | PMC545059 | CC BY | 2021-01-04 16:27:56 | no | BMC Med. 2005 Jan 1; 3:1 | utf-8 | BMC Med | 2,005 | 10.1186/1741-7015-3-1 | oa_comm |
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BMC MedBMC Medicine1741-7015BioMed Central London 1741-7015-3-21562906110.1186/1741-7015-3-2Research ArticleAn international comparative study of blood pressure in populations of European vs. African descent Cooper Richard S [email protected] Katharina [email protected] Amy [email protected] Adebowale [email protected] José R [email protected] Terrence [email protected] Simona [email protected] Michel [email protected] Mika [email protected] Paola [email protected] Birgitta [email protected] Michael [email protected] Department of Preventive Medicine and Epidemiology, Loyola University Stritch School of Medicine, Maywood, IL, USA2 Department of Pediatrics, University College Hospital, Ibadan, Nigeria3 Departamento de Medicina Preventiva y Salud Pública, Facultad de Medicina. Universidad Autónoma de Madrid, Spain4 Tropical Medicine Research Institute, University of the West Indies, Kingston, Jamaica5 Istituto Superiore di Sanità, Laboratorio di Epidemiologia e Biostatistica, Rome, Italy6 Department of Community Health and Epidemiology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada7 Department of Public Health and General Practice, University of Kuopio, Finland8 Department of Epidemiology and Public Health, University College London Medical School, London, UK9 Department of Medicine, University Hospital, Umeå, Sweden10 Robert-Koch Institut, Berlin, Germany2005 5 1 2005 3 2 2 9 8 2004 5 1 2005 Copyright © 2005 Cooper et al; licensee BioMed Central Ltd.2005Cooper 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 consistent finding of higher prevalence of hypertension in US blacks compared to whites has led to speculation that African-origin populations are particularly susceptible to this condition. Large surveys now provide new information on this issue.
Methods
Using a standardized analysis strategy we examined prevalence estimates for 8 white and 3 black populations (N = 85,000 participants).
Results
The range in hypertension prevalence was from 27 to 55% for whites and 14 to 44% for blacks.
Conclusions
These data demonstrate that not only is there a wide variation in hypertension prevalence among both racial groups, the rates among blacks are not unusually high when viewed internationally. These data suggest that the impact of environmental factors among both populations may have been under-appreciated.
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Background
Population surveys in the US from early in the last century have consistently documented higher blood pressures and related cardiovascular sequelae in blacks compared to whites [1,2]. The enormous attention focused on this observation has resulted in a dichotomous view of hypertension risk: whereby populations of African origin are considered more susceptible than all other continental groupings and a strong genetic hypothesis of inherent predisposition to hypertension among blacks has become the conventional wisdom [3-5]. Since this research has been limited primarily to the US, the generalizability of these conclusions is open to question. Data on the prevalence of hypertension in other genetically-related populations of African and European descent constitute important evidence but have so far not been considered in the debate.
International comparative studies on hypertension have been seriously limited by the absence of a valid method of standardization. In the last decade, however, high quality population surveys have been conducted in a wide range of populations that used either careful internal standardization or sufficiently comparable methods [6-15]. We report here on the patterns of hypertension prevalence in a sample of 3 such surveys among blacks from Africa, the Caribbean and the US and 8 surveys among whites from the US, Canada and Europe.
Methods
Study design
Black populations were drawn from the International Collaborative Study on Hypertension (ICSHIB) and the National Health and Nutrition Survey III [6,16]. A primary report of ICSHIB demonstrated a gradient in hypertension risk from east to west, parallel to the gradient in socioeconomic development and associated lifestyle [6]. An extensive process of cross-standardization was incorporated into ICSHIB to ensure that measurement technique did not bias the survey results [7]. We subsequently identified surveys on hypertension conducted since 1986 that were national in scope in North America and Europe. Two North American and six European surveys were included, viz: US [8] and Canada, [9], England [10], Finland [11], Germany [12], Italy [13], Spain [14] and Sweden [15]. The US data from NHANES-III are available for public use through the National Center for Health Statistics [8]. Investigators in Canada and Europe were contacted and invited to join this project. More detailed methods for this component of the study were reported earlier [16]. In brief, after achieving consensus on the main goals and resolving the methodological issues, data collection forms were distributed. Each collaborator provided average gender- and age-specific data by 5-year age groups for BPs, body mass index (BMI), and counts of hypertensives by treatment and control status. A description of the key aspects of each survey, including the BP measurement procedure, was collected in a standardized format.
The surveys that formed the basis of ICSHIB were conducted in localized communities by door-to-door screening [6,7]. In summary, individual communities were chosen on the basis of apparent representativeness and census data were obtained. Sampling was based on probability proportional to size and was structured to lead to a sample equally balanced by gender and age group across the 10-year age. The studies of the European-origin populations and African Americans were larger in scope [16]. Some were based on a random probability sample of the entire nation, while others were a series of regional samples; none were restricted mainly to a single province or sub-region within the country (Table 3). Collectively the studies enrolled 85,000 participants and the number of subjects in individual studies ranged from 1,800 to 23,000. Participation rates varied from 61% to 88%. Sampling was conducted mainly on population registries.
Table 3 Hypertension Prevalence (%) among Persons 35–64 Years, in African- and European-Origin Populations *
Total (%) Men (%) Women (%)
African-Origin Populations
Nigeria 13.5 13.9 13.1
Jamaica 28.6 23.4 31.8
US – Black 44.0 43.1 44.8
European-Origin Populations
US – White 26.8 29.7 23.9
Canada 27.4 31.0 23.8
Italy 41.5 48.0 35.1
Sweden 38.4 44.8 32.0
England 41.7 46.9 36.5
Spain 46.8 49.0 44.6
Finland 48.6 55.7 41.6
Germany 55.3 60.2 50.4
* Age-adjusted
Data collection methods
The examination methods have been reported in detail previously [6,7,16]. In brief, the mercury sphygmomanometer was used for BP measurements in every country except England, where the Dinamap 8100 oscillometric device was used. All studies had at least 2 measurements and the 2nd BP from the clinic visit was used to create the mean for the age-gender groups, except for England where the 2nd home BP was used. Hypertension was defined as BP ≥ 140/90 mmHg or current use of antihypertensive medication.
Data analysis
BP, body mass index (BMI), and hypertension prevalence were calculated for 5-year age-gender groups and aggregated as the primary data file. To achieve maximum overlap we restricted the analysis to 35–74 years for age-specific estimates of BP and hypertension prevalence, and 35–64 years for age-adjusted results. In the US NHANES whites and blacks were analyzed separately with the appropriate weighting for population size. As previously reported, the prevalence estimates obtained for US blacks from ICSHIB were virtually identical to those from NHANES [6]; to enhance generalizability, however, we used the NHANES data to represent the US black population. Hypertension prevalence and control was age-adjusted by age-averaging the 5-year age groups combining the data for men and women. For comparison of all white vs. all black populations the mean BP's and prevalences were averaged, considering each country as a single unit (i.e., without weighting by population size).
Results
Patterns of blood pressure
The age-averaged BPs and BMIs are presented for each survey, by gender, in Table 2. It must be recognized that where treatment is common these data may understate the true values, although this effect is likely to be small when the population is considered as a whole. Trends in BP with age showed considerable heterogeneity within population groups of both continental ancestry (i.e. African and European) (Figures 1, 2). In rural Nigeria, mean BPs were low and rose only modestly with age (Figure 1). Intermediate levels of BP were observed in Jamaica, while the US blacks had higher BPs at all ages. As previously reported, whites in the US and Canada had substantially lower BPs over the entire life span than did the Europeans (Figure 2). For greater clarity, the age-specific patterns are presented for all black and all white groups combined (Figure 3).
Table 2 Mean Systolic and Diastolic Blood Pressure and Body Mass Index among Persons 35–74 Years, in African- and European-Origin Populations*
Total Sys / Dias Men Sys / Dias Women Sys / Dias BMI, All
(mmHg) (mmHg) (mmHg) (kg/m2)
African-Origin Populations
Nigeria 121.5/72.4 122.2/73.0 121.0/71.9 22.9
Jamaica 122.9/71.7 122.5/72.0 123.2/71.5 27.0
US – Black 129.7/78.5 130.3/80.8 129.1/76.3 28.5
European-Origin Populations
US – White 120.9/75.2 123.4/78.2 118.3/72.2 27.3
Canada 128.2/80.8 131.2/83.2 125.1/78.5 26.8
Italy 129.8/83.1 132.4/85.4 127.2/80.7 26.4
Sweden 130.6/80.9 133.0/83.4 128.3/78.4 26.5
England 135.0/77.2 137.3/80.3 132.7/74.2 27.1
Spain 131.4/83.2 132.3/83.9 130.5/82.5 27.4
Finland 134.3/83.8 136.9/86.0 131.6/81.5 27.1
Germany 138.0/86.4 139.5/88.5 137.3/84.3 27.3
* Age-adjusted
Figure 1 Mean Systolic Blood Pressure, African Descent Populations; By Age Group
Figure 2 Mean Systolic Blood Pressure, European Descent Populations; By Age Group
Figure 3 Mean Systolic Blood Pressure, African and European Descent Populations; By Age Group
Hypertension prevalence
Hypertension prevalence, which accounts for the effect of treatment, follows a similar pattern although the east-west gradient among the African-origin groups is more consistent (Table 3, Figure 4). Among the 14 populations, US blacks fall near the middle in terms of prevalence (mean prevalence = 37%, U.S. blacks = 44%). Among those above the mean, all but one is of European origin. Important differences are apparent in the gender-specific prevalence hypertension in these groups. Among Jamaican women hypertension was substantially more common than among Jamaican men (32% vs. 23%), and relative gender equality existed for US blacks. In Europe, however, the prevalence of hypertension was higher among men in every country (range 5–10%).
Figure 4 Hypertension Prevalence (140/90 mmHg or Treatment), African and European Descent Populations; Ages 35–64, Age Adjusted
Hypertension prevalence and obesity
The only etiological factor on which standardized information was available was obesity, measured by its proxy BMI. The correlation between average BMI and hypertension prevalence was 0.6 (p < 0.01), all populations combined. Within the black populations the same correlation was observed between mean BMIs and hypertension prevalence (r = 0.6). Among whites, however, the relationship was weaker (r = 0.3). Of course, since obesity will be correlated with many other aspects of lifestyle, it is difficult to infer whether weight gain itself is playing a less important role in determining the variation among white populations. The contrasts noted above in hypertension prevalence by gender are consistent with the relative excess of obesity in women compared to men among Jamaicans and US blacks [2,6].
Discussion
Comparisons of BP distributions across populations are made difficult by the requirement of comparability of the survey methods. In the last two decades, however, adoption of standardized protocols along with rigorous training have greatly improved the quality of epidemiological studies of hypertension [6,17-19]. A number of countries now conduct recurring national surveys that monitor both secular trends and regional variation within the country [10-12,19,20]. While independent surveys from the same base population had given divergent results in the past, at least two recent single-community studies conducted in the US provided estimates virtually identical to NHANES [21,22]. Although this evidence does not diminish the requirement of careful assessment of survey methodology before making comparisons, it does demonstrate that reliable information can be obtained from independent studies.
The data presented here demonstrate a two-fold variation in prevalence of hypertension in both European- and African-origin populations. The prevalences are similar in blacks in the US and whites in Europe, although important gender differences are apparent. Although not a systematic sample, the populations that are included generally reflect the characteristic social setting in which these groups are found around the world. Summed across all groups, the white populations on average have a substantially higher burden of hypertension. This result can be attributed in large part to the inclusion of several black samples from developing countries where risk factors for hypertension are presently at a lower level. In the only head-to-head comparison within the same survey, US blacks have a prevalence that is 50% higher than among whites. Data from the UK, including the national survey, also demonstrate higher BPs and more hypertension among blacks of Caribbean and African descent [23-27]. On the whole, however, the published literature on racial disparities in hypertension from the UK is less consistent than in the US, where essentially every study has reported higher rates among blacks [28]. Surveys from Cuba, Trinidad and Brazil have also shown a smaller black-white gradient in BP than found in North America [29-31].
Are these findings merely artifactual, reflecting either methodological error or the sampling process? The most unexpected features of the data presented here are the high rates of hypertension in Europe, when contrasted to whites in Canada and the US. These results have been reported in greater detail in an earlier publication [16]. It is beyond the usual standard of statistical significance for the six European surveys to be higher by chance than both of those in North America (p < 0.05). As previously demonstrated, mortality rates for stroke – the most sensitive vital statistics indicator of uncontrolled high BP – are strongly correlated with the prevalence of hypertension among these countries ('r' = 0.8) [16]. Although the data are more limited, hypertension appears to be even more common in Eastern Europe [32-34]. In a comparison of Pol-MONICA with the US-based ARIC study, systolic BPs in Poland were 20 mmHg higher than in the US [3].
The primary purpose of this analysis was to provide descriptive results and very limited information was available on factors that might explain the findings we observed. The gradient among the black populations is consistent with the transition to an industrialized lifestyle and is thereby collinear with most known risk factors [6]. BMI is serving as an effective proxy for this relationship, although its independent contribution cannot be quantified. The explanation of the European-North American contrasts among the white populations is not as apparent. As we have discussed elsewhere, either known risk factors other than obesity are having a larger impact at the population level than usually appreciated, or unknown factors are at work [16]. In either case, further examination of this question seems justified.
Treatment guidelines and practice patterns vary widely among these countries [16-19]. Widespread treatment could, of course, alter the mean BPs in a population, although this effect would be confined to persons over 55 where hypertension is common. The US has the highest rate of treatment, with about 25% of hypertensives controlled, compared to 10% in Europe and less than 1% in Africa (with hypertension defined as 140/90 mmHg)[16]. Any biases that would be introduced into the cross-national comparisons by differential treatment and control are insufficient to alter the primary conclusions, however. The virtual absence of treatment in rural Africa would mean that the natural distribution has essentially been observed unaltered. The effect of treatment in the US or Canada would not be apparent in younger individuals, where contrasts in BPs with Europe and Africa are equally large.
If the North American-European contrasts are occurring in genetically homogeneous populations, large environmental influences must be at work that are not apparent on the surface. A similar process could be taking place across the social environments into which persons of African origin are assorted within societies such as the US and the UK. The debate over inherent susceptibility cannot be resolved with these data since neither the genetic nor the environmental influences can be held constant, allowing a test of the relative influence of the other factor. In fact, the question of inherent susceptibility is probably non-testable under any circumstances [35-37]. While the assumption is often made that contrasting environmental influences between blacks and whites can be adjusted by using proxy measures such as education, that assumption does not hold up under close examination [38]. Perhaps more to the point, however, these data demonstrate that the consistent emphasis given to the genetic elements of the racial contrasts may be a distraction from the more relevant issue of defining and intervening on the preventable causes of hypertension, which are likely to have a similar impact regardless of ethnic and racial background [39]. Once the problem of ethnic/racial contrasts is characterized more closely as a special instance of environmental influences at the population level, it could become more tractable in both the realms of research and practice.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
RC, KWM, AL and JB were responsible for study concept, design and supervision. RC, KWM, JB, SG, MJ, AA, TF MK, PP, BS and MT were involved in data acquisition. RC, KWM, JB, MJ, PP and AL were responsible for analysis and interpretation of data. RC and KWM drafted the manuscript. RC, JB, SG, AL, AA, TF, MJ, MK, PP, BS and MT were involved in critical revision of the manuscript for important intellectual content. Statistical expertise was provided by MJ. Administrative, technical and material support was provided by KWM, MK, and BS.
Table 1 Characteristics of the Surveys
Country Survey Yr(s) Population N Male (%) Participation Rate (%) Age Range Sampling Method*
Nigeria 1991–93 Local 1931 45 NA 25–74 Multistage, address
Jamaica 1993–95 Local 2573 41 65 25–74 Multistage, address
USA, NHANES Black 1988–94 National 5283 45 82 18–80+ Multistage, population registry
Canada 1986–92 National 23129 49 77.5 18–74 Multistage, medical insurance registries
England 1998 National 11884 45 87.5 16–80+ Multistage, post code address
Finland 1997 National 7064 47 72 25–64 Population registry
Germany 1997–99 National 7047 49 61.4 18–79 Population registry
Italy 1998 National 8233 50 - 35–74 Multistage, population registry
Spain 1990 National 2021 40 73 35–65 Multistage, national registry
Sweden 1999 Regional 1823 49 72 25–74 Population registry
USA NHANES White 1988–94 National 7252 46 82 18–80+ Multistage, population registry
* All stratified sampling methods
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The authors are grateful for the use of the data from the Osservatorio Epidemiologico Cardiovascolare, Italy. We would like to thank Guichan Cao for assistance in data management and analysis at Loyola University. Funding was provided by the Centers for Disease Control and Prevention, USA.
This work was supported by a grant from the Cardiovascular Branch of the US Centers for Disease Control and Prevention, Atlanta, GA (Cooperative agreement 0755).
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| 15629061 | PMC545060 | CC BY | 2021-01-04 16:27:56 | no | BMC Med. 2005 Jan 5; 3:2 | utf-8 | BMC Med | 2,005 | 10.1186/1741-7015-3-2 | oa_comm |
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BMC MedBMC Medicine1741-7015BioMed Central London 1741-7015-3-41564413110.1186/1741-7015-3-4Research ArticleSudden Infant Death Syndrome and prenatal maternal smoking: rising attributed risk in the Back to Sleep era Anderson Mark E [email protected] Daniel C [email protected] Holly A [email protected] Department of Community Health Services, Division of Pediatrics, Denver Health and Hospitals Authority, Denver, Colorado, USA2 Division of General Internal Medicine, University of Colorado Health Sciences Center, Denver, Colorado, USA3 Department of Community Health Services, Division of General Internal Medicine, Denver Health and Hospitals Authority, Denver, Colorado, USA2005 11 1 2005 3 4 4 26 5 2004 11 1 2005 Copyright © 2005 Anderson et al; licensee BioMed Central Ltd.2005Anderson 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
Parental smoking and prone sleep positioning are recognized causal features of Sudden Infant Death. This study quantifies the relationship between prenatal smoking and infant death over the time period of the Back to Sleep campaign in the United States, which encouraged parents to use a supine sleeping position for infants.
Methods
This retrospective cohort study utilized the Colorado Birth Registry. All singleton, normal birth weight infants born from 1989 to 1998 were identified and linked to the Colorado Infant Death registry. Multivariable logistic regression was used to analyze the relationship between outcomes of interest and prenatal maternal cigarette use. Potential confounders analyzed included infant gender, gestational age, and birth year as well as maternal marital status, ethnicity, pregnancy interval, age, education, and alcohol use.
Results
We analyzed 488,918 birth records after excluding 5835 records with missing smoking status. Smokers were more likely to be single, non-Hispanic, less educated, and to report alcohol use while pregnant (p < 0.001). The study included 598 SIDS cases of which 172 occurred in smoke-exposed infants. Smoke exposed infants were 1.9 times (95% CI 1.6 to 2.3) more likely to die of SIDS. The attributed risk associating smoking and SIDS increased during the study period from approximately 50% to 80%. During the entire study period 59% (101/172) of SIDS deaths in smoke-exposed infants were attributed to maternal smoking.
Conclusions
Due to a decreased overall rate of SIDS likely due to changing infant sleep position, the attributed risk associating maternal smoking and SIDS has increased following the Back to Sleep campaign. Mothers should be informed of the 2-fold increased rate of SIDS associated with maternal cigarette consumption.
Sudden Infant Death SyndromeSIDSsmokinginfant deathattributed risk
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Background
Previous literature has shown a relationship between maternal smoking and the Sudden Infant Death Syndrome (SIDS). Published studies vary in size and methodology but consistently demonstrate a two-fold increased odds of SIDS with both prenatal and postnatal maternal smoking [1-10], including one study of over 300,000 infants in an analysis of birth registry data from the late 1970s [4,6]. A recent secular change, namely the Back to Sleep campaign, has had a major role in reducing SIDS rates. This public health campaign encourages parents to place infants in a supine rather than prone sleeping position. Early studies investigating the effects of supine sleeping revealed significantly reduced SIDS rates [11], but smoking among mothers, which was not targeted as the primary intervention, remained unchanged.
The aim of this study was to confirm the relationship between reported prenatal maternal smoking and SIDS and to examine the effect of sleeping position changes on the attributed risk of SIDS and smoking. The analysis spans the rollout of the Back to Sleep campaign in the United States for the ten-year period from 1989 to 1998. We hypothesized that maternal prenatal smoking confers a clinically significant risk of SIDS and that an increased attributed risk of SIDS associated with smoking could be identified in the wake of the supine sleeping campaign.
Methods
Study setting/population
Since 1969, the State of Colorado has collected data on multiple items through a birth registry. This registry includes extensive demographic data such as maternal report of the number of cigarettes smoked per day during pregnancy. We used the Colorado Infant Death Registry over a 10-year period (1989–1998) to identify causes of infant death.
Study design
We conducted a retrospective cohort study utilizing Cochran Mantel-Haenszel univariate analysis of risk factors and multiple logistic regression on the outcomes of infant death, SIDS, and respiratory deaths. We excluded non-singleton births (14978 records), infants born at less than 2500 grams (36779 records), and birth records where a mother's smoking status was unknown (5835 records). Additional records with missing data were coded so as to place the record in the presumed lower risk group: 42 records with unclear marital status were coded as "married," 67 records missing a maternal age were coded as age "18–34 years," 865 records with unclear gestational age were coded as "term" infants, and 4 records missing a gender assignment were coded as "female" in the analysis. Missing data for education (9659 records) and ethnicity (582 records) were coded as such and included in the analysis directly. We linked the birth and death registries using a unique birth number present in both registries. Utilizing multiple logistic regression, we modeled the exposure of interest, maternal smoking, as both a dichotomous and a continuous variable. Other variables analyzed included infant gender and gestational age (less than 37 weeks, 37 weeks or older), as well as maternal marital status, ethnicity, time between pregnancies (less than 12 months or 12 months and greater), maternal age (<18 years, 18 to 34 years, and >34 years), education, and self-reported use of alcohol in pregnancy. In the analyses for SIDS and respiratory causes of death, deaths from other causes were excluded from the analysis.
Power to detect a 20% difference in a baseline disease occurrence of 3 per 1000 was calculated at 99% for a population of 500,000. SAS version 8.0 (SAS Institute) on a PC was utilized for statistical analyses. We included the interaction between ethnicity and cigarette use in the modeling and requested the Hosmer-Lemeshow statistic from the model.
Study outcomes
We compared cases of SIDS (ICD 9 codes 798.0 to 798.9) between cohorts of infants born to mothers who reported prenatal smoking versus mothers who reported no prenatal smoking. Secondary outcomes included total infant mortality and respiratory deaths (ICD 9 codes 033.0 to 033.9 and 460 to 490). We used unadjusted annual rates to calculate the attributable risk of SIDS associated with maternal cigarette consumption.
Results
Over the 10-year study period, 1573 infants died, 598 SIDS cases occurred (1.2/1000 live born infants), and 34 infants died due to a respiratory etiology. Table 1 describes characteristics of the exposed and unexposed infant cohorts. Smoking mothers were statistically more likely to be single, non-Hispanic, less educated, and to report alcohol use in pregnancy.
Table 1 Demographic characteristics of 488,918 births according to maternal smoking habits during pregnancy, Colorado Birth Registry, 1989 to 1998.
Number (%) Reported maternal smoking (%)
Sex
male 252,066 (51.6) 13.4
female 236,852 (48.4) 13.1
Gestational age
<37 weeks 16,057 (3.3) 14.7
"term" 472,861 (96.7) 13.2
Marital status of mother
married 376,222 (77.0) 10.1
single 112,696 (23.0) 23.9
Time between births
12 months or less 19,987 (4.1) 16.0
>12 months 468,931 (95.9) 13.2
Maternal Age
17 years or less 21,362 (4.4) 17.1
18 to 34 years 407,801 (83.4) 13.7
35 years or more 59,755 (12.2) 8.9
Maternal Ethnicity
caucasian 351,051 (71.8) 14.3
hispanic 97,911 (20.0) 10.4
black 21,989 (4.5) 14.4
other/missing 17,967 (3.7) 8.5
Maternal Educational level
<high school 85,284 (17.4) 23.9
high school 157,852 (32.3) 17.9
some college 111,824 (22.9) 10.5
college 112,384 (23.0) 3.0
more than college 13,840 (2.8) 1.7
missing 7734 (1.6) 12.5
Maternal Alcohol use
none 477,733 (97.7) 12.6
1 to 50 drinks/week 11,185 (2.3) 42.8
We calculated adjusted odds ratios by analyzing smoking as a dichotomous exposure (mother smoked or did not smoke) and as a continuous variable (number of reported cigarettes per day during the pregnancy). Dichotomous outcomes of reported smoking during pregnancy yielded adjusted odds ratios of 1.9 (95% CI: 1.6 to 2.3) for death due to SIDS, 1.5 (95% CI: 1.3 to 1.7) for infant deaths from all causes, and 3.0 (95% CI: 1.4 to 6.3) for deaths due to respiratory etiologies (p < 0.01 for all outcomes). Table 2 shows results for the model for each of the outcomes. The final logistic regression model for each outcome was slightly different, but smoking remained in each as a significant factor. The interaction between ethnicity and cigarette use did not contribute significantly to the final model and thus was excluded.
Table 2 Adjusted odds ratios for SIDS, all infant deaths, and respiratory deaths with dichotomous cigarette use and other potential confounders*
SIDS p All deaths p Respiratory deaths p
Reported smoking <0.0001 0.0001 0.0032
No cigarette use 1.0 1.0 1.0
Cigarette use 1.9 1.5 3.0
Gender <0.0001 <0.0001 Not significant
Female 1.0 1.0
Male 2.0 1.4
Marital status <0.0001 <0.0001 0.0127
Married 1.0 1.0 1.0
Not married 1.7 1.5 2.5
Pregnancy interval 0.0033 0.0016 0.0056
12 months or more 1.0 1.0 1.0
Less than 12 mo. 1.6 1.4 3.8
Gestational age 0.0053 <0.0001 Not significant
Term 1.0 1.0
Less than 37 weeks 1.6 3.7
Any alcohol use Not significant Not significant Not significant
Education 0.0002 <0.0001 Not significant
More than college 1.0 1.0
College 0.6 1.0
Some college 0.9 1.2
High school 0.9 1.2
Some high school 1.3 1.4
Unknown 0.7 2.1
Ethnicity 0.0204 Not significant 0.0337
White 1.0 1.0
Hispanic 0.8 1.8
Black 1.4 0.6
Other 0.7 4.5
Year of birth <0.0001 <0.0001 Not significant
1998 1.0 1.0
1997 1.7 1.0
1996 1.7 0.8
1995 1.6 0.8
1994 1.7 0.9
1993 2.7 1.0
1992 3.0 1.1
1991 3.6 1.3
1990 3.0 1.3
1989 3.1 1.4
*Logistic regression modelling with corresponding p-values for each variable
Analyzing cigarette consumption as a continuous variable shows the associated odds per cigarette are 1.042 (95% CI: 1.030 to 1.054; p < 0.0001). In this model, consumption of 10 cigarettes per day is associated with a 51% increased odds of SIDS (95% risk limit 35% to 70%). The Hosmer-Lemeshow test statistic was non-significant (p > 0.1948) suggesting adequate model fit.
Table 3 shows crude rates of SIDS in the exposed and unexposed cohorts of infants for the years 1989 to 1998. Included is a calculated percent attributed risk (PAR) which, when multiplied by the number of SIDS deaths in the exposed cohort, yields the number of SIDS infants whose death is associated with maternal smoking 12. In this analysis, 101 infant deaths due to SIDS bear an association with maternal smoking among the exposed cohort of 172 infants. Figure 1 demonstrates the increasing PAR associating maternal prenatal smoking and SIDS during the study period, suggesting a stronger link between SIDS and maternal smoking. The average rate for each 2-year period is plotted in the Figure as well. Time was a highly significant variable in the study and SIDS rates decreased markedly over the study time period. The remaining SIDS deaths show a greater relative association with maternal smoking in the years following the Back to Sleep campaign. In the final year of the analysis, 80% of the SIDS deaths in the smoke-exposed infant cohort are attributed to maternal smoking.
Table 3 SIDS in smoke exposed and unexposed infants including attributed risk and deaths among smoke exposed infants by birth year, State of Colorado 1989–1998.
Birth year SIDS deaths in: Total SIDS SIDS rate (SIDS/1000) in: Relative Risk PAR* Deaths Attributed to smoking
smoke exp unexp smoke exposed unexp
1989 21 51 72 3.21 1.33 2.4 58 12
1990 24 53 77 2.96 1.35 2.2 54 13
1991 27 69 96 3.32 1.72 1.9 48 13
1992 21 59 80 2.93 1.41 2.1 52 11
1993 22 49 71 3.28 1.17 2.8 64 14
1994 10 34 44 1.65 0.79 5.6 52 5
1995 12 28 40 2.19 0.65 3.4 70 8
1996 9 36 45 1.61 0.82 2.0 49 4
1997 16 29 45 3.21 0.64 5.0 80 13
1998 10 18 28 1.80 0.37 4.9 79 8
TOTAL: 172 426 598 101
* Percent Attributed Risk in smoke-exposed cohort
Figure 1 Percent attributed risk (PAR) between maternal prenatal smoking and sudden infant death, Colorado Birth Registry 1989–1998.
Discussion
Smoking is deleterious and has negative effects on children born to mothers who smoke [12-16]. We confirm a two-fold increased risk of SIDS in infants born to smoking mothers. In addition, as one cause of SIDS (prone sleeping) was reduced through the Back to Sleep public health campaign in the United States, we noted an increasing attributed risk of SIDS with smoking. As a major etiology of SIDS, prone sleeping position, decreased over the study period due to the national educational campaign, the remaining SIDS deaths may now be due to the more isolated effects of tobacco exposure. Due to a decreased overall rate of SIDS, almost 80% of SIDS among smoke-exposed infants now bear a relationship to cigarette smoking.
Taylor and Sanderson, using data prior to the Back to Sleep campaign in the United States, suggest that up to 30% of SIDS deaths may be attributed to maternal smoking [17]. We note an increasing percent attributed risk in our cohort of 489,000 infants after the roll out of the campaign. Using the attributed risk to calculate an absolute mortality among the exposed cohort, 101 of 172 infant SIDS cases over the 10-year study period are linked to maternal smoking, which is greater than half of the infant deaths due to SIDS in the smoke-exposed cohort.
Our study cannot establish a causal role for maternal smoking in SIDS. Others have argued for a causal relationship based on prospective data, demonstrable dose-response relationships, and analysis using multiple potential confounders [18]. The causal path appears more likely on the basis of multiple studies that support such a link, consistent findings of a dose-response relationship in the literature, and a potential biological basis for the association between the exposure and the outcome [19]. In this study, removing one causative factor for SIDS, prone sleeping, may have increased the relative effect of another factor, maternal smoking, as an agent associated with SIDS.
The published literature reports a 20% to 30% smoking rate among pregnant women [20,21]. Approximately 20% of pregnant smokers will deny smoking, but when tested with urine cotinine will be positive [22]. A recent report suggests that 21% of women in Colorado are smokers [23]. In our cohort, 15% of women reported some smoking during pregnancy, which may indicate a degree of under-reporting consistent with the published literature. While about one-fourth of women quit smoking during pregnancy, the recidivism rate is high and women are most likely to continue smoking into the postnatal period [24-26]. Furthermore, as most individuals who smoke begin before the age of 18 years [27], the results reported here provide yet another clarion call to eliminate smoking initiation and remove this preventable risk factor for infant death and SIDS. Expectant mothers, women desiring pregnancy, and health care providers who care for them need to be reminded about this strong association of infant death with the preventable risk factor of maternal smoking. For example, physicians can counsel women desiring or planning pregnancy that if they smoke, the child will have a markedly increased risk of SIDS due to the smoking.
This study has several important limitations related to design and the nature of the data set used. Given the rich nature of the Colorado Birth Registry, we controlled for many potential confounding variables, but others may exist. This analysis likely represents an under-estimate of the actual associated risk with prenatal smoking, given that it relies on maternal report of a behavior widely known to be harmful. We suspect that self-report of maternal smoking underestimates the true rate of maternal smoking. Fetal and infant exposure to tobacco smoke occurs in manner ways, including prenatal maternal smoking, prenatal maternal second-hand smoke exposure, and postnatal smoke exposure by one or more care providers in the home. In this study we cannot distinguish among these exposure pathways, which may result in an erroneous under or over estimate of risk. The time period spanned by this study includes the rollout of the Back to Sleep campaign in the United States and our dataset did not assess whether parents positioned their infants prone or supine. We therefore could not control for this ecological association with SIDS. The very nature of retrospective data creates limitations in that we cannot carefully control the setting in which the data are collected, or the individual collecting the data. Missing data records are a third limitation, although this appears to have not been a major issue with this dataset.
Conclusions
As prone infant sleeping has decreased, other causes of SIDS such as maternal smoking assume increased importance in the effort to protect infants from SIDS. Spanning the rollout of the Back to Sleep campaign in the United States we examined the increasing attributed risk associating smoking with SIDS. As SIDS rates declined, the attributed risk of SIDS with smoking has increased. In the final year of analysis, we demonstrated a link between 80% of the SIDS deaths and maternal smoking. A major, preventable exposure remains for infants in the United States and providers should redouble counseling efforts toward reducing this exposure.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
MA participated in the study conception, background research, study design, statistical analysis, and drafting of the manuscript. DJ participated in the study conception, study design, and proofreading of the manuscript. HB participated in the study conception, study design, statistical analysis, and proofreading of the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgments
Initial results for this study were presented at a plenary session in the Ambulatory Pediatric Association's annual meeting in May 2003, held in Seattle, Washington. The authors would like to thank Dr. Ned Calonge, Acting Head of the State of Colorado Department of Public Health and Environment, who participated in the project design, and Dr. Allison Kempe, who provided critical review on the manuscript; project activities were made possible by The Faculty Development in Primary Care: 1 D 14 HP 00153-01; CFDA #93.895
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| 15644131 | PMC545061 | CC BY | 2021-01-04 16:27:56 | no | BMC Med. 2005 Jan 11; 3:4 | utf-8 | BMC Med | 2,005 | 10.1186/1741-7015-3-4 | oa_comm |
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BMC GenetBMC Genetics1471-2156BioMed Central London 1471-2156-6-11563436010.1186/1471-2156-6-1Research ArticleThe Prader-Willi syndrome murine imprinting center is not involved in the spatio-temporal transcriptional regulation of the Necdin gene Watrin Françoise [email protected] Meur Elodie [email protected] Nathalie [email protected] Marie-Anne [email protected] Luisa [email protected] Françoise [email protected] Centre National de la Recherche Scientifique UMR 6156, IBDM, Parc scientifique de Luminy, Case 907, 13288 Marseille Cedex 9, France2 Institut National de la Santé et de la Recherche Médicale UMR491, IPHM, Faculté de Médecine de la Timone, 27 Bd. J. Moulin, 13385 Marseille Cedex 5, France3 Département de Développement, Génétique et Pathologie Moléculaire, Institut Cochin, 24 rue du Faubourg St Jacques, 75014 Paris, France2005 5 1 2005 6 1 1 20 9 2004 5 1 2005 Copyright © 2005 Watrin et al; licensee BioMed Central Ltd.2005Watrin 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 Prader-Willi syndrome (PWS) domain and its mouse orthologue include a cluster of paternally expressed genes which imprinted expression is co-ordinately regulated by an imprinting center (IC) closely associated to the Snurf-Snrpn gene. Besides their co-regulated imprinted expression, two observations suggest that the spatio-temporal expression of these genes could also be co-regulated. First, the PWS genes have all been reported to be expressed in the mouse nervous system. Second, Snurf-Snrpn and its associated IC are the most ancient elements of the domain which later acquired additional functional genes by retrotransposition. Although located at least 1.5 megabases from the IC, these retroposons acquired the same imprinted regulation as Snurf-Snrpn. In this study, we ask whether the IC, in addition to its function in imprinting, could also be involved in the spatio-temporal regulation of genes in the PWS domain.
Results
We compared the expression pattern of Snurf-Snrpn and C/D-box small nucleolar RNAs (snoRNAs) MBII-85 and MBII-52 to the expression pattern of the two evolutionary related retroposons Ndn and Magel2, in the developing mouse embryo. We show that these genes have highly similar expression patterns in the central nervous system, suggesting that they share a common central nervous system-specific regulatory element. Among these genes, Ndn and Magel2 display the most similar expression patterns. Using transgenic mice containing the Ndn and Magel2 genes, we show that the transgenic Ndn gene whereas not imprinted is correctly expressed. Search for DNase I hypersensitive sites in the Ndn-Magel2 genomic region and comparative genomic analyses were performed in order to identify potential transcriptional cis-regulatory elements.
Conclusions
These results strongly suggest that paternally expressed genes of the PWS domain share a common central nervous system-specific regulatory element. We proposed that this regulatory element could co-localize with the IC. However, we demonstrate that the IC, if required for imprinted regulation, is not involved in the spatio-temporal regulation of distantly located retrotransposed genes such as the Ndn gene in the PWS domain.
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Background
Genomic imprinting in mammals is a process that leads to the preferential mono-allelic expression of specific genes in diploid cells, depending on whether they are inherited from the sperm or from the oocyte. To date, approximately 70 mammalian imprinted genes have been identified which map to at least 11 regions of the mouse genome [1]. Most imprinted genes are therefore located in clusters, which are generally conserved between human and mouse. For some of these clusters, coordinate imprinted gene regulation has been shown to be controlled by imprinting centers (IC) [2]. Imprinted genomic regions from the two parents are differentially marked by heritable epigenetic modifications including DNA methylation and histone acetylation and/or methylation. These epigenetic modifications or imprints are established at least for some of them in the germ line of either parent [2].
The Prader-Willi syndrome (PWS) domain on human chromosome 15q11-q13 and its ortholog on mouse chromosome 7C-D1 are large chromosomal domains containing paternally expressed genes [3]. PWS results from the loss of expression of several of them including SNURF-SNRPN, NDN, MAGEL2, MKRN3 and the C/D-box small nucleolar RNAs(snoRNAs). In humans, mini-deletions, upstream the SNURF-SNRPN transcriptional unit, have led to the characterization of an IC which coordinates their imprinted expression [4]. One sub-region of this IC defined as the shortest region of deletion overlap in PWS patients (PWS-SRO) and encompassing the SNURF-SNRPN promoter, has been shown to be required for maintaining the paternal imprint [5,6]. Its deletion in the germ line or post-zygotically on the paternal chromosome leads to silencing of all paternally expressed genes of the PWS domain [4,6]. In mice, although the Snrpn promoter is not required for genomic imprinting [7], deletion of a 35 kb region including 16 kb of sequences upstream Snurf-Snrpn exon 1 to Snurf-Snrpn exon 7 unit also leads to silencing of the paternally expressed genes, indicating that both the position of the IC and its role in the coordinate imprinted expression of genes is conserved between human and mouse [8,9].
Recently, examination of tissue-specific pattern of mRNA expression at a genomic scale allowed the identification of several chromosomal regions harbouring tissue-specific co-regulated genes defined as regions of correlated transcription (RCTs) [10]. Noticeable, some of these RCTs overlap with known imprinted loci and it suggests that transcriptional regulatory elements controlling imprinted expression might also regulate tissue-specific expression. Therefore, one might hypothesize that this type of regulation is applied at the PWS domain. Two arguments support this hypothesis. First, all the murine orthologues of the PWS genes have been reported to be expressed in the developing nervous system [11]. Second, Nicholls proposed an evolutionary model for the origin of the human and mouse PWS domains [12,13]. In this model, the Snurf-Snrpn locus and its associated IC would be the ancestral imprinted transcriptional unit of the domain. The other genes, which are intronless, would have later and sequentially been acquired by retrotransposition and adopted the same imprinted regulation. Altogether, these data suggest that a spatio-temporal co-regulation of PWS genes could exist and have been acquired through the evolution of the domain. Thus, it is tempting to speculate that the cis-regulatory element(s) initially involved in the spatio-temporal regulation of the Snurf-Snrpn locus might influence the spatio-temporal regulation of retrotransposed genes in the PWS region. The IC could be a good candidate to play this role.
In this study, we investigate this hypothesis. First, we compared the expression pattern of the Snurf-Snrpn, MBII-85, MBII-52, Ndn and Magel2 in the developing mouse embryo. We show that their expression in the embryo is restricted to neural tissues and that these genes display strikingly similar expression patterns in the central nervous system. Second, we created transgenic mice with a BAC containing the Ndn and Magel2 genes. We showed that the Ndn transgene is not imprinted but is correctly expressed in the developing embryo. These results demonstrate that if the IC is required for imprinted regulation, it is not involved in the Ndn spatio-temporal regulation. Finally, since we have shown an almost identical spatio-temporal expression profile of Ndn and Magel2 in the developing embryo, it suggests that these two genes might share common cis-acting regulatory elements which should be present in the BAC transgene. We therefore searched for DNaseI hypersensitive sites in the Ndn-Magel2 genomic region and performed comparative genomic analyses in order to identify potential transcriptional cis-regulatory elements.
Results
Nervous system tissue-specific expression of mouse PWS genes in the embryo
In order to determine if mouse PWS genes might be transcriptionally co-regulated, we compared the expression profiles of Snurf-Snrpn, MBII-85, MBII-52, Ndn and Magel2 by in situ hybridization in the developing mouse embryo (Fig. 1, 2 and 3). In the 10.5 and 13.5 mouse embryos, Snurf/Snrpn, MBII-85, MBII-52, Ndn and Magel2 are almost exclusively expressed in the developing nervous system, and although expressed at different levels, they display strikingly similar expression patterns in the central nervous system (brain and spinal chord). As previously reported for Ndn [14], they are predominantly expressed in all the ventral parts of the neural tube, mostly or even exclusively in marginal areas where differentiating neurons reside. In the peripheral nervous system, Ndn and Snurf/Snrpn are both expressed at high levels in cranial and dorsal root ganglia, sympathetic and parasympathetic ganglia, structures in which MBII-85, MBII-52 and Magel2 trancripts are expressed at much lower levels or not at all (Fig. 2 and 3). Magel2 when compared to Ndn, Snurf-Snrpn and MBII-85/ -52 seems to have a more highly restricted expression domain. In the 12.5 embryo, Magel2 transcripts are detected at high levels and predominantly in the hypothalamus (Fig. 3). Careful examination shows that although found at low or very low levels, Magel2 transcripts are present in similar domains as the four other PWS genes studied. Ndn and Magel2 also share expression domains in some non-neuronal tissues such as in the muscles (skeletal muscles and tongue) and in some non-neuronal neural crest cells derived structures such as the branchial arches (Fig. 3; data not shown). Magel2 transcripts are detected at very low levels in the dorsal root ganglia and not detected in cranial ganglia.
Figure 1 Imprinted cluster on mouse chromosome 7C. Upper and lower boxes represent genes expressed from the paternal and maternal alleles respectively. Small nucleolar RNAs (snoRNAs) MBII-13, MBII-85 and MBII-52 genes lie within introns of long primary transcripts initiated at the U exons (U9 to U1 exons). Arrows indicate the transcriptional orientation of the genes. Frat3, Mkrn3, Magel2 and Ndn lie within 120 kb and around 1.5 to 2 Mb from the Snurf-Snrpn gene. Location of BAC109 used for the transgenic study is represented.
Figure 2 Comparison of Ndn and Snurf-Snrpn transcriptional unit expression patterns in the E13.5 mouse embryo. Comparison of Ndn (A, E, I), Snurf-Snrpn (B, F, J), MBII-85 (C, G, K) and MBII-52 (D, H, L) RNA expression on E13.5 mouse embryo adjacent sagittal sections, from lateral to more medial sections (rows I to II). Ndn as well as Snurf/Snrpn, MBII-85 and MBII-52 are almost exclusively expressed in the developing nervous system and display strikingly similar expression patterns in the developing brain and spinal chord. In the peripheral nervous system, Ndn and Snurf-Snrpn are expressed at similar levels in cranial (Ndn: A; Snurf-Snrpn: B) and dorsal root ganglia (Ndn: I; Snurf-Snrpn: J) whereas MBII-85 (C, K) and MBII-52 (D, L) are expressed at much lower levels or not at all. Note that Ndn is additionally expressed in muscle tissues such as the skeletal muscles (A) and the tongue (E), and in the adrenal primordium (I). ap, adrenal primordium; drg, dorsal root ganglia; gn, gonad; hp, hypothalamus; ht, heart; lg, lung; lv, liver; mb, midbrain; mo, medulla oblongata; mt, metanephros; ms, muscles; poa, post-optic area; pp, telencephalic epithelium preplate; ps, pons; sa, septal area; tg, tongue. Asterics indicate cranial and dorsal root ganglia in A, B, C, D.
Figure 3 Comparison of Magel2 and Ndn expression patterns in the E10.5 and E12.5 mouse embryo. Comparison of Magel2 (A, C, E, G) and Ndn (B, D, F, H) RNA expression in E10.5 (A, B, C, D) and E12.5 (E, F) mouse embryos on adjacent sagittal sections, and in E12.5 (G, H) mouse embryos on adjacent transversal sections. In the E10.5 embryo, the predominant expression in the ventral parts of the neural tube is particularly evident (A, B, C, D) for both genes. Magel2 and Ndn display a strikingly similar profile of expression in the infundibulum recess (C, D). Asterics indicate expression of both genes in symetrical ventral stripes of cells in the neural tube (A, B). Note that Magel2 as Ndn are expressed in the mandibular component of the first branchial arch (C, D), in the tongue (E, F). In the E12.5 embryo, Magel2 and Ndn are expressed in identical structures such as the ganglionic eminence, the pons, the medulla oblongata, in the preplate of the telencephalic vesicle, the hypothalamus, the ventral thalamus, the zona limitans intrathalamica and in the basal plate (E, F, G, H). drg, dorsal root ganglia; ge, ganglionic eminence; hp, hypothalamus; mcba, mandibular component of the first branchial arch; mo, medulla oblongata; pp, telencephalic epithelium pre-plate; ps, pons; rp, rathke pouc;h so, somites; tg, tongue; vt, ventral thalamus; zi, zona limitans intrathalamica.
In conclusion, all the PWS genes that we studied were expressed mainly (Ndn and Magel2) or even exclusively (Snurf-Snrpn, MBII-85/-52) in the mouse developing nervous system at E12.5 and E13.5, with strikingly similar identical patterns in the central nervous system (brain and spinal chord). More divergent expression patterns were observed in the peripheral nervous system (cranial and dorsal root ganglia, sympathetic ganglia), some genes being expressed at high levels (Ndn and Snurf-Snrpn) and the others being expressed at low levels or not at all (MBII-85, MBII-52 and Magel2). Our in situ hybridization data suggests the presence in the PWS domain of a central nervous system-specific neural element which coordinates the PWS genes expression.
Transgenic experiments
In order to determine if the spatio-temporal transcriptional regulation of PWS genes is dependant upon the IC, we initiated BAC transgenic analyses and chose to investigate the transcriptional regulation of the Ndn gene outside its natural genomic context. Since Ndn and Magel2 are co-regulated and the intergenic region between these two genes is around 30 kb only, we chose a bacterial artificial chromosome (BAC 109) containing both genes to generate transgenic mice (Fig. 4A). Three transgenic male founders were obtained and crossed with C57BL/6 females. Only one male transmitted the BAC109 transgene to its progeny and gave rise to the transgenic line 92 (Tg92) described in this study. The presence of BAC vector sequences allowed us to discriminate the transgenic from the wild type DNA. Genomic DNA from Tg92 mice was thoroughly analyzed by PFGE, Southern blot and PCR, to precisely determine the structure and the number of copy of the integrated transgene (Fig. 4A, data not shown). The transgene integrated in one full copy along with a truncated copy containing the whole 5' region upstream Ndn up to the Ndn promoter (Fig. 4A), near the centromeric region of chromosome 2, as determined by DNA FISH analysis (data not shown).
Figure 4 Transgenic analyses. (A) Structure and copy number of the BAC109 transgene in line Tg92. The BAC109 transgene, the relative positions of the Ndn and Magel2 genes, and the transgenic EagI and PmeI restriction fragments detected with probes 5' and 3' are represented on the upper diagram. Genomic DNA was isolated from wild type (WT) or transgenic (TG) mice, digested by EagI or PmeI, separated by PFGE, blotted and hybridized to the 5' or 3' probes. The presence of the 54 and 50 kb Eag1 transgenic fragments detected by the 5' and the 3' probes respectively demonstrate the integrity of the transgene. Eag1 is a methylation sensitive enzyme which explains the presence of a higher hybridization band corresponding to undigested methylated DNA (Und). Hybridization of PmeI digested genomic DNA with either the 5' or 3' Ndn probes, indicated that the BAC integrated in one full copy along with a truncated copy containing the whole 5' region upstream Ndn up to the Ndn promoter.(B) Non imprinted brain-specific expression of transgenic Ndn. Ndn expression was tested by Northern blot analysis. RNAs were isolated from Tg92mat-Ndn+/-pat or wild type littermate tissues: Br, brain, Kd, kidney, Lv, liver, Sp, spleen, Th, thymus, Ht, heart, Mu, muscles. Note that Ndn transgenic mRNAs are not detected in adult muscles whereas Ndn endogenous mRNAs are. After being hybridized to an intragenic Ndn probe, Northern blots were stripped and rehybridized to a β-actin probe to check for the presence and integrity of RNAs.
Transgenic Ndn expression analysis was performed by Northern blot hybridization, on adult tissues of mice inheriting the transgene either maternally or paternally, on an Ndn null background (Tg92mat-Ndn+/-pat and Tg92pat-Ndn+/-pat, respectively). Whether maternally or paternally inherited, the transgenic Ndn gene was expressed with the same tissue specificity as the endogenous Ndn gene (Fig. 4B). Transgenic Ndn transcripts were only detected in the brain, although at a slightly lower level than endogenous transcripts. It should be noted that no transgenic Ndn gene expression was detected in muscle, a tissue in which endogenous Ndn transcripts are detected although at low levels as compared to the brain. In situ hybridization experiments were then performed on transgenic embryos carrying a paternal deletion of the Ndn allele (Tg92mat-Ndn+/-pat) and wild type mouse embryos, to compare the transgenic and endogenous profile of Ndn expression. Transgenic Ndn transcripts were detected in embryos from E10.5 as endogenous Ndn transcripts, and in the same structures, namely the central and peripheral nervous system (Fig. 5), at a slightly lower level in transgenic embryos as it was noted in transgenic adult tissues. No transgenic Ndn expression was detected in the dermo-myotome, the muscles or the tongue, although endogenous Ndn transcripts were detectable in these tissues (Fig. 5: H and I). Since Magel2 null mice were not available, Magel2 imprinting and expression from the BAC109 transgene could not be analyzed. However, no ectopic site of Magel2 expression was noted during embryogenesis.
Figure 5 Comparison of the wild type and transgenic Ndn expression profiles in E10.5 and E12.5 mouse embryos. In situ hybridization was performed with an Ndn riboprobe on sagittal sections of wild type (A, B, C, H), Tg92mat-Ndn+/-pat (D, E, F, I) and Ndn+/-pat (G) embryos. The absence of Ndn signal in the E10.5 Ndn+/-pat embryo (G) demonstrates the specificity of the Ndn riboprobe. Similar expression profiles are shown in the infundibulum recess (ir) of E10.5 embryos (A, D), in the preplate of the telencephalic epithelium (pp) and in the dorsal root ganglia (drg) of E12.5 embryos. Note that no hybridization signal is detected in the transgenic Tg92mat-Ndn+/-pat E12.5 muscle (ms) (I) whereas endogenous Ndn is expressed in this tissue (H). rp, rathke pouch; tv, telencephalic vesicle.
Since the transgenic Ndn gene was not imprinted, methylation studies of the transgenic Ndn promoter region and CpG island were omitted.
Search for Ndn transcriptional regulatory elements
Since Ndn was correctly expressed at least in the nervous system from the BAC transgene, Ndn regulatory elements must be present in the BAC109 genomic sequences. With the increasing availability of genomic sequence and the recent development of powerful global alignment algorithms, interspecies genomic sequence comparisons are becoming an efficient mean to identify conserved non-coding sequences (CNS) which regulatory potential can further be assessed experimentally [15,16]. However, since the whole human and mouse PWS domains are rather highly conserved which might render difficult to discriminate between functional and non functional CNS, we first undertook an experimental approach. We analyzed the chromatin DNase I sensitivity of the genomic sequences present in the BAC transgene. In the adult brain, hybridization of BglII- or BamHI-cut DNA with a series of probes described in Fig. 1 led to the characterization of several major DNaseI hypersensitive sites associated to the Ndn gene (HS1/2, HS3 and 4) and of one site upstream the Magel2 gene (HS5) (Fig. 6). The probes used allowed a systematic analysis of the Ndn/Magel2 genomic sequences present in the BAC transgene, from -17.5 kb upstream the Ndn gene to17 kb downstream the Magel2 gene. Additional upstream Ndn sequences which were present in the BAC could not be analyzed since their sequences are not yet available.
Figure 6 Mapping of DNase I hypersensive sites in the Ndn and Magel2 genomic region. (A) The diagram shows a map of the sequenced genomic region present in the BAC109. Localisation of HS sites is indicated by arrows in relation to the Ndn and Magel2 genes, and to the AK086725 EST. Position of BglII and BamH1 sites and probes 1 to 10 are indicated. Asterics indicate polymophic BglII restriction sites present on castaneus alleles detected with probes 1/2 and probes 7/8. A, B and C design three non-coding regions of homology between the mouse and the human genomes.(B) Southern blot analysis of BglII or BamHI cut genomic DNA isolated from brain nuclei treated with increasing concentrations of DNase I (from 0 to 100 U). Maternal (mat) and paternal (pat) alleles are indicated when discrimination is possible.(C) Sequence comparison between mouse and human Ndn/Magel2 genomic regions. Alignments were peformed with the genome VISTA program (window size 100 bp, homology threshold 70%). Conserved non-coding sequences (CNS) are depicted in pink, untranslated regions (UTR) and ORF in pale and dark blue respectively. Genes are indicated by blue arrows. Repeated elements are depicted above the sequences comparison. Mapped HS sites are indicated by vertical arrows. A, B and C design three non-coding regions of homology between the mouse and the human genomes. Although the C region is depicted as a non-gening sequence, recent isolation of ESTs corresponding to this region suggests that it might belong to the Magel2 transcription unit. The asterisk above the C region indicates the beginning of the ESTs.
Hybridization of BglII-cut DNA with probe 1 revealed two adjacent (~50–70 bp apart) and strong DNase I hypersensitive sites (HS1 and HS2) in the Ndn promoter region of the paternal allele (Fig. 6B). Hybridization with probe 2 revealed two additional but weaker DNase I hypersensitivity sites on the paternal allele (HS3 and HS4), localized in the Ndn 5'UTR and at the beginning of the coding region, respectively. In contrast to the paternal allele, the maternal allele was highly resistant to DNase I digestion. In the adult kidney, a tissue in which Ndn is not expressed, the two parental alleles were highly and almost equally resistant to DNase I digestion (data not shown). The presence of DNase I hypersensitive sites HS1/2, HS3 and HS4 is therefore closely linked to the transcriptional activity of the Ndn gene since they were present on the paternal allele and only in Ndn-expressing tissues.
Hybridization of brain BamH1-cut DNA with probe 6 or BglII-cut DNA with probes 7 and 8 revealed one relatively strong DNase I hypersensitive site (HS5) 3.5 kb upstream the Magel2 gene, which was present on both parental alleles and co-localized with a polymorphic BglII site present on the CAST/Ei allele. HS5 was localized 1 kb upstream newly isolated full length Magel2 ESTs (AK086725, AK082944, AK086725; see Fig. 6A) which initiate around 2.5 kb 5' to the described Magel2 mRNA (NM013779) [17]. HS5 was present on both maternal and paternal alleles and in tissues in which Magel2 and/or Ndn are not expressed such as the adult kidney (data not shown) which suggests that this DNase I hypersensitive site is not linked to the transcriptional activity of these two genes.
No prominent DNase I hypersensitive site were detected in the Ndn-Magel2 intergenic region (excepted HS5). Multiple faint hybridization signals could however be detected with probes 3 and 4 on BglII-cut DNA (data not shown) and with probe 5 on BamH1-cut DNA, revealing regions of low DNase I hypersensitivity in the Ndn-Magel2 intergenic region (Fig. 6B). Finally, hybridization of probe 10 on brain BamH1-cut DNA allowed the detection of a lower hybridization band, revealing one or potentially two DNase I hypersensitive sites (HS6a or/and HS6b). We were not able to precisely localise the HS6 site(s) as well as its (their) parental origin.
We next mapped HS1 to 6 DNase I hypersensitive sites on a mouse/human sequence alignment (Fig. 6-C). The available mouse sequences used for this comparison start from -17.5 kb upstream the Ndn gene to the Magel2 extremity of BAC109. Several CNS defined as orthologous sequences greater than 100 bp and greater than 70% identity [15] were found and mainly gathered into three regions: upstream the Ndn gene (region A), in the intergenic Ndn-Magel2 region (region B) and in a 2.8 kb upstream the Magel2 gene (region C). None of the hypersensitive sites identified in this study mapped to region A, B or C. HS1/2 which are closely linked to the transcriptional activity of the Ndn gene are localized in a CNS immediately upstream the Ndn gene. HS5 co-localized to an isolated CNS localized 0.7 kb upstream the highly conserved C region and around 1 kb upstream the newly described full length Magel2 ESTs. However, the functional significance of HS5 in the Magel2 and/or Ndn gene transcriptional regulation remains to be determined since this site was neither allele nor tissue-specific. It should be noted that there was almost no conservation of sequence in the region downstream the Magel2 gene. In contrast, the intergenic Ndn-Magel2 region was relatively well conserved and regions of low DNase I hypersensitivity were detected in this region which might have functional importance.
Discussion
Coordinated central nervous system-specific expression of PWS genes
Study and comparison of Snurf-Snrpn, MBII-85/-52 snoRNAs, Ndn and Magel2 expression profiles brought new information on the transcriptional regulation of these genes. Snurf-Snrpn, the snoRNAs MBII-52 and MBII-85 and the distantly located Ndn and Magel2 genes were expressed in the mouse developing nervous system at E10.5 and E13.5 with strikingly similar patterns in the central nervous system.
More divergent expression patterns were observed in the peripheral nervous system and non-neuronal tissues.
Accordingly, Snurf-Snrpn, MBII-85 and MBII-52 snoRNAs were exclusively expressed in the developing nervous system, and in identical structures in the central nervous system. Marked differences of expression between Snurf-Snrpn and the snoRNAs were found in cranial and dorsal root ganglia, structures in which Snurf-Snrpn was highly expressed whereas the snoRNAs were expressed at very low levels (MBII-85) or not at all (MBII-52). These results reinforce the proposal that the snoRNAS derive from the processing of long primary transcripts initiated at the U exons, upstream Snurf-Snrpn [18] (Le Meur et al., submitted). It is interesting to note that Snurf-Snrpn was previously reported to be ubiquitously expressed in adult tissues [12], whereas we found it to be exclusively expressed in the nervous system at E10.5 and 13.5 of mouse embryogenesis.
As previously reported [14,11], Ndn and Magel2 mRNAs were detected almost exclusively in the nervous system of the mouse embryo from E9.5/10.5 onwards, and although Magel2 transcripts were detected at much lower levels than Ndn transcripts, these two genes displayed almost identical expression patterns in the central nervous system (brain and spinal chord). Particularly striking was their identical pattern of expression in the E10.5 embryo. At later developmental stages, Magel2 expression domain became more restricted but remained included in Ndn expression domain. Magel2 transcripts were however detected at very low levels in almost all the structures in which Ndn was expressed, including non neuronal structures such as the mandibular component of the first branchial arch, the tongue and the muscles. Magel2 transcripts have been reported to be particularly instable due to the presence of AU-rich elements (ARE) in their 3' untranslated region [17] and this could explain why they were detected at such low levels.
Our expression analysis in the mouse embryo indeed suggests the existence of a central nervous system-specific neural regulatory element coordinating the PWS genes expression. Ndn and Magel2, specifically, could share additional regulatory elements.
Co-expression of PWS genes and the IC
We further emitted the hypothesis that the central nervous system-specific regulatory element could be physically associated to the PWS IC because of the proposed evolutionary history of the PWS domain and because the IC is involved in coordinating long range chromatin modifications leading to imprinted expression of genes in the whole domain. The Snurf-Snrpn locus with its associated IC has been proposed to be the most ancestral transcriptional unit of the domain [12,13]. Other PWS genes (Ndn, Magel2, Mkrn3 and Frat3) would have later been acquired by retroposition and/or local cis-duplication of retroposed genes and would have adopted the same transcriptional regulation as the Snurf-Snrpn transcriptional unit. Unless retroposons arise from reverse-transcriptase-mediated processing of aberrant or alternative transcripts including endogenous promoter elements, their functionality is dependent upon regulatory elements present at their site of insertion. Retroposons in the PWS region could have therefore adopted the Snurf-Snrpn regulation both for imprinted and spatio-temporal expression. Finally, the fact that paternal deletions of the IC abolish the expression of all the PWS genes [8,7] also suggests a possible intertwining between regulatory elements controlling both imprinting and spatio-temporal expression.
Our study did not however confirm that the IC might coordinate the spatio-temporal expression of PWS genes. Our transgenic analysis clearly showed that Ndn does not rely on the IC for its spatio-temporal expression. Ndn was correctly regulated from the BAC transgene lacking the IC both in the developing embryo and in adult tissues. These results demonstrate that the cis-regulatory sequences involved in both the developmental and tissue-specific expression of Ndn were present in the transgene and were not associated to the IC. The fact that the IC was not involved in the spatio-temporal regulation of the Ndn gene does not however exclude its putative involvement in the spatio-temporal regulation of the Snurf-Snrpn transcriptional unit. No transcriptional regulatory elements or sequences excepted those involved in the imprinted expression [19] have yet been characterized in the Snurf-Snrpn transcriptional unit.
Search for Ndn and Magel2 regulatory elements
Since the cis-regulatory sequences involved in both the developmental and tissue-specific expression of Ndn were present in the transgene and since we showed that Ndn and Magel2 expression was coordinated both in the central nervous system and in some non neuronal tissues of the mouse embryo, we searched for Ndn and Magel2 regulatory elements in the BAC109 genomic sequences. Ndn and Magel2 are two evolutionary related retrotransposons, which belong to the MAGE D gene family [20]. Phylogenetic studies could not predict whether these two genes arose from two distinct retroposition events or whether they arose from the initial retroposition of one of these two genes followed by a cis-duplication event (Blanc M., unpublished observations). Their close genomic localisation, within 30 kb in the mouse genome, would however be in favour of this second hypothesis. The co-expression of Ndn and Magel2 could therefore result from regulatory elements sharing and/or duplication. Intraspecies genomic comparisons of mouse or human Ndn and Magel2 upstream sequences did not reveal any significant homology which does not exclude the possibility that one of these two genes arose by a cis- duplication. Our experimental search for regulatory elements elements in the Ndn/Magel2 genomic sequences was limited to sequences available in the databases and was therefore not exhaustive. However, we found several DNase I hypersensitive sites (HS1/2, HS3 and 4) linked to Ndn transcriptional activity and associated to the Ndn promoter. Previous transgenic studies in the zebrafish using a series of hybrid transgenes containing various mouse Ndn promoter sequences associated to the reporter LacZ gene suggested that the mouse Ndn promoter from -845 bp to +63 bp functioned in the zebrafish embryo in a temporal, spatial and tissue-specific manner [21]. In particular, a cis-acting element driving the neuronal-specific expression was located into an 87 bp sequence from -173 to -87 bp of the Ndn gene which exactly co-localizes with HS1/2. No identified transcriptional factor brain-specific binding site could however be identified in this sequence. Deletion of promoter sequences in Ndn-KO mutant mice [22] did not affect Magel2 transcriptional regulation (Watrin F., data not shown) which suggests that if the coordinated Magel2 and Ndn expression results from the sharing of a putative enhancer, this enhancer is not associated to Ndn promoter sequences.
The human NDN gene has been shown to be expressed in a larger panel of tissues than the mouse Ndn gene [23,24] but our data suggest that these two genes might have similar expression profiles in the nervous system [23] (data not shown). An interspecific (mouse/human) comparison of available genomic sequences was therefore performed in order to identify genomic sequences involved in central nervous system expression. This comparison did not allow the identification of such sequences but nevertheless brought some new information on the Magel2 transcriptional unit, showing the existence of a 2.5 kb highly conserved region upstream the described Magel2 gene. Identification of full length embryonic and brain specific Magel2 ESTs including this region further confirms that this region belong to the Magel2 transcriptional unit.
In view of our results, expression of the Ndn and Magel2 genes could be coordinated by a common enhancer distinct from Ndn promoter sequences and which could be localized in sequences upstream the Ndn gene that we could not analyze. It should be noted that the Ndn/Magel2 intergenic region was rather well conserved and harbored several regions of low DNase I hypersensitivity which might deserve further investigation.
The transgene is not imprinted
As expected, our transgene which was physically separated from the IC was not imprinted. When outside of its natural genomic context, Ndn was expressed from either parental allele. Whether particular genomic sequences around or associated to Ndn such as the 5' CpG island are necessary to respond to the primary imprinting signal established at the IC remains to be determined. An imprinted brain-specific Ndn non coding (nc) antisense RNA (PX00010K13; DDBJ accession no. AK14392) initiated in (or including) the 5' part of Ndn coding sequence and extending in Ndn upstream sequences has recently been described and according to the authors, this antisense transcript could be involved in the imprinted expression of Ndn mRNA [25]. This transcript could potentially be transcribed from our transgene. We did not detect this antisense nc transcript in transgenic brain RNA which is coherent with the fact that the transgene was not imprinted. However, we did not detect it in wild type brain either (data not shown). In one of the mouse models in which part of the Ndn CDS -and therefore the region in which the antisense transcript is initiated- was deleted [26], the LacZ gene which replaced the Ndn was monoallelically expressed, further strengthening an absence of role of this hypothetical antisense transcript in Ndn imprinting.
Conclusions
Our analysis strongly suggests the existence of a central nervous system-specific regulatory element which would coordinate expression of the PWS genes in the developing mouse embryo and we proposed that this element could be associated to the IC. However, our transgenic studies clearly demonstrate that when physically separated from the IC, a transgenic Ndn gene, although not imprinted anymore, can still be correctly expressed in the developing nervous system of the mouse embryo. The BAC transgene therefore contained the regulatory elements needed for the spatio-temporal expression of Ndn (and most likely Magel2) in the developing nervous system. Three hypotheses can be made: 1) the PWS genes co-expression that we observed is incidental, 2) the element(s) localised in genomic sequences surrounding the Ndn/Magel2 and which regulate(s) Ndn (and Magel2) expression could also regulate other PWS genes, 3) several central nervous system-specific regulatory elements resulting from duplication events might be present in the PWS domain. The evolutionary history of the PWS domain is more in favour of the third hypothesis but will need further investigations to be confirmed.
Methods
Mice
Adult C57BL/6 and C57BL/6 X CBA mice were purchased from IFFA CREDO, and M. musculus castaneus (CAST/Ei) male mice from the Jackson Laboratory. Mice carrying an Ndn null mutation on a 129/Sv genetic background [22] and transgenic Tg92 mice were bred in-house.
In situ hybridization
All in situ hybridization experiments for the study of PWS genes expression were performed on C57BL/6 embryos. In situ hybridization was performed on 14 μM paraformaldehyde-fixed cryosections with antisens digoxigenin-labeled riboprobes. Sections were washed in 1X PBS, treated with RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% Na deoxycholate, 0.1% SDS, 1 mM EDTA, 50 mM Tris pH 8), post-fixed in paraformaldehyde, acetylated and further washed in PBST before the hybridization step. All hybridization and post-hybridization washes were performed at 70°C. Hybridization was performed in 50% formamide, 5X SSC, 5X Denhart's solution (Sigma), 0.5 mg/ml herring sperm DNA, 0.25 mg/ml yeast RNA. Washes were performed in 50% formamide, 2X SSC, 0.1% Tween 20. Digoxigenin labelling was detected using anti-digoxigenin Fabs (Roche Biochemicals) coupled to alkaline phosphatase and NBT/BCIP (Sigma). The Ndn riboprobe (290 bp) hybridizes to the 3' UTR of the Ndn mRNA (nt 2130 to 2420; accession number D76440). The Magel2 riboprobe (318 bp) hybridizes to the 3' part of the Magel2 ORF (nt 3730 to 4048; accession number AK086725). The snoRNAs MBII-85 and MBII-52 riboprobes were synthesized from plasmids given by J. Cavaillé. The Snurf riboprobe (277 bp) hybridizes to U/Snurf-Snrpn transcripts and recognizes sequences from the 3' end of the Snurf exon 1 to the 3' end of the Snurf exon 3 (nt 3639 to nt 77687; accession number AF332579).
Generation and breeding of transgenic mice
BAC109 was isolated by hybridization of mouse bacterial artificial (BAC) high-density membranes (Research Genetics, Inc, USA) with a genomic Ndn probe, and contains a 103 kb NotI insert comprising the Ndn and Magel2 genes. The Ndn gene is localized in the center of BAC109, the EagI intragenic site being situated 54 and 50 kb away from the vector NotI sites and the 3' extremity of the Magel2 gene is localized 17 kb from one of the vector NotI site (Fig. 3). Unlinearized cesium chloride gradient purified BAC109 DNA was resuspended in injection buffer (10 mM Tris-HCl pH 7.5, 0.1 mM EDTA pH 8.0, 100 mM NaCl, 30 μM spermine, 70 μM spermidine) at 3 ng/μl and microinjected in the pronucleus of C57BL/6 X CBA fertilized eggs. Founders were identified by Southern blot analysis using a PCR probe amplified from the pBeLoBAC11 vector and a BglII digestion, and subsequently bred on a C57BL/6 genetic background. Transgene copy number and integrity were determined by PFGE and Southern blot analysis. The 5' and 3' probes used in the Southern blot analysis correspond to the probes 1 and 2 described in the "DNase I Hypersensitivity Mapping" section. Transgenic Ndn expression analysis (Northern blot and in situ hybridization) was performed on embryos or adult mice inheriting the transgene either paternally or maternally, on a Ndn null background. These embryos or adult mice were obtained by mating transgenic males carrying the Ndn null mutation to wild type C57BL/6 females (Tg92pat-Ndn+/-pat) or transgenic females to males carrying the Ndn null mutation (TgBAC92mat-Ndn+/-pat).
DNase I hypersensitivity mapping
Tissues were dissected either from inter-specific F1 hybrids between M. musculus C57BL/6 females and M. musculus castaneus males (B6 x CAST/Ei) F1 or from inbred C56BL/6 mice (B6). Nuclei were isolated from adult mouse tissues as described [27]. Nuclei were resuspended in DNase I digestion buffer (0.3 M sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 15 mM Tris-HCl pH 7.5, 1 mM DTT, 0.2 mM PMSF, 0.4 mM CaCl2, 5% glycerol) at a concentration of 5.107 nuclei/ml. Increasing amounts of DNase I (bovine pancreas grade I; Boehringer Mannheim) from 0 to 100 units diluted in 5 μl of DNase I digestion buffer were added to 95 μl aliquots of nuclei. After a 1 min incubation at 25°C, the digestion was stopped by addition 100 μl of stop solution (20 mM EDTA, 1% SDS, 0.1 mg/ml proteinase K) and samples were incubated overnight at 37°C. Genomic DNA was purified by multiple phenol/chloroform extractions and precipitated with ethanol. After resuspension, the DNA was digested with the appropriate restriction enzymes, separeted by gel electrophoresis, and transferred to Hybond N+ membranes. The membranes were hybridized by random hexamer- radiolabeled probes, in church solution (0.5 M NaPi pH 5.5, 7% SDS), at 65°C. Filters were washed three times in 0.2X SSC, 0.1% SDS at 65°C and exposed to X-ray film at -70°C. When necessary, blots were stripped by incubation in 0.1X SSC, O.1% SDS at 95°C. Most probes were prepared by PCR amplification of BAC109 sequences, using the following primers: Probe 1: S-5'-AGATCTGAAGACATAATG-3', AS-5'-GCTCTCCATTTCTAT TAGGTC-3'; Probe 2:S-5'-ATAAGTATTTGGTACTTTCAC-3', AS-5'-TGCTAAGTGCCTACACTGAG3'; Probe 3: S-5'-GAGCGAAACTATTCTGACAG3', AS-5'-AAGCTTCCTCCTCTATGGCAA-3'; Probe 4: S-5'-GATTTCTGCTAAGATTGG-3', AS-5'-ATGTTCCCTCTAGAAACC-3'; Probe 6: S-5'-AGTTAGAGACAAGCCTAG-3', AS-5'-TTCTGGGATGTCTCAGGA-3'; Probe 7: S-5'-GCATTTTGAGGAAGTACCCA-3', AS-5'-CATGGCCATTTCTAACTGTG-3'; Probe 8: S-5'-CCAAGGAGCTTGGAGGGC-3', AS-5'-CTCGTAGAGTGCGGCCAA-3'; probe 9: S-5'-ACATCAATAGTTTGATAC-3', AS-5'-GGGTGTGGCTGTGCATTGTT-3'
Authors' contributions
FW drafted the manuscript, designed the study, and carried out the PWS genes expression analysis, the transgenic analysis and the search for transcriptional regulatory elements. ELM contributed to the PWS gene expression analysis. NR carried out molecular studies on the transgenic lines and participated to the search for DNase I hypersensitive sites. LD was involved in the design of the transgenic studies. MR injected the transgene and established the transgenic lines. FM coordinated the project and participated to the manuscript draft.
Acknowledgements
This work was supported by grants from Prader-Willi France, the Anber foundation, the association pour la Recherche sur le Cancer (ARC) (grant #4329) and by the CNRS. We thank Marie-Geneviève Mattéi for the chromosomal location of transgenes, Mathieu Blanc and Pierre Pontarotti for their help in the analysis of the MAGE D genes phylogenetic relationships, Jérôme Cavaillé for the gift of the MBII-85 and -52 riboprobe vectors.
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| 15634360 | PMC545062 | CC BY | 2021-01-04 16:38:18 | no | BMC Genet. 2005 Jan 5; 6:1 | utf-8 | BMC Genet | 2,005 | 10.1186/1471-2156-6-1 | oa_comm |
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BMC Fam PractBMC Family Practice1471-2296BioMed Central London 1471-2296-6-31564413410.1186/1471-2296-6-3Research ArticleAccuracy of parents in measuring body temperature with a tympanic thermometer Robinson Joan L [email protected] Hsing [email protected] Donald W [email protected] Department of Pediatrics and Stollery Children's Hospital, 2C3 Walter MacKenzie Centre, 8440-112 St., Edmonton, Alberta, T6G 2B7 Canada2005 11 1 2005 6 3 3 10 8 2004 11 1 2005 Copyright © 2005 Robinson et al; licensee BioMed Central Ltd.2005Robinson 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 is now common for parents to measure tympanic temperatures in children. The objective of this study was to assess the diagnostic accuracy of these measurements.
Methods
Parents and then nurses measured the temperature of 60 children with a tympanic thermometer designed for home use (home thermometer). The reference standard was a temperature measured by a nurse with a model of tympanic thermometer commonly used in hospitals (hospital thermometer). A difference of ≥ 0.5 °C was considered clinically significant. A fever was defined as a temperature ≥ 38.5 °C.
Results
The mean absolute difference between the readings done by the parent and the nurse with the home thermometer was 0.44 ± 0.61 °C, and 33% of the readings differed by ≥ 0.5 °C. The mean absolute difference between the readings done by the parent with the home thermometer and the nurse with the hospital thermometer was 0.51 ± 0.63 °C, and 72 % of the readings differed by ≥ 0.5 °C. Using the home thermometer, parents detected fever with a sensitivity of 76% (95% CI 50–93%), a specificity of 95% (95% CI 84–99%), a positive predictive value of 87% (95% CI 60–98%), and a negative predictive value of 91% (95% CI 79–98 %). In comparing the readings the nurse obtained from the two different tympanic thermometers, the mean absolute difference was 0.24 ± 0.22 °C. Nurses detected fever with a sensitivity of 94% (95 % CI 71–100 %), a specificity of 88% (95% CI 75–96 %), a positive predictive value of 76% (95% CI 53–92%), and a negative predictive value of 97% (95%CI 87–100 %) using the home thermometer. The intraclass correlation coefficient for the three sets of readings was 0.80, and the consistency of readings was not affected by the body temperature.
Conclusions
The readings done by parents with a tympanic thermometer designed for home use differed a clinically significant amount from the reference standard (readings done by nurses with a model of tympanic thermometer commonly used in hospitals) the majority of the time, and parents failed to detect fever about one-quarter of the time. Tympanic readings reported by parents should be interpreted with great caution.
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Background
Parents and health care workers use temperatures measured by parents at home to determine if a child requires medical assessment. Health care workers sometimes make decisions about the need for investigations and hospital admission based on the temperature that parents report. This is especially true in the newborn or in the immunocompromised patient, where any temperature above the normal range can be indicative of bacterial infection.
Health care workers have used commercial tympanic thermometers in hospitals for over 15 years. These thermometers are quick and easy to use and minimize the risk of nosocomial infection. Tympanic thermometers are now available for home use at a cost of about $50 US. There are no published studies comparing measurements obtained with tympanic thermometers designed for home use to measurements obtained with other types of thermometers, or comparing readings obtained by parents with these instruments to those obtained by health care workers. The primary objective of this study was to determine the diagnostic accuracy of readings obtained by parents with a tympanic thermometer designed for home use.
Methods
The Health Research Ethics Board of the University of Alberta approved this study and parents and older children signed a consent form.
Reference standard
Traditionally, rectal temperatures have been used as the reference standard in pediatrics. However, readings done by a nurse using a model of tympanic thermometer commonly used in hospitals were chosen as the reference standard for this study as they are easier to obtain than are rectal temperatures and tend to be closer to PA temperatures when the patient is febrile [1] or when the temperature is changing [2,3], although the opposite may be true in the steady state [4].
Subjects
Parents of patients from the General Pediatric Clinic, the Emergency Department, or on the inpatient units of the Stollery Children's Hospital were eligible for the study. Parents of febrile or afebrile children aged 6 months to 16 years were approached on the days when the study nurse was available, but preference was given to patients who were being assessed because of fever. Parents were not approached if it would be inconvenient for them to participate in the study, or if they did not communicate in English, but there were no other exclusion criteria.
Study procedures
After informed consent was obtained from the parent (and the child when practical), the parent was given the instruction sheet from the Braun Thermoscan (Thermoscan Inc., San Diego, CA) tympanic thermometer (home thermometer) to read. They were then asked to measure their child's temperature when they felt they understood the instructions. They then recorded the temperatures they measured on a sheet of paper. Without looking at this sheet of paper, one of three trained research nurses then measured the child's temperature using the same thermometer. The nurse then also measured the child's temperature with CORECHECK (ALARIS Inc., San Diego, CA) tympanic thermometer (hospital thermometer). All readings were done in the same ear with a new probe cover.
Outcome measures
The mean absolute difference between the parents' reading and the reading by the nurse using the same thermometer was calculated. Readings were then analyzed separately for the febrile children. A temperature of ≥ 38.5 °C on the reading taken by the nurse using the hospital thermometer was defined as a fever. The percentage of times that the readings differed by 0.5 °C or more was calculated as this was considered a clinically significant difference.
Even if the parent and the nurse obtained similar readings from the home thermometer, it would still be possible that these readings are inaccurate, as there are no published studies demonstrating the accuracy of this thermometer. Therefore, the readings obtained by the nurse and the parent from the home thermometer were compared to those obtained by the nurse using the hospital thermometer (the reference standard for the study). If the readings obtained by the parent differed considerably from those obtained by the nurse, but the nurse then obtained very consistent readings with the two different thermometers, one could conclude that the parents were inaccurate because of human error. On the other hand, if the nurse obtained inconsistent readings with the two different thermometers, this would suggest that either instrument or human error could be the problem.
The measures of central tendency were determined, and the intraclass correlation coefficient determined for the three sets of readings. The three sets of measurements were also compared using the Bland-Altman method [5] where the differences between two measurements are plotted against the average of the measurements.
Sample size calculation
It was not possible to calculate a sample size, as the expected standard deviation between the three sets of readings could not be predicted. We estimated that a sample size of 60 children would be adequate.
Results
Characteristics of study subjects
Sixty parents were approached and all agreed to participate in the study. Their children were 35 males and 25 females aged 5 months to 15 years (median 3 years). Although the majority of patients were being assessed because of fever, only 17 children were febrile at the time of the study. All 3 measurements were obtained within 5 minutes for 55 of the 60 cases.
Main results
Table 1 shows the results of the study. The mean absolute difference between the temperature recorded by the parent and the temperature recorded by the nurse using the home thermometer was 0.44 ± 0.61 °C (Figure 1). The difference was similar in febrile and afebrile children. The higher reading was from the parent in 20 of the 60 cases. Parents detected fever with a sensitivity of 76% (95% CI 50–93%), a specificity of 95% (95% CI 84–99%), a positive predictive value of 87% (95% CI 60–98%), and a negative predictive value of 91% (95% CI 79–98 %) (Table 2).
Table 1 Comparison of results of temperatures measured by parents using a tympanic thermometer designed for home use, a nurse using the same thermometer, and the same nurse using a tympanic thermometer designed for hospital use
Mean absolute difference (°C) Range of absolute differences (°C) % of time where absolute difference ≥ 0.5 °C
Parent versus nurse using home tympanic thermometer
All children n = 60 0.44 ± 0.61 0.0 – 3.1 33%
Parent versus nurse using home tympanic thermometer
Afebrile children n = 43 0.44 ± 0.65 0.0–3.1 35%
Parent versus nurse using home tympanic thermometer
Febrile children n = 17 0.45 ± 0.51 0.10 – 1.9 29%
Nurse using home thermometer versus same nurse using hospital thermometer n = 60 0.24 ± 0.22 0.0 – 1.0 13%
Parent using home thermometer versus nurse using hospital thermometer N = 60 0.51± 0.63 0.0 – 3.4 72%
Figure 1 Comparison of readings done by a parent to readings done by a nurse using a home tympanic thermometer
The mean absolute difference between the temperatures measured by the nurse using the home thermometer versus the hospital thermometer was 0.24 ± 0.22 °C. The higher reading was from the hospital thermometer in 25 of the 60 cases. There was agreement between the readings taken by the nurse from the two thermometers with regard to the presence of fever, except in one case where one reading was 38.4 °C and the other was 38.6 °C (Table 2). Nurses detected fever with a sensitivity of 94 % (95 % CI 71–100 %), a specificity of 88 % (95% CI 75–96 %), a positive predictive value of 76% (95% CI 53–92 %), and a negative predictive value of 97% (95% CI 87–100 %) (Table 2). In comparing the readings obtained by the parent using the home thermometer to those obtained by the nurse using the hospital thermometer, the mean absolute difference was 0.51 ± 0.63 °C, with 72% of the readings differing by ≥ 0.5 °C.
Table 2 Ability of parents and nurses to detect fever using a home tympanic thermometer, with a hospital tympanic thermometer being the reference standard
Nurse reported fever on hospital thermometer Nurse reported no fever on hospital thermometer
Parent reported fever on home thermometer 13 2
Parent reported no fever on home thermometer 4* 41
Nurse reported fever on home thermometer 16 5
Nurse reported no fever on home thermometer 1** 38
*Readings by parents were 37.8 °C, 37.9 °C, and 38.4 °C twice with corresponding hospital thermometer readings being 38.6 °C, 38.5 °C, 38.6 °C, and 39.6 °C
**Reading was 38.4 °C on home thermometer and 38.6 °C on hospital thermometer
The intraclass correlation coefficient for the three sets of readings was 0.80. Figures 2, 3, and 4 show that the variation between the readings was consistent at different temperatures.
Figure 2 Scatter plot of the difference between temperature measured by a parent and that measured by a nurse using a thermometer designed for home use
Figure 3 Scatter plot of the difference between temperature measured by a parent using a thermometer designed for home use and a nurse using a thermometer designed for hospital use
Figure 4 Scatter plot of the difference between temperature measured by a nurse using a thermometer designed for home use and a a thermometer designed for hospital use
Discussion
It is now common for parents to report a child's body temperature as measured by a tympanic thermometer designed for home use. The only published study looking at the reliability of parents at measuring tympanic temperatures reported on the consistency of readings, rather than comparing the temperatures measured by parents to those measured by health care workers or by another method [6].
The current study showed that readings obtained by parents differ from those obtained by a nurse using the same instrument by a mean of 0.44°C, with 33% of the readings differing by a clinically significant amount (0.5°C or more). The absolute differences were similar for febrile and afebrile children. Using the reference standard of a tympanic temperature measured by a nurse with a model of thermometer commonly used in hospitals, the parents did not detect a fever in four of 13 cases (although in two of these cases, the reading by the parent was just 0.1 °C below our definition of fever). This suggests that the readings obtained by a parent with a tympanic thermometer are sometimes not reliable. In fact, previous studies showed that parents' subjective assessment of whether their child had a fever had a sensitivity of 81.8% [7] and 88.9% [8], which is similar to the 76 % sensitivity in the current study when parents used a tympanic thermometer.
In comparing the readings taken by a nurse with a home tympanic thermometer to a hospital tympanic thermometer, the mean absolute difference was 0.24 °C, with 13 % of the readings differing by a clinically significant amount. It is not clear if this degree of discrepancy is to be expected when a skilled operator takes two tympanic readings, or if it was a true difference between the two types of tympanic thermometer. The nurse would have identified all but one of the 17 children with fever as being febrile with either thermometer, and that child had a temperature of 38.4 °C on one thermometer and 38.6 °C on the other thermometer. Therefore, it appears that with a skilled operator, the tympanic thermometer designed for home use is reasonably reliable, and is likely to detect fever even if the actual reading is not always accurate.
The chief limitation of this study is that readings obtained by a nurse from the hospital tympanic thermometer must be reliable for the results of this study to be accurate. Some studies have concluded that tympanic thermometers do not give an accurate measurement of body temperature [7,9,10]. However, the reference standard in these studies (axillary or rectal temperature) may not be ideal. Studies have shown that when used by a skilled operator on a cooperative patient with rapidly changing body temperature, the readings from models of tympanic thermometers commonly used in hospitals more closely approximate pulmonary artery or esophageal temperature than do rectal or axillary temperatures [2,3]. Therefore, a tympanic temperature measured with a hospital tympanic thermometer was considered the best available reference standard for this study. However, if the nurses used incorrect technique, this would affect the results of the study. Falsely high readings with a properly calibrated tympanic thermometer should only occur if the thermometer itself has been stored above room temperature, so the fact that in 25 of the 60 cases the parent obtained a higher reading than did the nurse suggests the possibility that the nurses were not using ideal technique. Another limitation is that we could not blind the nurses for comparing their own two temperature readings. There is some evidence that falsely low readings can be obtained if tympanic readings are obtained in rapid succession as placement of the thermometer results in local cooling, so it is possible that readings would have been more comparable had we waited at least 2 minutes between readings [11]
It is possible that parents would have performed better had they leisurely read the instructions in their own home rather than doing so in a hospital-based setting, or had they had time to practice. However, it is also possible that they performed better than they would have in the home, as they knew evaluation of their performance was occurring. It is also possible that parents who would choose to buy a tympanic thermometer will differ from our study population. It is not clear what advice to give to parents about purchasing a thermometer. The ability of parents from the inner city to correctly use and read a mercury glass thermometer was poor in previous studies [12,13]. An electronic thermometer is easier to read and is now relatively inexpensive, but may not be as accurate as a mercury thermometer [14]. The results of the current study suggest that readings obtained by parents with a tympanic thermometer often differ by a clinically significant amount from readings obtained by nurse. Despite their ease of use, there are reports of parents inserting tympanic thermometers in the rectum [15]. It is not clear if parents could more accurately use an infrared temporal artery thermometer. A study showed that using the highest of three temporal artery temperature obtained by a parent or nurse, a reading of ≥ 37.8 °C was 97% sensitive and 84% specific for detecting a rectal temperature of ≥ 38.5 °C, and that the limits of agreement for temperatures obtained by parents versus nurses using this thermometer were -0.6 °C to 0.7 °C [16]. This is lower than the limits of agreement for the current study, but it is possible that the parents would have performed better had we recorded the highest of three readings.
Conclusions
With the recent outbreak of Severe Acute Respiratory Syndrome, rapid measurement of body temperature (sometimes by personnel who are not health care workers) has become an important tool to indicate if people can board airplanes and if staff can work. It is therefore important to determine the accuracy of non-health care workers at measuring body temperature. This study showed that the temperatures measured by parents with a tympanic thermometer often differ by a clinically significant amount from those measured by a nurse with a tympanic thermometer. There is not a consistent gradient or direction of the gradient between the two temperatures, which makes it difficult to interpret the temperatures reported by parents. There was a smaller gradient between the readings taken by a nurse with a tympanic thermometer designed for home use compared with those taken with a model of tympanic thermometer commonly used in hospitals, but there were still potentially clinically significant discrepancies 14% of the time. This makes it difficult to conclude if the poor performance of the parents relates entirely to human error, or if there is also an element of instrument error. The fact that in comparison with our reference standard (tympanic temperature measured by the nurse with a hospital thermometer) the readings taken with the home thermometer by the nurse correlated much better than those taken by the parent suggests that the parents did not use ideal technique. In any case, one should interpret temperatures taken by parents with a tympanic thermometer with great caution. Although parents will detect the majority of fevers with these instruments, the absolute numbers obtained may not be accurate. Again, we find that in pediatrics, the clinical picture is a more useful piece of information than are "the numbers"!
Abbreviations
°C – degrees Celsius
PA: pulmonary artery
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
JLR wrote the protocol and the manuscript. HJ helped with patient recruitment and reviewed the manuscript. DWS did the statistical analysis and reviewed the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgments
The authors would like to thank Ann Roth, Gwen Alton, and Kate Mackintosh, who collected the data.
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| 15644134 | PMC545063 | CC BY | 2021-01-04 16:29:12 | no | BMC Fam Pract. 2005 Jan 11; 6:3 | utf-8 | BMC Fam Pract | 2,005 | 10.1186/1471-2296-6-3 | oa_comm |
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BMC Infect DisBMC Infectious Diseases1471-2334BioMed Central London 1471-2334-4-591560147510.1186/1471-2334-4-59Research ArticleThird generation cephalosporin use in a tertiary hospital in Port of Spain, Trinidad: need for an antibiotic policy Pinto Pereira Lexley M [email protected] Marjorie [email protected] Hema [email protected] Karen [email protected] P [email protected] Department of Paraclinical Sciences, Faculty of Medical Sciences, The University of the West Indies, St Augustine, Trinidad2 Pharmacy, General Hospital, Port of Spain, Trinidad3 The Caribbean Epidemiology Center, Port of Spain, Trinidad2004 15 12 2004 4 59 59 9 8 2004 15 12 2004 Copyright © 2004 Pinto Pereira 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
Tertiary care hospitals are a potential source for development and spread of bacterial resistance being in the loop to receive outpatients and referrals from community nursing homes and hospitals. The liberal use of third-generation cephalosporins (3GCs) in these hospitals has been associated with the emergence of extended-spectrum beta- lactamases (ESBLs) presenting concerns for bacterial resistance in therapeutics. We studied the 3GC utilization in a tertiary care teaching hospital, in warded patients (medical, surgical, gynaecology, orthopedic) prescribed these drugs.
Methods
Clinical data of patients (≥ 13 years) admitted to the General Hospital, Port of Spain (POSGH) from January to June 2000, and who had received 3GCs based on the Pharmacy records were studied. The Sanford Antibiotic Guide 2000, was used to determine appropriateness of therapy. The agency which procures drugs for the Ministry of Health supplied the cost of drugs.
Results
The prevalence rate of use of 3GCs was 9.5 per 1000 admissions and was higher in surgical and gynecological admissions (21/1000) compared with medical and orthopedic (8 /1000) services (p < 0.05). Ceftriaxone was the most frequently used 3GC. Sixty-nine (36%) patients without clinical evidence of infection received 3Gcs and prescribing was based on therapeutic recommendations in 4% of patients. At least 62% of all prescriptions were inappropriate with significant associations for patients from gynaecology (p < 0.003), empirical prescribing (p < 0.48), patients with undetermined infection sites (p < 0.007), and for single drug use compared with multiple antibiotics (p < 0.001). Treatment was twice as costly when prescribing was inappropriate
Conclusions
There is extensive inappropriate 3GC utilization in tertiary care in Trinidad. We recommend hospital laboratories undertake continuous surveillance of antibiotic resistance patterns so that appropriate changes in prescribing guidelines can be developed and implemented. Though guidelines for rational antibiotic use were developed they have not been re-visited or encouraged, suggesting urgent antibiotic review of the hospital formulary and instituting an infection control team. Monitoring antibiotic use with microbiology laboratory support can promote rational drug utilization, cut costs, halt inappropriate 3GC prescribing, and delay the emergence of resistant organisms. An ongoing antibiotic peer audit is suggested.
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Background
Appropriate use of antibiotics is central to limiting the development and the spread of resistant bacteria in hospitals and communities. Use of broad-spectrum antibiotics, in particular the 3GCs in nosocomial infections have been linked to the emergence of antibiotic resistance and increase in costs [1]. The hospital setting is particularly conducive to the development of antibiotic resistance as patients who are severely ill, immuno-compromised or have devices and/or implants in them are likely to receive frequent courses of empirical or prophylactic antibiotic therapy [2]. Furthermore, the absences of guidelines for antibiotic use, protocols for rational therapeutics and infection control committees have led to overuse and misuse of antimicrobials in different specialized units in hospitals.
The increasing resistance to 3GCs accompanied by an increasing cost burden has raised concerns about the detection, prevalence, and clinical implications of infections with Escherichia coli and Klebsiella spp. An important source of this resistance results from the production of extended-spectrum beta-lactamases (ESBLs) by bacteria. ESBLs are modified beta-lactamase enzymes mainly derived from the ubiquitous TEM1/2 and SHV-1 plasmid-mediated enzymes, which hydrolyse expanded spectrum cephalosporins to varying degrees. Many beta-lactamases result in resistance to 3GCs in Enterobacteriaceae. Genera such as Enterobacter, Citrobacter and Serratia posses chromosomal broad spectrum beta-lactamases which are normally repressed, and when induced result in resistance to 3GCs. Klebsiella and E. coli usually have the SHV- or Tem- type beta-lactamases, and key mutations in these result in true "ESBLs". ESBLs have received attention in the last decade because although penicillins, cephalosporins, or aztreonam appear to be susceptible in vitro, ESBL producing E. coli or Klebsiella spp. may demonstrate clinical resistance to these antibiotics leading to treatment failures. Liberal use of the 3GC antibiotics has resulted in the ESBLs conferring resistance among Enterobacter[3] and Enterobacteriacae worldwide [4-6] compromising their clinical use. Prior antibiotic use is an important risk factor for colonization and bacterial infection and though generally antibiotic use cannot always be correlated with emergent antibiotic resistance, studies have reported the association of resistant K pneumoniae and other Enterobacteriaceae and vancomycin-resistant enteroccocci with cephalosporin use [7-11]. Recent increases in multidrug resistant gram-negative bacilli, particularly ESBLs is of great concern. The association between emergent ESBL-mediated infections and 3GC use emphasizes the importance of better describing 3GC drug utilization to best optimize their use. Few data are available in this regard from developing countries. In the Caribbean 3GC resistance amongst the Enterobacteriaceae has been reported from Barbados [12], and extended spectrum beta-lactamase producing Enterobacteriaceae has recently been observed in tertiary care in Jamaica [13] and Trinidad [14]. From unpublished reports of drug procurements for the public sector, the third generation cephalosporins are widely used at the General Hospital Port of Spain, which is the largest tertiary health care institution in the country. In order to assess the appropriateness of prescribing 3GCs and to determine the direct cost of treatment, an audit of prescriptions of these agents was undertaken at the POSGH, between January to June 2000.
Methods
Setting
In Trinidad and Tobago, health care is delivered through the public sector, though several private facilities are also available. At the publicly-financed health care institutes patients undergo investigations and receive treatment without cost. There are 2 tertiary general hospitals in Trinidad, the General Hospitals at Port of Spain and San Fernando and several secondary and primary health care facilities where patients can access medical care. The POSGH is the country's major health care institute and receives referrals from all over the country including the sister isle of Tobago. This hospital is a 900-bed institute providing out-patient services and health care for warded patients for over one third of Trinidad and Tobago's population of 1.3 million. Medical students undergoing training at the University of the West Indies, also attend clinical rotations at this hospital.
Patients
Between January to June 2000, we conducted a cross sectional study of adult in- patients (over 13 years) who had received one or more courses of treatment with one of the 3GCs (cefotaxime, ceftriaxone, ceftazidime) available at the POSGH. The Hospital Pharmacy identified warded patients from the respective services based on the prescriptions dispensed. Patient characteristics, clinical data and laboratory investigations were obtained from the hospital records. While we did consider the course of antibiotic duration in defining appropriateness of therapy, we did not analyse data based on use per patient days because these data were not clearly available from the records. Specific data on the category of service, concomitant disease and drug therapy, organ system with infection, invasive/indwelling devices and the 3GC used were collected using a standardized instrument.
Case definition of infection
An infection was deemed to be present when the physician's diagnosis of infection was a differential diagnosis stated in the chart and/or in a patient who had a fever (>100.4 C) and elevated WBC Count >12,000/cmm.
On review all patients with diabetes and HIV had a clinical diagnosis of infection stated in the records.
Criteria for appropriate prescription
The appropriateness of antibiotic therapy was determined using the criteria described in the Sanford Antibiotic Guide, 2000. Therapy was deemed to be inappropriate based on any of the following parameters: the type of therapy (prophylactic and empiric), combination of antibiotics, the route of administration, the dose, and the duration of therapy. The Sanford guide is widely accepted in Trinidad and the Caribbean as a reference for making appropriate therapeutic recommendations and is also used as a reference manual by medical microbiologists. No other guidelines are currently available in Trinidad. The course of therapy was considered a parameter of appropriate therapy, but a separate analysis based on this parameter was not possible since the information was not always available from the patient records.
Cost of antibiotic treatment
The direct cost of total antibiotic treatment was computed using the data obtained from the National Insurance Property Development Corporation, which is the agency contracted by the Ministry of Health for procurement and distribution of drugs in the public sector health institutes.
Statistical analysis
Data were analysed using EPI Info 6.4 (CDC, Atlanta GA) and categorical variables were compared using the Odds Ratio (95% CI) and the chi-square (X2) tests.
Results
One hundred and ninety two (192) adult patients admitted to the POSGH were treated with 3GCs during the first six months of 2000, providing a prevalence rate of use of 9.5 patients per 1000 admissions, (192/20146). The rate of use was higher (p < 0.05) in those patients utilizing the surgical and gynecological services (21 per 1000 admissions) compared with those admitted in the medical and orthopedic services (8 per 1000 admissions). One hundred and twenty seven (66%) patients received ceftriaxone, cefotaxime was prescribed for 51 (26.5%) patients, and ceftazdime was administered to 14 (7.5%) patients.
The demographic characteristics of patients who received a 3GC and factors related to infection and treatment in the study population are shown in Table 1. Fifty eight percent (58%) of patients were females. The mean age of patients was 45 ± 20.56 (SD), years. The use of the third generation cephalosporins was highest in patients utilizing the surgical facilities (44%), followed by patients in the medicine (27.5%), gynecology (15%), and orthopedic (12.5%) divisions. Ceftriaxone was the most widely used agent particularly in surgical patients (61, 48%) compared with those admitted to the medicine 24 (19%), gynecology 17(13%)and orthopedic wards 13(10%). In at least one third of patients (63, 32.8%) factors, which could predispose to infection, were identified, diabetes mellitus and HIV/AIDS were diagnosed in 24.5% and 4.7% of patients respectively.
There were 107(56%) patients who had fever and a high WBC count with leucocytosis and who were considered to have a clinically suspected infection. Thirty six percent of patients who had no clinical evidence of infection had received treatment with a 3GC. The most common site of suspected infection (20.3%) was the skin and soft tissue followed by the respiratory (10.9 %), gastrointestinal (10.7%), and urinary (9.3%) tracts.
Sixty eight percent (131) of patients in this study received empirical antibiotic treatment and in 29.7% of patients these antibiotics were prescribed as prophylactic therapy. Only 4% of patients received 3GCs based on recommended therapeutic regimens. More patients (87%) who were admitted to the medical services received empirical antibiotic treatment compared with patients in the surgical services (68%), (p < 0.05). Prophylactic antibiotic regimens were most frequently prescribed in the orthopedic (50%) and gynecological services (45%) in contrast to patients in surgery (31%) who received 3GCs as prophylactic therapy. However patients in the surgical units (61%) were more likely to be administered two or more antibiotics compared with those receiving medical care in the orthopedic (33%) or gynecological (45%) services (p < 0.01). Single antibiotic therapy was prevalent in the medical, orthopedic, and gynecological services (66%, 55%, and 55% respectively). Seventy-eight percent of those patients who did not display any clinical evidence of infection were treated with one antibiotic. Patients with infections of the skin and soft tissue (75%) and the urinary tract (61%) were more likely to receive treatment with two or more antibiotics compared with patients admitted with respiratory tract infections (48%) (p < 0.05).
Biological samples were not sent to the laboratory in as many as 73% of patients to determine a bacteriological etiology of suspected infection. (Table 2). Of the 52 (27%) patients who were submitted to laboratory investigation, a bacteriological etiology could be established in 25 (48 %) patients. The organisms which were isolated were; Pseudomonas spp (7), Acinetobacter spp (3), Enterobacter spp (2), Klebsiella spp (2), Proteus (2) S. aureus (3), and others (8).
An analysis of factors that were associated with the inappropriate use of 3GCs in the patient sample is shown in Table 3. There was an independent association between the type of service and inappropriate use of third generation cephalosporins and the odds of inappropriate therapy with these agents was six times more for patients in the gynecology services (odds ratio 6.58, p < 0.003) compared with patients utilizing the orthopedics (odds ratio, 2.47) and medical (odds ratio 1.80)hospital services More patients received inappropriate antibiotic treatment when the site of infection was not determined (odds ratio 5.05) compared with those in whom the site of infection was located to the skin and soft tissue infection (odds ratio 3.10), and the respiratory tract (odds ratio 2.74). The 3GCs were significantly associated with inappropriate use (odds ratio. 3.50 CI 1.73–7.11) when used as single therapy than when used with multiple antibiotics.
The average duration of patient stay in the hospital was 14 ± 22 days (range 1 day -150 days). Patients in the orthopedic wards were hospitalized for more days (24.5 ± SD 35.28) compared with the number of days patients spent in the medical (12.45), surgical (13.7) and gynecology (7 days) services. Interestingly, the mean duration of stay of patients in whom antibiotic treatment was appropriate, was not significantly different from the number of days that patients who were treated inappropriately were warded. The overall direct costs from use of these antibiotics in this study was TT$ 117,432 (US$ 19251.00). The cost of treatment with these antibiotics in patients who were inappropriately treated was (TT $79,487.00, US$ 13, 30.00) and was twice as high as the cost of treatment for those patients who were treated appropriately (TT$ 35, 138.00, US$ 5767)
Discussion
The extensive use of third generation cephalosporin antibiotics has caused the emergence of extended spectrum beta-lactamases in Gram-negative bacteria worldwide [3-6]. More third generation cephalosporins are being widely used in hospitals for empirical and prophylactic therapy, and as their use extends across the board, more organisms will develop resistance to them presenting the threat of antimicrobial ineffectiveness in life threatening infections. Several investigators have developed and evaluated cost effective programs and adherence to hospital antibiotic guidelines to control antibiotic abuse [15-17]. The active promotion of guidelines increased appropriate prescribing of 3GCs from 21–52% over three years [18]. Though an Infection Control Committee at the POSGH had developed an antibiotic policy earlier for rational use of antimicrobial agents, the Committee did not formally approve the guidelines, they were not disseminated to all physicians and several were unaware of their formulation. A systematic review and evaluation of the guidelines was never conducted despite increasing antimicrobial resistance to third generation cephalosporins in this hospital [14]. It was therefore believed to be timely to undertake a pharmacy audit of use and cost of third generation cephalosporins in this teaching hospital.
In our study, the prevalence rate of use of 3GCs was lower than the reported rate of 43 per 1000 admissions for ceftriaxone in 51 Victorian Hospitals in Australia of which 82% was prescribed as empirical treatment [19]. Our data reveals extensive overall inappropriate use of these drugs (62%) at the POSGH in Trinidad, with a rate that is higher than an earlier literature report (31%) [20]. Inappropriate use of the cephalosporins was seven times more common in the gynecological services compared with other services (odds ratio 6.58 P < 0.003) in this hospital which provides clinical clerkship teaching for graduating medical students from the University of the West Indies. Inappropriate antibiotic use has been reported from teaching hospitals in Aberdeen with significant empirical overuse [21] in New York [22] in the surgical practice [74%], in China [23] where inappropriate 3GC use was an independent risk factor for significant high mortality, in Malaysia [24] for patients in the medical wards (22–65%), in South Africa [25] for patients in the gynaecology ward (54%), and in Thailand (91%) for all departments [26]. There were twice as many patients who received inappropriate prophylactic or empirical therapy with the 3GCs compared with those who received appropriate treatment. We found higher inappropriate use of cefotaxime and ceftriaxone when these agents were used as single agents rather than in combination therapy with other antimicrobial agents. We believe this practice may have followed from the convenience of a single daily dose by intramuscular injection particularly for ceftriaxone, which was the most frequently used 3GC.
Third generation cephalosporins were introduced in the Caribbean between the late eighties and the early nineties and soon after in 1993, the first isolation of resistant Gram-negative bacilli resistant to them was demonstrated in Barbados [12]. In Jamaica, K. pneumoniae isolates confirmed to be ESBL producers have recently been reported in the University Hospital of the West Indies [13]. The 3GCs were introduced to the POSGH formulary in 1990 and their use increased by 48% and 22% from 1995 to1998 and 1998 to 2001 respectively. Cefotaxime was then, the most widely used member of the class and the only member to be available in the public sector health institutes. Resistance to cefotaxime and ceftriaxone in Trinidad, has been demonstrated for isolates of enterobacter (35%,15%), proteus (28%, 8%), acinetobacter (75%, 61%), providencia (75%, 50%) and klebsiella (6%,5%) for all specimens between January to June 2001 at the Port of Spain General Hospital (unpublished data), but blood culture isolates such as klebsiella (25%) and pseudomonas (90%) were resistant to cefotaxime and/or ceftriaxone. The resistance rate of Enterobacter spp to cefotaxime and ceftriaxone was 53% and 37% respectively in the Intensive Care Unit at this hospital. It is interesting that only 4% of prescriptions were based on therapeutic regimen following culture and sensitivity results in our study, and in 73% of patients with suspected infection bacteriological confirmatory tests were never done and 3GCs were prescribed. We believe high inappropriate use of these antibiotics has contributed to increasing multiple antibiotic resistance and extended spectrum beta-lactamase producing enterobacteriaceae observed in this hospital [14].
The cost of inappropriate antibiotic use was twice as much for patients who were treated appropriately and highlights irrational antibiotic consumption at the hospital, prompting protocols for rational antibiotic prescribing and utilization review. The World Health Organization has recommended multifaceted strategies to improve hospital-prescribing practices such as the development of consensus guidelines, educational activities and rapid feedback to prescribers about inappropriate use to reduce overuse of antibiotics in the hospital [27]. Restriction of cephalosporin use has demonstrated significant cost savings and improved antibiotic susceptibility with reduced infection-related hospital mortality in critically ill patients [28]. Restructuring the formulary to rationalize antimicrobial use with restricted use of third generation cephalosporins would impact positively in curtailing antibiotic resistance and decrease selective pressure from the overuse of these agents. Moreover the spiraling costs of unwanted antibiotic therapy will be limited with resultant benefits for the consumer and the health care provider in the public health sector. The study highlights the need for an antibiotic audit and invites an ongoing peer audit.
Conclusions
This first audit on 3GC use from the Caribbean demonstrates inappropriate use of these drugs in tertiary care and presents opportunities to develop consensus guidelines for rational use of these drugs in hospitals. We recommend microbiological services in the hospitals undertake continuous surveillance of resistance patterns, to guide in the development of prescribing guidelines. Such guidelines should be widely disseminated and implemented in these institutions, so that prescribers remain informed of rational therapeutics. This study highlights the need to constitute an antibiotic monitoring team comprising a pharmacist, physician, medical microbiologist and infection control nurse to periodically review and evaluate the use and cost of antibiotics at the major tertiary care hospital in Trinidad, to assist clinicians in optimizing clinical care of patients with Gram-negative infections.
List of abbreviations
3GCs Third generation cephaosporins
ESBLs Extended spectrum Beta lactamses
POSGH General Hospital, Port of Spain
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
LMPP conceived the study, the design, co-ordinated it and prepared the final manuscript. MP, HR and KT did the data collection. PP assisted with the co-ordination, did the analysis and prepared the draft manuscript. All authors saw the final manuscript and made contributions.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
Mr Nicholas George, Senior Pharmacist/Manager, NIPDEC Central Stores, Trinidad provided information on drug costs and use in the public sector.
Figures and Tables
Table 1 Characteristics of patients treated with 3rd generation cephalosporins at the POSGH
Characteristics No (%)
Gender
Male 84 (42)
Female 111(58)
Age: Mean SD (Range) 45 ± 20.56(13–90 years)
Category of service
Surgery 84(43.8)
Medicine 53(27.6)
Gynaecology 29(15.1)
Orthopaedics 24(12.5)
Others (CNS, Genital, Septicaemia) 2
Predisposing Factors
None 129(67.2)
Diabetes Mellitus 47(24.5)
HIV/AIDS 9 (4.7)
Infection characteristics
Clinically suspected 108(56)
Clinically absent 84(44)
Site Unknown 69(35.9)
Skin and Soft tissue 39(20.3)
Respiratory tract 21(10.9)
Gastrointestinal tract 20(10.7)
Urinary tract 18(9.3)
Others 25(13.0)
Antibiotic Use
Prophylactic 57 (29.7)
Empiric 131(68.2)
Therapeutic 4(2.0)
Single drug 94(49.6)
Two or more drugs 94(49.6)
Appropriate 62(32.3)
Inappropriate 124(64.6) 6(3.1)
Undetermined
Bacteriological Investigations
Done 52 (27)
Not done 140 (73)
Table 2 Microbiology investigations in patients (192) prescribed third generation cephalosporins
Type of Service No. Investigations Not done (%) Investigations Done (%) Positives (n = 52) (%)
Medicine 53 30 (55.7) 23 (44.3) 9 (39.0)
Surgery 84 69 (81.1) 15 (18.9) 10(66.6)
Gynecology 29 23 (79.3) 6 (20.7) 2 (33.3)
Orthopedics 24 16 (66.6) 8 (33.4) 3 (37.5)
Others 2 2
Total 192 140 (73) 52 (27) 25(48.0)
Table 3 Factors associated with appropriate 3rd generation cephalosporin use at the POSGH, Jan- June 2000.
Variables Appropriate Use (n = 62) Inappropriate Use (n = 124) Odds ratio (95% CI) p Value
Gender
Male 25 37
Female 53 71 0.91 (0.46–1.76) 0.88
Age (mean ± SD) 42 (18.73) 46.61(21.63) NS
Service
Gynecology 3 24 6.58 (1.69–29.89) p < 0.003
Orthopedics 6 18 2.47 (0.081–7.00)
Medicine 16 35 1.80 (0.81–4.01)
Surgery 37 45 1.00
Infection
Present 44 60 2.61 (1.30–8.28) p < 0.003
Absent 18 64
Type of infection
Site unknown 15 53 5.05 (1.45–18.10) p < 0.007
Skin and soft tissue 14 24 4.29 (0.94–20.71
Respiratory 6 13 3.13(.0.88–11.35)
Urinary tract 10 7 1.00
Therapy
Prophylactic 17 39
Empiric 45 81 1.27 (0.62–2.65) p < 0.48
Multiple therapy
Single antibiotic 18 73
2 or more antibiotics 44 51 3.50(1.73–7.11) p < 0.001
==== Refs
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Sanders CC Sanders WE beta-lactam resistance in Gram negative bacteria: global trends and clinical impact Clin Infect Dis 1992 15 824 839 1445981
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Goossens H MYSTIC (Meropenam Yearly Susceptibility Test Information Collection) results from Europe: comparison of antibiotic susceptibilities between countries and centre types. MYSTIC Study Group (European centres only) J Antimicrob Chemotherapy 2000 46 39 52 10.1093/jac/46.suppl_2.39
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Kim BN Lee SO Choi SH Kim NJ Woo JH Ryu J Kim YS Outcome of antibiotic therapy for third-generation cephalosporin-resistant Gram-negative bacteraemia an analysis f 249 cases caused by Citrobacter, Enterobacter and Serratia species Int J Antimicrob Agents 2003 22 106 111 12927949 10.1016/S0924-8579(03)00094-3
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| 15601475 | PMC545064 | CC BY | 2021-01-04 16:03:30 | no | BMC Infect Dis. 2004 Dec 15; 4:59 | utf-8 | BMC Infect Dis | 2,004 | 10.1186/1471-2334-4-59 | oa_comm |
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BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-4-971561757610.1186/1471-2407-4-97Research ArticlePlasma prolactin in patients with colorectal cancer Soroush Ahmad Reza [email protected] Hosein Mahmood [email protected] Mehrnush [email protected] Behnam [email protected] Sara [email protected] Department of surgery, Tehran University of Medical Sciences, Shariati hospital, kargar shomali st, Tehran 14114, Iran2 Research Development Centre, Tehran University of Medical Sciences, Shariati hospital, kargar shomali st, Tehran 14114, Iran3 Students' Scientific Research Centre (SSRC), Tehran University of Medical Sciences, Tehran, Iran2004 23 12 2004 4 97 97 23 6 2004 23 12 2004 Copyright © 2004 Soroush 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
Colorectal cancer is a common malignancy of the gastrointestinal tract. It is the second cancer cause of death in females and third in males. Production of prolactin has been reported with several tumours. However, elevated prolactin plasma levels in colorectal cancer patients remained unclear.
Methods
In this cross sectional study serum prolactin and carcinoembryonic antigen (CEA) concentrations were assayed using immunoradiometric assay kits, preoperatively in 47 patients, and the results were compared with 51 age and sex matched controls.
Results
Prolactin and CEA concentration in patients were significantly more as compared with controls. Hyperprolactinemia was found in 36 (76.6%) patients, while 28 (59.6%) had high level of CEA.
Conclusions
Prolactin may be a better tumour marker than CEA in patients with colorectal malignancy.
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Background
Colorectal cancer is the third highest cause of cancer mortality [1]. In Australia, the United Kingdom and the United States, it is the second common cancer for women after breast cancer (age-standardised incidence 22–33 per 100,000), and men after prostate or lung cancer (age-standardised incidence 31–47 per 100,000). About 148,300 new cases are reported each year in the USA and 56,600 Americans die annually from this cause [2]. Prognostic factors are very important for the evaluation, judgment and optimal treatment of patients with cancer.
Carcinoembryonic antigen (CEA) has been recognized as a serum marker for colorectal cancer in the past three decades [3] and has an important role in the management of colorectal cancer [4].
Prolactin (PRL) is a hormone with multiple biological actions, synthesized by the anterior pituitary gland [5] and is best known for its roles in the mammary gland. However, it is now revealed that PRL is able to exert its effects on additional cells and tissues (decidual cells of the placenta, bone, brain, lymphocytes and breast epithelial cells) [6,7]. PRL is secreted not only by lactotrophic cells of the pituitary gland but also by a variety of other normal tissues and human tumours [8] including malignant tumours of the lung[9], kidney[10], uterine[11], ovary [12], and breast[13]. Increase of PRL in colorectal cancer is unclear. Bhatavdekar, et al reported a significantly higher preoperative prolactin levels in patients with colorectal carcinoma [14], While, Indinnimeo et al and Carlson et al could not confirm hyperprolactinemia in patients with colorectal cancer. [15,16] These conflicting results do not support the hypothesis of increasing level of PRL in colorectal carcinoma.
The aim of this study was to see whether PRL levels are increased in colorectal cancer and to compare the preoperative serum levels of PRL and CEA concentrations in colorectal cancer patients; also we intended to find the possible correlation between hyperprolactinemia and the stage of colorectal cancer.
Methods
This cross-sectional study included 47 patients with colorectal cancer (18 women, 29 men; mean age 55.4 years, range 32–81) and 51 non-cancer patients (21 women, 30 men; mean age 58.5 years, range 20–81), who were candidates of colectomy. All patients were admitted in Shariati general hospital affiliated to Tehran University of Medical Sciences (TUMS) from April 2001 to January 2003. All women in the study were postmenopausal. None of the patients were on any drugs known to increase plasma prolactin levels in the last 6 months prior to the study and were not affected by endocrine, renal, and psychiatric disorders. There was no history of rectal bleeding and family history of colorectal cancer. Peripheral venous blood samples were daily collected from seven days before the operation to measure serum PRL and CEA levels. Blood samples were collected between 07:30 a.m. and 09:30 a.m. to avoid diurnal variation of PRL. Serum prolactin levels were measured with immunoradiometric assay (IRMA) kits produced by Kavoshian co (Tehran-Iran). Serum CEA levels were assayed by IRMA, who used kits from IMMUNOTECH co (Marseille-France). The PRL and CEA assays were carried out within 10 days of sampling. The cut off levels for PRL and CEA were 20 ng/ml and 5 ng/ml serum respectively [17,18]. Stages of cancer were determined by pathology assessment of neoplastic specimens.
The study was approved by the Vice-Chancellor of ResearchEthic Committee of TUMS.
The data evaluated by Chi-square test and T test using the Statistical Package of Social Science (SPSS Inc., Chicago, IL) for Windows version 11.5. A P-value of <0.05 was considered statistically significant.
Results
The number of male and female participants was 29 and 18 in case and 30 and 21 in control group.
We compared serum PRL and CEA level of 47 patients with 51 healthy controls. There was no significant difference between the age and the sex distribution of the patients and control groups.
Mean PRL serum level in colorectal cancer group was 21.6 ± 8.1versus 10.5 ± 4.6 in control group, which was significantly different (p < 0.0001). In other words 36 patients showed hyperprolctinemia (76.7%) while there was only one such patient in the control group (1.9%).
CEA level in 59.6% of the cancer group was above the normal compared with 1.9% of controls (P < 0.0001).
Out of 47 patients in the cancer group 9 were in stage I (19.1%), 19 in stage II (40.4%), 10 in stage III (21.3 %) and 9 in stage IV (19.1%). Hyperprolactinemia and high CEA concentrations were found in all stages (figure 1).
Discussion
Prognostic factors are crucial for the evaluation and optimal treatment of patients with cancer [17]. Therefore tumour markers have a greater role in the assessment of therapeutic response. Measurements of serum tumour markers may be helpful in detecting the metastatic process while still in the sub clinical phase [15]. The ectopic secretion of hormones like PRL by non-endocrine neoplasms is well recognized and used as therapeutic monitoring [15].
The main source of PRL is the pituitary gland, but in recent years, some studies have reported hyperprolactinemia in patients with breast [13], lung [9], prostate and ovary tumours [12].
Elevated circulatory levels of this hormone have also been detected in colorectal cancer. Bhatavdekar et al reported a high preoperative serum concentration of PRL in patients with colorectal cancer [14]. Ilan et al. reported elevated levels of PRL in 53% of the patients with colorectal malignancy [19]. Baert et al, in contrast didn't determine preoperative hyperprolactinemia in a series of 32 patients and their study didn't support the hypothesis of ectopic prolactin production by colorectal cancer [20]. Indinnimeo, et al found no hyperprolactinemia and prolactin positive immunostaining in colorectal cancer [15]. Previous studies, that reported normal levels of PRL [20,15], have suggested such factors as renal, endocrine and psychiatric disorders, medications and premenopausal situation may be the cause of hyperprolactinemia in patients with colorectal neoplasms. In the present study, patients with above factors were excluded and our results confirmed preoperative hyperprolactinemia in colorectal cancer. We found no correlation between the plasma PRL concentration and the stage of the tumour.
CEA present in blood during fetal life, falls to very low levels in most adults, and circulates in high concentrations in patients with some cancers [21]. CEA is over expressed in most colorectal cancers and is an important tumour marker in the management of colorectal cancer. A preoperative high CEA value suggests advanced disease either locally or with a distant metastasis. The preoperative serum CEA level can be a useful predicting factor regarding the outcome of the surgical operation [4]. In the present study we found that serum PRL and CEA levels are increased in patients with colorectal cancer but the greater portion of the patients had an increased level of PRL compared with elevated level of CEA. Considering the fact that laboratory cost for detecting CEA is higher than PRL, We suggest PRL as a valuable tumour marker in colorectal cancer. There are studies that support the tumour marker value of PRL [18,22] and studies that do not [16,20].
Conclusions
This study demonstrates that PRL, a pituitary gland hormone, is elevated in colorectal cancer. Moreover, our results show that PRL may be a better tumour marker than CEA in patients with colorectal malignancy. However, further prospective studies are warranted to better understand the evaluation of PRL as a prognostic factor, and to follow up patients after operation to determine the possible correlation between PRL levels, survival and response to therapy.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
Dr Ahmad Reza Soroush and Dr Hosein Mahmood zadeh participated in the design of study, acquisition of data and interpretation of data. Dr Mehrnush Moemeni participated in the design of study, acquisition of data, interpretation of data, and drafting the article. Behnam Shakiba participated in acquisition of data, interpretation of data, drafting the article and final approval of this version. Sara Elmi participated in analysis and interpretation of data, drafting the article and final approval of this version. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgments
This study was funded by the Research and Development Centre of Shariati Hospital, Endocrinology and Metabolism Research Centre and Surgery Research Centre of Tehran University of Medical Sciences. The authors wish to thank Dr Mohammad Reza Mohebbi, Dr Mahmoud Ghazi-Khansasi and Mr. Shahram Sharabyani who kindly edited the manuscript.
Figures and Tables
Figure 1 Hyperprolactinemia and high CEA levels in stages of colorectal cancer.
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| 15617576 | PMC545065 | CC BY | 2021-01-04 16:03:02 | no | BMC Cancer. 2004 Dec 23; 4:97 | utf-8 | BMC Cancer | 2,004 | 10.1186/1471-2407-4-97 | oa_comm |
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BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-5-41564413310.1186/1471-2407-5-4Research ArticleMycophenolate mofetil modulates adhesion receptors of the beta1 integrin family on tumor cells: impact on tumor recurrence and malignancy Engl Tobias [email protected]ć Jasmina [email protected] Borna [email protected] Iyad [email protected]üller Iris [email protected] Wolf-Dietrich [email protected] Dietger [email protected] Roman A [email protected] Zentrum der Chirurgie, Klinik für Urologie und Kinderurologie; Johann Wolfgang Goethe-Universität; Frankfurt am Main, Germany2005 11 1 2005 5 4 4 6 8 2004 11 1 2005 Copyright © 2005 Engl 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
Tumor development remains one of the major obstacles following organ transplantation. Immunosuppressive drugs such as cyclosporine and tacrolimus directly contribute to enhanced malignancy, whereas the influence of the novel compound mycophenolate mofetil (MMF) on tumor cell dissemination has not been explored. We therefore investigated the adhesion capacity of colon, pancreas, prostate and kidney carcinoma cell lines to endothelium, as well as their beta1 integrin expression profile before and after MMF treatment.
Methods
Tumor cell adhesion to endothelial cell monolayers was evaluated in the presence of 0.1 and 1 μM MMF and compared to unstimulated controls. beta1 integrin analysis included alpha1beta1 (CD49a), alpha2beta1 (CD49b), alpha3beta1 (CD49c), alpha4beta1 (CD49d), alpha5beta1 (CD49e), and alpha6beta1 (CD49f) receptors, and was carried out by reverse transcriptase-polymerase chain reaction, confocal microscopy and flow cytometry.
Results
Adhesion of the colon carcinoma cell line HT-29 was strongly reduced in the presence of 0.1 μM MMF. This effect was accompanied by down-regulation of alpha3beta1 and alpha6beta1 surface expression and of alpha3beta1 and alpha6beta1 coding mRNA. Adhesion of the prostate tumor cell line DU-145 was blocked dose-dependently by MMF. In contrast to MMF's effects on HT-29 cells, MMF dose-dependently up-regulated alpha1beta1, alpha2beta1, alpha3beta1, and alpha5beta1 on DU-145 tumor cell membranes.
Conclusion
We conclude that MMF possesses distinct anti-tumoral properties, particularly in colon and prostate carcinoma cells. Adhesion blockage of HT-29 cells was due to the loss of alpha3beta1 and alpha6beta1 surface expression, which might contribute to a reduced invasive behaviour of this tumor entity. The enhancement of integrin beta1 subtypes observed in DU-145 cells possibly causes re-differentiation towards a low-invasive phenotype.
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Background
With the improved long-term outcome of allograft recipients in the cyclosporine or tacrolimus era, malignant tumors have become increasingly important. Malignant tumours develop in 15–20% of graft recipients after 10 years, and thus contribute substantially to the morbidity and mortality of these patients [1]. Malignancies can develop in three ways: de-novo occurrence in the recipient, recurrent malignancy in the recipient or transmission of malignancy from the donor. In all cases, the post-transplant treatment regimen and the level of immunosuppression are high risk factors due to the long-term modification of the immune system.
During the last years, the novel immunosuppressive drug mycophenolate mofetil (MMF) has been introduced into the clinical protocol to overcome severe side effects associated with cyclosporine or tacrolimus. Meanwhile, it has become part of the immunosuppressive regimen after liver, kidney or heart transplantation [2]. Still, the influence of MMF on tumor recurrence or de novo malignancy has not been explored.
MMF effects are based on the inhibition of inosine monophosphate dehydrogenase (IMPDH) and prevention of guanosine monophosphate synthesis from inosine monophosphate, a rate-limiting step in the purine biosynthesis in lymphocytes. Consequently, MMF blocks the proliferation and clonal expansion of T and B lymphocytes, and prevents the generation of cytotoxic T cells, as well as other effector T cells [3]. Additional mechanisms may also contribute to the efficacy of MMF in preventing allograft rejection. By depleting guanosine nucleotides, MMF suppresses glycosylation and the expression of some adhesion molecules, thereby decreasing the recruitment of lymphocytes and monocytes into sites of inflammation and graft rejection [3]. Immunoprecipitation studies have shown that one of the glycoproteins affected is the lymphocytic alpha4beta1 integrin, the ligand for VCAM-1 on activated endothelial cells. Further experiments have revealed inhibition of the integrin LFA-1, the counter-receptor of ICAM-1, after MMF administration [4,5].
The integrins constitute a family of transmembrane receptor proteins composed of heterodimeric complexes of noncovalently linked alpha and beta chains. Integrins function in cell-to-cell and cell-to-extracellular matrix (ECM) adhesive interactions and transduce signals from the ECM to the cell interior and vice versa. For various types of cancers, different changes in integrin expression are closely associated with tumor growth and metastasis. Based on the knowledge that MMF modulates integrin expression, we postulated that MMF might not only suppress leukocyte recruitment to the donor graft, but also prevent integrin-dependent tumor dissemination. To explore how far MMF might serve as a metastasis-blocking agent, we investigated the beta1 integrin subunit expression pattern of colon, kidney, pancreas and prostate tumor cells before and after MMF treatment, as well as MMF effects on tumor cell adhesion to human endothelium in vitro.
The present study indicates that MMF possesses anti-tumoral properties particularly to colon and prostate carcinoma cells. Alterations of the beta1 integrin profile are responsible for blocking tumor cell adhesion to vascular endothelium.
Methods
Cell cultures
Kidney carcinoma Caki I cells, pancreatic carcinoma DanG cells and colonic adenocarcinoma HT-29 cells G were obtained from the tumor cell bank of Johannes Gutenberg University, Mainz, Germany. Prostate carcinoma DU-145 cells were purchased from DSMZ (Braunschweig, Germany). Tumor cells were grown and subcultured in RPMI1640 medium (Seromed, Berlin, Germany) supplemented with 10% FCS, 100 IU/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified, 5% CO2 incubator.
Endothelial cells (HUVEC) were isolated from human umbilical veins and harvested by enzymatic treatment with chymotrypsin. HUVEC were grown in Medium 199 (Biozol, Munich, Germany), 10% fetal calf serum (FCS; Gibco, Karlsruhe, Germany), 10% pooled human serum (Blood Bank of The German Red Cross, Frankfurt am Main, Germany), 20 μg/ml endothelial cell growth factor (Boehringer, Mannheim, Germany), 0.1% heparin (Roche, Basel, Switzerland), 100 ng/ml gentamycin (Gibco) and 2% 1 M HEPES-buffer (Seromed, Berlin, Germany). To control the purity of HUVEC cultures, cells were stained with fluorescein isothiocyanate (FITC)- labelled monoclonal antibody against Factor VIII-associated antigen (Von Willebrand factor; clone F8/86; Dako, Hamburg, Germany) and analyzed microscopically or by FACscan (Becton Dickinson, Heidelberg, Germany; FL-1H (log) channel histogram analysis; 1 × 104 cells/scan). Cell cultures with a purity > 95% were serially passaged. Subcultures from passages 2–4 were selected for experimental use.
Mycophenolate mofetil (MMF)
Tumor cells were pretreated with MMF (Roche Bioscience, Grenzach-Wyhlen, Germany) (0.1 μM, 1 μM). Before adding the MMF-treated tumor cells to HUVEC (monolayer adhesion assay), cell cultures were washed to remove MMF from the medium. Results were compared to untreated controls.
Viability of tumor cells in presence of MMF was assessed by propidium iodide dsDNA-intercalation or quantitative fluorescence analysis of enzyme-catalyzed fluorescein-diacetate metabolism.
Monolayer adhesion assay
HUVEC were transferred to six-well multiplates (Falcon Primaria; Becton Dickinson, Heidelberg, Germany) in complete HUVEC-medium. When confluency was reached, 0.5 × 106 tumor cells of each entity/well were carefully added to the HUVEC monolayer for 60 min. Subsequently, non-adherent tumor cells were washed off using warmed (37°C) Medium 199. The adherent cells were fixed with 1% glutaraldehyde and counted in five different fields (5 × 0.25 mm2) using a phase contrast microscope (20 × objective) to calculate the mean cellular adhesion rate.
Evaluation of integrin surface expression
Tumor cells were washed in blocking solution (PBS, 0.5% BSA) and then incubated for 60 min at 4°C with the FITC-conjugated monoclonal antibody anti-alpha2beta1 (Becton Dickinson; clone AK-7), anti-alpha4beta1 (Cymbus Biotechnology, Hofheim, Germany; clone HP2I1), anti-alpha5beta1 (Cymbus Biotechnology; clone SAM-1), anti-alpha6beta1 (Becton Dickinson; clone GOH3), or with the PE-conjugated monoclonal antibody anti-alpha1beta1 (Becton Dickinson; clone SR84), or anti-alpha3beta1 (Becton Dickinson; clone C3II1). Integrin expression of tumor cells was then measured using a FACscan (Becton Dickinson; FL-1H (log) channel histogram analysis; 1 × 104 cells/scan) and expressed as mean fluorescence units (MFU).
A mouse IgG1-FITC was used as an isotype control for FITC conjugated antibodies. To evaluate background staining of PE conjugated antibodies, goat anti mouse IgG-PE was used (all: Cymbus Biotechnology).
To analyze integrin beta1 distribution on the cell membrane, tumor cells were transferred to round cover slips (pretreated with 2% 3-aminopropyl-triethoxysilan) placed in a 24 well multiplate. Upon reaching confluency, cell cultures were washed and fixed in cold (-20°C) methanol/acetone (60/40 v/v). Subsequently, cells were incubated for 60 min with unconjugated anti-integrin monoclonal antibodies. Indocarbocyanine (Cy 3™; Dianova; working dilution: 1:50) conjugated goat-anti-mouse IgG was then added as the secondary antibody. To prevent photobleaching of the fluorescent dye, cover glasses with stained cells were taken out of the wells and the residual liquid was removed. These were then embedded in an antifade reagent / mounting medium mixture (ProLong™ Antifade Kit, MoBiTec, Göttingen, Germany) and mounted on slides. The slides were viewed using a confocal laser scanning microscope (LSM 10; Zeiss, Jena, Germany) with a plan-neofluar ×100 / 1.3 oil immersion objective.
mRNA expression of beta1 integrins
mRNA expression of beta1 integrins was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Tumor cells were seeded in 50 ml culture flasks (25 cm2 growth area; Falcon Primaria, Becton Dickinson) and cultured with or without MMF. Total RNA was extracted by using RNeasy kit (Qiagen, Hilden, Germany) and RNA samples were then treated with 80 U/ml of Rnase-free Dnase I (Boehringer Mannheim, Mannheim, Germany) for 60 min at 37°C, to eliminate amplifiable contaminating genomic DNA. Subsequently, samples were incubated for 10 min at 65°C to inactivate Dnase. Complementary DNA was synthesized from 1 μg of total RNA per sample with a 60 min incubation at 42°C, using the Moloney murine leukaemia virus reverse transcriptase (Invitrogen, Karlsruhe, Germany) and oligo-(dT) priming (Boehringer Mannheim). Amplification was carried out using gene specific primers and Platinum-Taq polymerase (Invitrogen) in a Mastercycler Gradient thermocycler (Eppendorf, Hamburg, Germany). Reactions were performed in the presence of 0.5 μl cDNA, with an initial incubation step at 95°C for 2 min. Cycling conditions consisted of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 30 sec over a total of 30 cycles. The reaction was completed by another 10 min incubation step at 72°C. The specific sequences for sense and anti-sense primers are shown in table 1. The PCR products were subjected to electrophoresis in 1.5% agarose gel and visualized by ethidium bromide.
Statistical analysis
All studies were performed 3–6 times. Statistical significance was investigated by the Wilcoxon signed rank test showing two-sided probabilities and using normal approximation. Differences were considered statistically significant at a p value less than 0.05.
Results
MMF modulates tumor cell adhesion to HUVEC
The 60 min adhesion rates of tumor cells were calculated at 22.5 ± 4.1 DanG cells/0.25 mm2, 39,8 ± 10.5 DU-145 cells/0.25 mm2, 55,3 ± 11.7 Caki I cells/0.25 mm2, or 80,5 ± 17.2 HT-29 cells/0.25 mm2. MMF differentially modulated the adhesive capacity of the tumor cells which was strongly dependent on the drug concentration and the cell line used (figure 1). Adhesion of DanG cells was weakly reduced by 1 μM MMF. A modest down-regulating effect was seen on Caki I cells at a MMF concentration of 0.1 μM. Strong and significant adhesion blockade was achieved when HT-29 cells were treated with 0.1 μM MMF (p = 0.0079). This effect was reverted at a dosage of 1 μM. Furthermore, MMF dose-dependently and significantly reduced the adhesive capacity of DU-145 cells with a maximum effect at 1 μM (p = 0.0079). In all experiments, cell viability was not impaired by MMF.
beta1 integrin expression pattern
Figures 2 and 3 depict the integrin beta1 surface expression pattern on untreated tumor cell cultures. Figure 2 is related to FITC-labelled antibodies, figure 3 to PE-labelled antibodies. Each tumor entity was characterized by a specific integrin pattern. Integrins were expressed in the following order (MFU ± SD; n = 4):
DanG: alpha3beta1 (455.6 ± 71.0) > alpha2beta1 (175.7 ± 24.3) > alpha6beta1 (54.8 ± 9.2) > alpha1beta1 (27.3 ± 3.2).
Caki I: alpha3beta1 (601.3 ± 82.0) > alpha1beta1 (139.2 ± 24.0) > alpha5beta1 (22.9 ± 4.2).
HT-29: alpha3beta1 (202.0 ± 33.8) > alpha6beta1 (119.6 ± 14.5) > alpha2beta1 (69.2 ± 9.0) > alpha1beta1 (25.4 ± 3.4).
DU-145: alpha3beta1 (942.5 ± 112.9) > alpha2beta1 (95.4 ± 12.4) > alpha5beta1 (69.1 ± 8.9) > alpha6beta1 (50.9 ± 7.2) > alpha1beta1 (31.4 ± 4.8).
Mean IgG1-FITC isotype control was 8.6 ± 1.8 MFU, mean IgG1-PE isotype control was 9.9 ± 1.7 MFU.
Analysis of the mRNA expression level confirmed the flow cytometry data (see below).
The distribution pattern of those integrins which were predominantly expressed on the respective tumor cell line was further explored by confocal microscopy (figure 4). alpha2beta1 (DanG cells), alpha3beta1 (Caki I, DanG HT-29 cells), and alpha6beta1 integrins (HT-29 cells) were distributed homogenously among the cell surface. In contrast, alpha1beta1 integrins on Caki I cells accumulated mainly at the sites of cell-cell-contacts.
MMF modulates beta1 integrin surface expression
MMF evoked distinct alterations of the beta1 integrin expression pattern (figure 5). MMF only slightly changed alpha1beta1, alpha3beta1, and alpha5beta1 integrin surface levels on Caki I cells (figure 5A), and weakly down-regulated alpha2beta1 on DanG cells when applied at 1 μM (figure 5B). However, 0.1 μM MMF strongly and significantly diminished alpha3beta1 (p = 0.0022) and alpha6beta1 integrins (p = 0.035) on HT-29 cells (figure 5C). This effect was reverted at concentrations of 1 μM MMF. The alpha3beta1 receptor became even slightly enhanced, compared to control values. alpha1beta1, alpha2beta1, alpha3beta1, alpha5beta1 on DU-145 cells were up-regulated significantly by MMF in a dose-dependent fashion, whereby strongest effects were seen on alpha2beta1 surface level in the presence of 1 μM MMF (>70% fluorescence enhancement, compared to non-treated controls; p = 0.0022; figure 5D).
Influence of MMF on beta1 integrin coding mRNA
To allow a clear interpretation of the strong effects of MMF on adhesion and beta1 integrin surface expression of HT-29 and DU-145 cells, MMF evoked alterations of gene activity was also evaluated in these cell lines (figure 6). Control experiments using non-treated HT-29 cells revealed high alpha2beta1, alpha3beta1, alpha6beta1 mRNA expression level (figure 6A). Application of 0.1 μM MMF induced down-regulation of alpha3beta1 and alpha6beta1 coding mRNA, which paralleled MMF's influence on receptor surface expression. The effect was reverted at a dosage of 1 μM. alpha3beta1 and alpha6beta1 coding mRNA became even slightly enhanced, compared to control experiments. With respect to the prostate tumor cell line DU-145, alpha1beta1, alpha2beta1, alpha3beta1 and alpha6beta1 coding mRNA was clearly detected in untreated cell cultures. Both, 0.1 μM and 1 μM MMF reduced mRNA of beta1 integrin subtypes, although the effect was more pronounced in the presence of 0.1 μM MMF (figure 6B).
Discussion
Although MMF has become part of the standard regimen after organ transplantation its impact on tumor development and dissemination is still not clear.
Our adhesion experiments demonstrate that MMF down-regulates binding of tumor cells to endothelium, which might argue for anti-tumoral properties of this compound. Notably, HT-29 and DU-145 cells responded well to MMF (-70% adhesion reduction), while Caki I and DanG tumor cells were influenced only modestly. From a clinical viewpoint, distinct adhesion-blocking properties of MMF might be limited to colon and prostate carcinoma cells. Interestingly, HT-29 cells were more susceptible to MMF than DU-145 cells: 0.1 μM MMF was sufficient to significantly diminish adhesion of HT-29 cells, whereas 1 μM MMF was necessary to evoke maximum effects on DU-145 cells. The different sensitivity of the tumor cell lines to MMF might be caused by an unequal metabolic activity, coupled with variable IMPDH levels. Recent data have shown that the level of expression of IMPDH mRNA and protein differ among several cell lines [6], and that IMPDH is selectively up-regulated in neoplastic and replicating cells [7]. Although this has not yet been proven, MMF might be more effective in rapidly proliferating tumor cells than in tumors with a lower replicating activity. In this context, the average doubling times of HT-29 and DU-145 cultures during their exponential growth phase were calculated to be 13–16 h or 22 h, respectively [8-10], whereas the mean population doubling times of renal or pancreatic carcinoma cell lines ranged between 24–104 h or 16–40 h, respectively [11-14].
It should also be considered that MMF might switch on/off different intracellular signaling cascades in colon versus prostate tumor cells. Indeed, adhesion blockade of HT-29 cells was accompanied by reduced alpha3beta1 and alpha6beta1 surface expression, while adhesion blockade of DU-145 cells was accompanied by a dose-dependent up-regulation of integrins alpha1beta1, alpha2beta1, alpha3beta1, alpha5beta1 on the cell membrane.
Studies on integrin receptors presented evidence that beta1 integrin expression by colon carcinoma cells qualifies these cells to successfully adhere to secondary sites. Recent experiments have demonstrated that colon cancer cells adhere to endothelial cells via beta1 integrins and that addition of beta1 integrin blocking antibodies reduces tumor cell adhesion [15,16]. Based on a murine spleen injection-liver metastasis protocol, the alpha3beta1 integrin subtype was identified to predominantly facilitate the metastatic activity of colon cancer cells [17].
A converse scenario might be created during prostate carcinogenesis, as levels of beta1 integrins have been found reduced in neoplastic versus normal prostate tissue [18,19], and in malignant versus non-tumorigenic prostate cell lines [20]. An in vitro cell culture model revealed that TGF-beta stimulates the expression of alpha2beta1 integrin on prostate cancer cell lines and concomitantly reduces tumor cell adhesion to human bone marrow endothelium [21]. Down-regulation in the expression of the alpha3beta1 integrins may also allow prostate tumor cells to become more invasive and lead to an increased propensity for metastasis: When human alpha3beta1high and alpha3beta1low expressing prostate carcinoma cells were injected into immunocompromised SCID mice, only those cells with a drastically reduced integrin level were found to form tumors at the primary sites and to be highly invasive and metastatic [22]. This is in context with our data demonstrating beta1 integrin elevation on DU-145 prostate tumor cells in the context with diminished adhesion behaviour.
When discussing the relevance of integrins in tumor recurrence and malignancy, we should keep in mind that integrin receptors serve as mechanistic binding as well as differentiation triggering elements. Therefore, up-regulation of the same integrin type might either lead to enhanced cell adhesion by coupling the receptor to its ligand, or to a reduced cell adhesion by activating integrin driven differentiation signals. Based upon our in vitro assay, we conclude that MMF blocks adhesion of colon and prostate carcinoma cells by two different mechanisms: a) Loss of alpha3beta1 and alpha6beta1 surface expression directly contributes to the reduced adhesive behaviour of HT-29 cells, b) Enhancement of integrin beta1 subtypes might cause re-differentiation of DU-145 cells towards a low-adhesive phenotype. However, it still remains to be determined if MMF indeed acts as a differentiation inducing drug in prostate tumor cells. Beside the hypothesis that beta1 upregulation might activate differentiation inducing signals, selective inhibition of tumor-promoting pathways should also taken into consideration.
Presumably, down-regulation of alpha3beta1 and alpha6beta1 surface expression on HT-29 tumor cells might be caused by inhibition of receptor glycosylation and/or receptor de novo synthesis. The latter hypothesis seems to be more likely because MMF's effects at the cell surface were also observed at the mRNA level. This was not the case with DU-145 cells where MMF evoked up-regulation of membranous beta1 integrins was not paralleled by similar modifications of the beta1 integrin coding mRNA. There is still no clear concept why MMF causes integrin up-regulation in one tumor entity but down-regulation in another entity, both coupled with reduced tumor cell adhesiveness. Presumably, HT-29 and DU-145 tumor cells might be equipped with different enzyme systems, the intracellular signaling cascade might be activated differentially in colon versus prostate tumor cells, or sensitivity of specific pathways to MMF might differ between both tumor types.
Speculatively, alterations of post-translational events might change the receptor surface presentation in prostate carcinoma cells. Elegant experiments by Liang and coworkers demonstrated that over-expression of alpha5beta1 or beta1 integrin induced the decrease of protein kinase B (PKB) phosphorylation and subsequent accumulation of cyclin-dependent kinase inhibitor p21 [23]. A yeast-based two-hybrid system was employed which identified IMPDH as specifically interacting with PKB [24]. Furthermore, MMF treatment significantly increased p21 proteins, which could be reversed by the simultaneous addition of guanine or guanosine [25,26]. Hypothetically, p21 may act as an MMF triggered upstream signal (via PKB?), which contributes to enhanced beta1 integrin surface expression.
Conclusions
The present study indicates that MMF possesses anti-tumoral properties particularly to colon and prostate carcinoma cells. Alterations of the beta1 integrin profile are responsible for blocking tumor cell adhesion to vascular endothelium. MMF might also act on further adhesion proteins which are relevant for tumor recurrence and dissemination. An in vitro study published recently refers to the sLeX-selectin pathway targeted by MMF [27]. CD44 glycoproteins as well as receptors of the cadherin family might also be modulated under MMF-based immunosuppressive regimen. From a clinical viewpoint, further studies must be undertaken which evaluate the tumor recurrence rate and classify the tumor type in MMF versus non-MMF treated transplant patients.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
TE performed parts of the in vitro studies, contributed toward the design of the study and drafted the manuscript. JM carried out confocal microscopy, BR and IN performed FACS-analyses. IM designed PCR primers and carried out the PCR studies. WDB contributed to the manuscript design and finalisation. DJ participated in the conception and design of the study. RAB carried out the adhesion assays, participated in the conception and design of the study and its coordination. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This work was supported by the "Matthias Lackas-Stiftung", "Paul und Ursula Klein-Stiftung", the "Heinrich und Erna Schaufler-Stiftung", and the "Gisela Stadelmann-Stiftung".
Figures and Tables
Figure 1 Adhesion capacity of HT-29, DanG, Caki I, and DU-145 tumor cells. Each tumor cell type was pretreated with 0.1 or 1 μM MMF or remained untreated (control) and was then added for 60 min to human umbilical vein endothelial cell monolayers. Non-adherent tumor cells were washed off in each sample, the remaining cells were fixed and counted in five different fields (5 × 0.25 mm2) using a phase contrast microscope. Adhesion capacity is depicted as tumor cell binding and related to 100% binding of non-treated control cell lines (mean ± SD; n = 6). Asterix indicates statistically significant difference to the control.
Figure 2 FACS analysis of beta1 integrin surface expression on HT-29, DanG, Caki I, and DU-145 tumor cells. Tumor cells were washed in blocking solution and then stained with the FITC-conjugated monoclonal antibody anti-alpha2beta1 (CD49b; clone AK-7), anti-alpha4beta1 (CD49d; clone HP2I1), anti-alpha5beta1 (CD49e; clone SAM-1), or anti-alpha6beta1 (CD49f; clone GOH3). A mouse IgG1-FITC was used as an isotype control for FITC conjugated antibodies. Fluorescence was analyzed using a FACScan flow cytometer, and a histogram plot (FL1-Height) was generated to show FITC-fluorescence.
Figure 3 FACS analysis of beta1 integrin surface expression on HT-29, DanG, Caki I, and DU-145 tumor cells. Tumor cells were washed in blocking solution and then stained with the PE-conjugated monoclonal antibody anti-alpha1beta1 (CD49a; clone SR84), or anti-alpha3beta1 (CD49c; clone C3II1). To evaluate background staining of PE conjugated antibodies, goat anti mouse IgG-PE was used. Fluorescence was analyzed using a FACScan flow cytometer, and a histogram plot (FL1-Height) was generated to show FITC-fluorescence.
Figure 4 Confocal images of the distribution pattern of beta1 integrin receptor molecules on HT-29, DanG, and Caki I tumor cells. Integrin subtypes which were predominantly expressed on the respective tumor cell lines are shown. All: magnification × 100.
Figure 5 Dose-response analysis. Each tumor cell type was pretreated with 0.1 or 1 μM MMF and compared to untreated controls. beta1 integrin surface expression was evaluated by a FACscan using FITC-conjugated monoclonal antibody anti-alpha2beta1 (CD49b), anti-alpha4beta1 (CD49d), anti-alpha5beta1 (cd49e), anti-alpha6beta1 (CD49f), or PE-conjugated monoclonal antibody anti-alpha1beta1 (CD49a) or anti-alpha3beta1 (CD49c). A mouse IgG1-FITC or IgG-PE was used as isotype controls. Mean fluorescence values (MFU) of MMF treated cell cultures are related to non-treated controls which were set to 100% (MFU ± SD, n = 6). Asterix indicates statistically significant difference to the control.
Figure 6 Analysis of beta1 integrin subtypes mRNA levels in HT-29 and DU-145 tumor cell lines. HUVEC were either grown in standard medium or in medium enriched with 0.1 or 1 μM MMF. mRNA expression level was investigated by reverse transcriptase-polymerase chain reaction. The housekeeping gene GAPDH served as the internal control.
Table 1 The sequences of the primers used for RT-PCR
mRNA Sense primer sequence Antisense primer sequence bp
GAPDH atcttccaggagcgagatcc accactgacacgttggcagt 509
alpha1beta1 catgcgctcgttttggaa cggccacatctcgggaccaga 309
alpha2beta1 gcatctcagaagtctgttgcc cctgttgttaccttcagggag 335
alpha3beta1 tacgtgcgaggcaatgaccta tttgggggtgcaggatgaagct 306
alpha6beta1 tggaggtacagttgttggcg ctccgttaggttcagggagt 253
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| 15644133 | PMC545066 | CC BY | 2021-01-04 16:03:07 | no | BMC Cancer. 2005 Jan 11; 5:4 | utf-8 | BMC Cancer | 2,005 | 10.1186/1471-2407-5-4 | oa_comm |
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BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-5-51564712010.1186/1471-2458-5-5Research ArticleTetanus immunity in nursing home residents of Bolu, Turkey Karabay Oguz [email protected] Fatma [email protected] Ali [email protected] Kazım [email protected] Department of Infectious Diseases and Clinical Microbiology, Izzet Baysal University, Faculty of Medicine, Golkoy, Bolu, Turkey2 Department of Microbiology, Izzet Baysal University Faculty of Medicine, Golkoy, Bolu, Turkey3 Department of Internal Medicine, Izzet Baysal University Faculty of Medicine, Golkoy, Bolu, Turkey4 Department of Anaesthesiology and Reanimation Izzet Baysal University Faculty of Medicine, Golkoy, Bolu, Turkey2005 12 1 2005 5 5 5 10 8 2004 12 1 2005 Copyright © 2005 Karabay et al; licensee BioMed Central Ltd.2005Karabay 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
Tetanus is a serious but vaccine-preventable disease and fatality rate of the disease is high in the neonates and the elderly. The aim of this study was to detect the tetanus antibody prevalence in the over sixty-year age residents of the nursing homes in Bolu.
Methods
A voluntary-based study was done in the residents of two nursing homes in Bolu, Turkey. Blood samples were taken from 71 volunteers residing in there nursing homes. Tetanus IgG antibodies were measured by a commercial ELISA kit.
Results
Among overall subjects, only 11 (15.7 %) had the protective tetanus antibody titers at the time of the study. Totally, 10 subjects were examined in emergency rooms due to trauma or accidents within the last ten years and, four (40%) of them had protective antibody levels. Of the remaining 61 subjects only 7 (11%) had protective antibody levels (p < 0.05) [Relative Risk = 3.49, 95% Confidence Interval 1.24–9.77].
Conclusions
Tetanus antibody level is below the protective level in the majority of the over-sixty-year-age subjects residing in the nursing homes. Each over sixty-year age person in our country should be vaccinated. Until this is accomplished, at least, nursing home residents should be vaccinated during registration.
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Background
Tetanus is an acute disease caused by the tetanus toxin, released by the bacterium Clostridium tetani. Tetanus spores are present in soil and manure and may be introduced into the body through a puncture wound, burn or scratch. Tetanus can never be eradicated because the spores are widely spread in the environment, and found in the intestinal flora of domestic animals, horses, chickens, and humans. Tetanus is not spread from person to person [1].
Tetanus is preventable by proper immunization. However, the disease is still prevalent even in the most developed countries and it often emerges in the elderly [2]. The prevalence of neonatal tetanus is considered a criterion for developmental level of the country. Every year, the cases of neonatal tetanus are reported in four to six European countries; Turkey and Albania have the highest occurrence rates [3].
The mortality rate of tetanus is high in the neonates and the elderly patients [4]. Age-related mortality rates were found to be 79.4 % in the neonatal period [5], 11 % in adults younger than 50 years old and 54 % in adults older than 50 years old [4,6]. Totally, 280 cases were observed in Turkey between 1994 and 1995. The mortality rate was 29.8% for these cases [7]. Risk is greater in people over 60, particularly in the developed countries [8].
In the USA, the disease occurs at a six-times higher rate in this age group [8,9]. Of 99 tetanus patients whose complete information was reported to the Centers for Disease Control and Prevention (CDC) during 1987 and 1988, 68% were over 50, while only six were younger than 20. No cases of neonatal tetanus were reported. The disease continues to occur almost exclusively among people who are unvaccinated or inadequately vaccinated or whose vaccination histories are unknown or uncertain [10].
Bolu is a city in the Western Black Sea Region of Turkey and has a population of approximately 283.000. There are two nursing homes (Izzet Baysal and Neziha Baysal Nursing Homes) in the each with a boarding capacity of 60 people. Tetanus antibody level in such a specific group has not been researched in the country yet. The purpose of this study was to detect tetanus antibody prevalence in the voluntary residents of the nursing homes over 60 years old.
Methods
This study was performed May 1–30, 2004. Subjects were selected from Izzet Baysal and Neziha Baysal Nursing home residents who volunteered for antibody measurement.
A standard form was filled including subjects' clinical information, demographic traits along with vaccination and injury stories. 10 cc venous blood was taken from each subject and kept at -20°C until the day of study.
Tetanus Ig G antibodies were measured by a commercial ELISA kit [Novatec Dietzenbach, Germany] at a 450/620 nm wavelength. The results were evaluated in the way previously defined by Schoroder et al. [11]. Briefly, antitoxin levels below 0.1 IU/L were defined as "below protective level" and antitoxin levels above 0.1 IU/L were defined as "at protective level".
Students't-test was used to compare quantitative variables while chi-square and Fischer's exact tests (two-tailed) were used for qualitative data, p < 0.05 was considered to be significant. Statistical analyses were performed with Epi-info 6.0 (Center for Disease Control, Atlanta, USA).
Results
Subjects
We aimed to include all residents of the nursing homes. In the study period, 78 subjects were staying in the two nursing homes (40 residents in Izzet Baysal Nursing Home and 38 residents in Neziha Baysal Nursing Home). However, 7 subjects did not want to participate in the study. Therefore, these seven subjects could not be included.
Antibody results and properties of subjects
A total of 71 voluntary subjects [54 (76%) male and 17 (24%) female] were included in the study. The mean age was 71. Only 11 (15.4%) of the 71 subjects had a protective antibody level against tetanus. 15 subjects were aged between 60–65 (3 of them had protective antibodies), 22 subjects were aged between 66–75 (2 of them had protective antibodies) and 34 of 71 subjects were aged ≥ 76 years (6 of them had protective antibodies). Protective antibody level decreased with every age group (60–65, 66–75, and ≥ 76) above 60 years old, but there were no statistically significant difference between the five-year age periods above 60 (p > 0.05) (Fig. 1).
Figure 1 Antibody levels of subjects
Totally, 10 subjects were examined in emergency rooms due to trauma or accidents within the last ten years. While four (40%) of them had protective antibody levels, only 7 (11%) of 61 subjects who didn't get examined in the emergency room had protective antibody levels (p < 0.05) [Relative Risk = 3.49, 95% Confidence Interval 1.24–9.77].
Only 10 (14%) of the 71 subjects had been vaccinated in the last ten years. Five of them had been vaccinated as a result of emergency room visits. Antibody level and demographic properties of subjects were presented in the Table 1.
Table 1 Demographic characteristics and antibody levels of subjects according to age groups
Characteristics TOTAL n = 71 (%)
Age (mean ± SD) 71.1 ± 8.6
Female/Male 17/54
Diabetes mellitus 7 (9.8)
Emergency room visits due to injury within the last 10 years 10 (14)
Tetanus vaccine within last 10 years Emergency room visit/No emergency room visit 5 (7)/5 (7)
Occupation with soil / garden-work 20 (28.1)
Average tetanus Ig G level 0.080
Tetanus Ig G >0.1 IU/ml 11 (15.4)
Discussion
According to the records of the Ministry of Health, 2039 tetanus cases were detected in Turkey between 1980–2002, 462 (23%) of which died [12]. In our country, a vaccination campaign against tetanus was initiated in the mid 1960s. However, it was carried out irregularly until 1985. After 1985, 3 doses of tetanus toxoid were given to all neonates after birth and a booster dose was applied in the 16th month followed by vaccination of primary school children at aged 7 and 12. While 67 neonatal tetanus cases were detected in 1990 in a population of 57.582.244, 32 neonatal tetanus cases were detected in 2002 in a population of 70.415.244 [12]. The neonatal tetanus rate has decreased with an increased rate vaccination in the country. In Turkey, females are vaccinated during pregnancy and males are vaccinated during military service but there is no vaccination program for the elderly. Unfortunately, a falling could not be achieved in the prevalence of tetanus in the elderly. Similar results were obtained in the developed countries, too. For instance, it was established that, rate of occurrence of tetanus in the people older than 60 was ten times higher than the young in Italy [9]. Routine vaccination programs focus on the childhood period in the developing countries. However, according to researches, the majority of adult population is sensitive to tetanus [13].
In our study group, only 11 (15.4%) of 71 subjects had protective antibody levels while the rest were under the risk of tetanus. This resultsuggested that lack of protective immunity among nursing home residents was a significant public health concern. In industrialized countries tetanus has become a rare disease and an infrequent cause of death, mainly due to the implementation of comprehensive immunization programs. But, tetanus is still an important problem for the developing countries, due to poor immunization standards and inadequate hygiene [14]. Deaths due to tetanus in Turkey mostly occur in the elderly. The 27.4 % of the total deaths caused by tetanus in 1989 occurred in the patients over 40. But today mortality rate in this age group ranges between 48.8–60.6% [15]. Those who are not vaccinated and the elderly are at risk [16].
Individuals over 60 are usually retired people and spending most of their time in garden or land-work. Tetanus spores may be introduced into the body through a puncture wound, scratch during these works. So they face risk of getting tetanus. Also, 28 % of our subjects still were busy with soil and garden work.
According to our results, antibody levels in individuals who had been examined in an emergency room within the last ten years due to injury are significantly higher than who didn't have such a history (p < 0.05). This situation suggests that vaccination programs are not well established in countries like Turkey, thus injuries and accidents expose people over 60 to a great risk of getting the tetanus prophylaxis [17].
Once the existence of tetanus is suspected, intensive, and effective management is essential. The patient should receive intensive care aimed at prevention of muscle spasms, prevention of respiratory tract and metabolic complications, and neutralization of circulating toxins [18]. Assuming that one tetanus case stays in the intensive care unit for at least fifteen days, it costs $9.000 per case approximately. In Turkey, 30.000 people can be vaccinated with a booster dose of tetanus vaccine for this amount of money [19,20]. Moreover, vaccination will not only ensure economic benefits but also protect thousands of people against tetanus. Apart from being more profitable, such policy will improve the health statistics of the country [20]. It is imperative that every person over 60 should be vaccinated against tetanus.
Conclusions
The lack of protective immunity against tetanus among the nursing home residents is a significant public health concern. A vaccination program including every individual over 60 should be charted immediately. After the primary series of 3 doses, protection against tetanus should be sustained by scheduling booster doses routinely in every 10 years [21]. Until this campaign is accomplished, at least, nursing home residents should be vaccinated during registration.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
All authors read and approved the final manuscript. OK designed the study and drafted the manuscript. AT, KA, analysed the data. OK oversaw the microbiological research. OK and FO interpreted the results of the analysis and critically reviewed the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We wish to thank Professor Cihangir UYAN, and Chemist Tufan SAHAN. Their contributions to and critical review on our draft are greatly acknowledged.
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| 15647120 | PMC545067 | CC BY | 2021-01-04 16:28:55 | no | BMC Public Health. 2005 Jan 12; 5:5 | utf-8 | BMC Public Health | 2,005 | 10.1186/1471-2458-5-5 | oa_comm |
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BMC Med EducBMC Medical Education1472-6920BioMed Central London 1472-6920-5-21564212510.1186/1472-6920-5-2Research ArticleDesign and validation of the Health Professionals' Attitudes Toward the Homeless Inventory (HPATHI) Buck David S [email protected] F Marconi [email protected] Suzanne [email protected] Donna [email protected] Dana L [email protected] Allegra [email protected] Robert J [email protected] Department of Family and Community Medicine, Baylor College of Medicine, Houston, TX, USA2 Department of Family Practice, University of California San Francisco, San Francisco, CA, USA2005 10 1 2005 5 2 2 3 9 2004 10 1 2005 Copyright © 2005 Buck et al; licensee BioMed Central Ltd.2005Buck 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
Recent literature has called for humanistic care of patients and for medical schools to begin incorporating humanism into medical education. To assess the attitudes of health-care professionals toward homeless patients and to demonstrate how those attitudes might impact optimal care, we developed and validated a new survey instrument, the Health Professional Attitudes Toward the Homeless Inventory (HPATHI). An instrument that measures providers' attitudes toward the homeless could offer meaningful information for the design and implementation of educational activities that foster more compassionate homeless health care. Our intention was to describe the process of designing and validating the new instrument and to discuss the usefulness of the instrument for assessing the impact of educational experiences that involve working directly with the homeless on the attitudes, interest, and confidence of medical students and other health-care professionals.
Methods
The study consisted of three phases: identifying items for the instrument; pilot testing the initial instrument with a group of 72 third-year medical students; and modifying and administering the instrument in its revised form to 160 health-care professionals and third-year medical students. The instrument was analyzed for reliability and validity throughout the process.
Results
A 19-item version of the HPATHI had good internal consistency with a Cronbach's alpha of 0.88 and a test-retest reliability coefficient of 0.69. The HPATHI showed good concurrent validity, and respondents with more than one year of experience with homeless patients scored significantly higher than did those with less experience. Factor analysis yielded three subscales: Personal Advocacy, Social Advocacy, and Cynicism.
Conclusions
The HPATHI demonstrated strong reliability for the total scale and satisfactory test-retest reliability. Extreme group comparisons suggested that experience with the homeless rather than medical training itself could affect health-care professionals' attitudes toward the homeless. This could have implications for the evaluation of medical school curricula.
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Background
In 2003, the Department of Health and Human Services reported that there are between two to three million people in the United States who experience homelessness each year [1]. According to Gelberg and Arangua [2], such estimates provide only a partial picture of the problem: among the U.S. population, 14% (26 million people) have been homeless at some time in their lives and 5% (8.5 million people) have been homeless within the past five years. Yet even as this number grows, the homeless continue to be subjected to broad stereotyping and stigmatization, both of which make it easier to ignore them. It is not surprising then that homeless persons are reluctant to obtain needed, continuous medical care within traditional outpatient settings. This is particularly problematic because they often have competing or immediate needs [3,4], multiple health problems [5,6], and increased morbidity and mortality [7,8].
The disinclination of the homeless to seek care may be due in part to the way in which many health-care workers respond to them. A less investigated but possibly equally important circumstance is the attitudes that health-care professionals have toward the homeless. As members of the larger society, these professionals often harbor the same preconceived ideas and biases toward the homeless that the rest of society does. Such judgmental attitudes can, and often do, emerge during the provider-patient encounter, thus limiting the effectiveness of medical treatment of the homeless [9].
As a countermeasure to prevailing negative attitudes and widespread stigmatization toward marginalized persons, recent medical literature has drawn on a well-known practice in the social sciences [10,11] and has begun calling for greater emphasis on the dimensions of compassion and humanism in medical education [12-14]. Humanism in psychology became popular in the 1950s when Rogers [15] began practicing client-centered therapy, which allows the relationship between therapist and client to develop so that the client can be guided within the framework of the therapeutic encounter. According to Branch [[16], p. 1067], humanism in medicine may be defined as "the physicians' attitudes and actions that demonstrate interest in and respect for the patient, and address the patient's concerns and values." As physicians interact with homeless patients, a heightened sense of these patients' vulnerability may evoke greater empathy and humanism, two attitudes that can only have a positive impact on the quality of care that the physicians provide.
Although it would be helpful to have a better understanding of physicians' current opinions about homeless patients, only two instruments have been designed and validated to measure attitudes toward homeless persons: the Attitudes Toward the Homeless Inventory (ATHI) [17], and the Attitudes Towards the Homeless Questionnaire (ATHQ) [18]. The ATHI surveys American college students' attitudes toward homelessness but does not specifically address the attitudes of health-care providers. In addition, it was not designed to measure a health-care provider's desire or confidence in his or her ability to deliver health care to homeless persons. Nevertheless, Buchanan et al. [19] recently used the ATHI to measure the attitudes of 12 primary care residents before and after a 2-week rotation in homeless health care and found that they felt more comfortable affiliating with homeless people after the course. The ATHQ, which was designed and validated in the United Kingdom, does assess the attitudes of health-care providers toward the homeless, but differences in health systems terminology (e.g., NHS for National Health Service, and terms related to homelessness, such as sleep rough) would make its use in its original form problematic in the United States. For this reason, we decided to use the ATHI for comparison purposes because its language is more consistent with American English.
The purpose of our study was to develop and validate a new measure, the Health Professionals' Attitude Toward the Homeless Inventory (HPATHI), an instrument that could be used to assess United States medical students' and physicians' attitudes toward homeless persons and to measure their level of interest and confidence in their ability to deliver health-care services to the homeless population. The objectives of this study were to: 1) describe the process of designing and validating the new instrument, and 2) discuss the usefulness of the instrument for assessing the impact of educational experiences working with the homeless on the attitudes, interest, and confidence of medical students and other health-care professionals.
Methods
The development and validation of the HPATHI occurred in three phases. The first phase consisted of the identification of items for the inventory; the second phase involved a pilot test of the initial instrument with a group of medical students; and the final phase entailed modifying the instrument and administering it in its revised form to our target population – health-care professionals, including primary care physicians, primary care residents, and third-year medical students.
Throughout this three-phase process, we sought to ascertain the reliability and validity of the HPATHI. Three types of validity were tested. Content validity was evaluated using the Delphi technique [20], which seeks consensus on instrument items among a panel of experts. Concurrent validity was computed by correlating the target population's responses to the HPATHI with their responses to the ATHI. Construct validity was established by using the extreme groups method [21], by conducting an exploratory factor analysis, and by conducting item analyses to assess the relationship of each individual item to the instrument's overall scale and hypothetical subscales.
Stages of development and validation of the HPATHI
In Phase 1, a group of physicians and nurse practitioners, all experts in homeless health care, was recruited through snowball sampling [22] to serve on a Delphi panel [20]. These individuals were identified as experts because they work with the homeless on a regular basis and are members of the National Health Care for the Homeless Clinicians' Network. They received a list of statements about the homeless, homelessness, and homeless health care that was compiled by selecting and adapting appropriate items from the AHTI [17] and the ATHQ [18]. We, as the authors of this study, added additional items that were based on our experiences and our review of the literature. The experts were asked to classify the statements into one of three categories ("essential," "interesting but not essential," and "irrelevant") and to generate other items that they considered to be necessary to explore health-care professionals' attitudes toward the homeless. The panel reached a consensus on the items to be used in the instrument through successive rankings of the items. The statements were then combined into an instrument with a five-point Likert scale (1 = strongly disagree; 2 = disagree; 3 = neither agree nor disagree; 4 = agree; and 5 = strongly agree) organized around three thematic areas: a) attitudes toward the homeless; b) interest in working with the homeless; and c) confidence in one's ability to work with the homeless.
During Phase 2, we administered the first draft of the HPATHI to a convenience sample of third-year medical students enrolled at Baylor College of Medicine (BCM) in Houston, Texas. These students were selected because they showed an interest in working with the homeless by registering for an elective course that allows students to provide health care to the homeless in a clinic setting [23]. Of the 100 students who attended class on the day that the instrument was administered, 72 completed it and were asked to provide contact information for the retest. Student responses to the instrument were analyzed using Cronbach's alpha coefficient to establish its internal consistency. Two weeks later, 34 of the 63 students who provided e-mail addresses completed the instrument a second time, thereby providing the data to determine the HPATHI's test-retest reliability. We then conducted an item analysis of redundant items or those with poor item-to-scale correlations.
In Phase 3, we prepared Web-based versions of the HPATHI and of the ATHI and sent the Internet link to our target population with a standard invitation to complete both instruments. Primary care physicians who serve as faculty in the BCM Department of Family and Community Medicine, family practice residents in the BCM Department of Family and Community Medicine, general internal medicine residents in the BCM Department of Family and Community Medicine, and students from the BCM Medical School served as a convenience sample of individuals completing the instrument over a six-month period. Subsequent data analysis consisted of:
1. Conducting an exploratory factor analysis using a Promax rotation to examine the structure of the HPATHI.
2. Estimating the internal consistency reliability of the online version of the HPATHI using Cronbach's alpha coefficient;
3. Determining the concurrent validity of the HPATHI against the ATHI;
4. Comparing extreme groups on their responses to the HPATHI. The extreme groups were determined according to their level of training (preclinical medical students vs. physicians) and according to their experience in working with the homeless (<1 month vs. >1 year).
This study was approved by the BCM Institutional Review Board for educational research with human subjects. Data analysis was conducted using SPSS Version 12.0 statistical software for Phases 1 and 2 and SAS® (V8.2 and V9.1) for Phase 3.
Results
Phase 1: instrument development
Of the 23 panel members who received the first list of 24 statements, 16 (70%) reviewed them, rank ordered them, and generated an additional 26 statements. We rearranged the combined list of 50 statements according to the proposed ranking ("essential," "interesting but not essential," and "irrelevant") and returned them to the 16 panel members for a second ranking. Based on their responses, six statements were eliminated and the remaining 44 were returned to the 16 panel members for a third ranking. Nine panel members returned responses after this iteration, and we eliminated nine additional items according to their recommendations. The remaining 35 items then constituted the first draft of the instrument we named the Healthcare Professionals' Attitude Toward the Homeless Inventory (HPATHI). Although seven panel members did not participate in the third iteration, their rankings in the second iteration corresponded to the final list of items.
Phase 2: pilot administration of the 35-item HPATHI
The sample population of third-year medical students completed a pilot administration of the 35-item HPATHI; the subset of students who responded two weeks later underwent a second administration of the same instrument. Table 1 displays the means and standard deviations of student responses to both administrations of the HPATHI. Items 2, 5, 6, 11, 15, 16, 20, and 23 were reverse-coded for the analysis so that a higher total mean on the instrument would indicate a positive attitude toward the homeless.
Table 1 Means and standard deviations for HPATHI* test and retest
Statement Test n = 76 Retest n = 34
M SD M SD
1. Homeless people are victims of circumstance. 3.26 .839 3.24 .819
2. Most homeless people are mentally ill. 2.90 .875 3.24 .955
3. Homeless people have the right to basic health care. 4.60 .620 4.68 .475
4. Homelessness is a major problem in our society. 4.36 .698 4.50 .707
5. Homeless people choose to be homeless. 3.71 .777 3.91 .712
6. Homeless people are lazy. 3.82 .738 3.94 .694
7. Health care dollars should be directed toward serving the poor and homeless. 3.83 .888 4.03 .717
8. Doctors should address the physical and social problems of the homeless. 4.17 .839 4.47 .563
9. Doctors have a duty to care for the homeless. 3.94 .977 4.18 .834
10. Caring for the homeless is pointless since they do not follow-up. 4.14 .612 4.24 .654
11. Providing medical care for the homeless is futile. 4.04 .740 4.21 .592
12. I am comfortable being a primary care provider for a homeless person with a major mental illness. 3.03 1.14 3.44 1.05
13. I feel comfortable being part of a team when providing care to the homeless. 4.32 .535 4.15 .643
14. I feel comfortable providing care to different minority and cultural groups. 4.42 .710 4.18 .673
15. I feel overwhelmed by the complexity of the problems that homeless people have. 2.97 .839 3.09 .996
16. I understand that my patients' priorities may be more important than following my medical recommendations. 3.97 .769 4.24 .741
17. I entered medicine because I want to help those in need. 4.42 .687 4.62 .551
18. I am interested in working with the underserved. 3.96 .941 3.88 .946
19. I enjoy addressing psychosocial issues with patients. 3.69 .973 3.74 1.08
20. I resent the amount of time it takes to see homeless patients. 3.79 .604 3.94 .547
21. I enjoy learning about the lives of my homeless patients. 3.58 .921 3.82 .834
22. I believe social justice is an important part of health care. 3.75 1.06 3.74 1.14
23. I believe caring for the homeless is not financially viable for my career. 3.24 .831 3.26 .864
24. I am too pressed for time to investigate psychosocial issues routinely. 3.37 .941 3.56 .894
25. I feel overwhelmed by the number of problems that homeless people have. 2.79 .844 2.88 .844
26. My knowledge regarding the problem of homelessness is adequate. 2.58 .931 2.65 .884
27. I can provide care for the homeless effectively. 2.97 .878 3.00 .921
28. Homeless people come from all walks of life. 4.42 .622 4.65 .485
29. Most homeless people tend to be drug addicts or alcoholics. 3.10 .735 3.38 .697
30. I think mentally ill homeless people refuse to get treatment. 3.67 .692 3.82 .797
31. Homeless people are dangerous, aggressive, and physically threatening. 3.97 .556 3.97 .627
32. There are only a few children among the homeless population. 4.31 .493 4.32 .684
33. All people have a right to basic health care. 4.50 .805 4.59 .657
34. I feel it is important to provide care to all socio-economic groups. 4.54 .670 4.62 .604
35. Most poor people have adequate access to health care through the public system. 2.15 1.02 2.03 .937
Totals 114 6.93 116 7.75
* Statements 24–35 were excluded from HPATHI after analysis.
Cronbach's alpha coefficient for the first administration was 0.87; the test-retest reliability coefficient (Pearson r) was 0.69. Through an item analysis we discarded 12 items that were either highly correlated with other items, and were thus considered repetitious, or that had item-scale correlations less than 0.20.
Phase 3: administration of the HPATHI to the target population
One hundred and sixty health-care professionals (24 primary care physicians, 15 primary care residents, 47 clinical medical students, 71 preclinical medical students, and 3 medical students who did not specify their education level) from one academic institution completed the HPATHI; 147 of them also completed the ATHI. Table 2 displays the means and standard deviations for both instruments by gender, by level of training (primary care physician; primary care resident; clinical medical student; preclinical medical student), and by experience with the homeless (no experience; <1 month, >1 month but <1 year, and >1 year).
Table 2 Comparison of the HPATHI and ATHI by gender, level of training, and experience
HPATHI ATHI
N Mean SD N Mean SD
Total 160 3.90 0.34 147 3.36 0.28
Gender
Female 103 3.95 0.30 96 3.38 0.26
Male 57 3.81 0.38 51 3.32 0.30
Level of Training†
MS1 55 3.89 0.32 54 3.34 0.26
MS2 16 4.10 0.23 16 3.47 0.21
MS3 28 3.91 0.41 26 3.38 0.36
MS4 19 3.84 0.30 18 3.33 0.25
Resident 15 3.73 0.22 13 3.30 0.25
Faculty 24 3.93 0.36 18 3.38 0.30
Experience
None 31 3.72 0.33 28 3.21 0.23
<1 month 64 3.91 0.30 62 3.37 0.27
>1 month & <1 year 30 3.88 0.32 25 3.37 0.22
1 – 3 years 17 4.10 0.28 16 3.49 0.31
>3 years 18 4.05 0.38 16 3.46 0.33
†On the HPATHI, 3 respondents failed to indicate a level of training and on the ATHI, 2 respondents failed to indicate a level of training.
The exploratory factor analysis (principal components), using a Promax rotation to account for the relationship among the factors, yielded a three-factor structure that explained 39% of the variance of the data. Factor 1 consisted of items 12, 13, 14, 17, 18, 19, 20, 21, 22, and 23 and was labeled Personal Advocacy; factor 2 consisted of items 1, 3, 4, 7, 8, 9, and 15 and was labeled Social Advocacy; and factor 3 consisted of items 5, 6, 10, and 11 and was labeled Cynicism. Table 3 presents the items with their loadings in each factor.
Table 3 Factor loadings for the 23-item HPATHI
Factor 1 Factor 2 Factor 3
1. Homeless people are victims of circumstance. -0.095 0.518 0.064
3. Homeless people have the right to basic health care. -0.096 0.644 0.154
4. Homelessness is a major problem in our society. -0.124 0.677 0.140
5. Homeless people choose to be homeless. 0.008 0.233 0.469
6. Homeless people are lazy. -0.026 0.330 0.559
7. Health-care dollars should be directed toward serving the poor and homeless. 0.180 0.575 0.079
8. I am comfortable being a primary care provider for a homeless person with a major mental illness. 0.329 0.466 -0.071
9. I feel comfortable being part of a team when providing care to the homeless. -0.015 0.514 0.188
10. I feel comfortable providing care to different minority and cultural groups. -0.007 0.152 0.725
11. I feel overwhelmed by the complexity of the problems that homeless people have. 0.028 0.056 0.748
12. I understand that my patients' priorities may be more important than following my medical recommendations. 0.469 -0.206 0.202
13. Doctors should address the physical and social problems of the homeless. 0.438 -0.046 0.395
17. I entered medicine because I want to help those in need. 0.485 0.003 0.088
18. I am interested in working with the underserved. 0.516 0.168 0.085
19. I enjoy addressing psychosocial issues with patients. 0.697 0.106 -0.224
20. I resent the amount of time it takes to see homeless patients. 0.613 -0.214 0.097
21. I enjoy learning about the lives of my homeless patients. 0.788 -0.039 -0.188
22. I believe social justice is an important part of health care. 0.509 0.404 -0.154
23. I believe caring for the homeless is not financially viable for my career. 0.504 -0.096 0.042
The HPATHI was further shortened to 19 items by the deletion of four more items, which either were not represented in the three-factor structure (items 2, 14, and 16) or had an adverse effect on the subscale's reliability (item 15). The three subscales were also significantly related to each other: factor 1 had Pearson's r correlations of 0.47 (n = 160; p < 0.001) with factor 2 and 0.43 (n = 160; p < 0.001) with factor 3; and factor 2 had a Pearson's r correlation of 0.48 (n = 160; p < 0.001) with factor 3. Table 4 displays the descriptive statistics and measurement properties for the 19-item HPATHI total and subscales. These three factors, if taken as subscales for the HPATHI, showed satisfactory Cronbach's alpha coefficients: 0.75, 0.72, 0.72, and 0.84 respectively for factor 1 (mean = 3.86; sd = 0.47), factor 2 (mean = 4.06; sd = 0.46), factor 3 (mean = 4.06; sd = 0.50), and total scale (mean = 3.96; sd = 0.38).
Table 4 Measurement properties for reduced HPATHI scale and subscales
Descriptive Statistics Subscale Statistics Full-scale Statistics
Item Mean SD Scale¶ Item-Total Correlation Cronbach's Alpha if Deleted Item-Total Correlation Cronbach's Alpha if Deleted
1 2.55 0.76 SA 0.28 0.73 0.27 0.84
3 1.45 0.61 SA 0.52 0.66 0.40 0.83
4 1.68 0.71 SA 0.48 0.67 0.39 0.83
5 2.20 0.80 C 0.42 0.73 0.40 0.83
6 2.14 0.65 C 0.53 0.65 0.50 0.83
7 2.15 0.79 SA 0.48 0.67 0.55 0.82
8 1.81 0.67 SA 0.49 0.67 0.50 0.82
9 2.01 0.72 SA 0.47 0.67 0.39 0.83
10 1.76 0.60 C 0.59 0.62 0.47 0.83
11 1.68 0.64 C 0.54 0.65 0.44 0.83
12 2.82 0.96 PA 0.31 0.75 0.31 0.84
13 1.79 0.58 PA 0.43 0.73 0.47 0.83
17 1.63 0.58 PA 0.38 0.73 0.37 0.83
18 1.84 0.78 PA 0.51 0.71 0.52 0.82
19 2.23 0.97 PA 0.51 0.71 0.47 0.83
20 2.09 0.74 PA 0.40 0.73 0.36 0.83
21 2.19 0.78 PA 0.55 0.71 0.46 0.83
22 2.00 0.85 PA 0.49 0.71 0.54 0.82
23 2.66 0.94 PA 0.34 0.74 0.31 0.84
¶PA = Personal Advocacy; SA = Social Advocacy; C = Cynicism
The Pearson's correlation coefficient between the HPATHI and the ATHI was 0.68 for the HPATHI's total scale (concurrent validity) (Table 4). For the extreme group comparisons, no significant difference was found between preclinical medical students and primary care physicians in their responses to the HPATHI (F = 1.05; df = 3, 156; p = 0.371). On the other hand, respondents who had more than one year of experience with the homeless scored significantly higher than those who had less than one month of experience (F = 6.19; df = 2, 157; p = 0.003) (Table 5). When the individual hypothetical subscales were considered, all items were either moderately or strongly correlated with their respective subscales (range of Pearson's correlation coefficients, 0.38 to 0.68). However, when the entire instrument was considered, the item analysis showed that items 1, 2, 15, and 16 had low item-scale correlations (Pearson's r < 0.24).
Table 5 Extreme group comparisons by level of training and experience with the homeless
N Mean SD
Level of Training
MS1 & MS2 71 3.94 0.31
Residents & Faculty 39 3.85 0.33
Experience with the Homeless‡
Less than 1 month 95 3.85 0.32
More than 1 year 35 4.01 0.33
‡Differences between the means of the two groups by experience were statistically significant (p < 0.01)
Discussion
Using the Delphi method to select the survey items helped ensure the HPATHI's content validity. Not only were the items selected and validated by homeless health-care experts, but the items chosen for inclusion in the HPATHI were based on findings from current literature on the issue. In addition, by correlating the HPATHI with the ATHI, we were able to demonstrate satisfactory concurrent validity. Construct validity for the HPATHI (i.e., attitudes toward the homeless) was determined by the extreme group comparisons, the item analyses, and the factor analysis.
Results of the extreme group comparisons showed that experience with the homeless, rather than medical training, is a significant factor that correlates with health-care professionals' attitudes towards the homeless and their interest in working with the homeless population. Moreover, individuals who had more extensive experience with the homeless showed more positive attitudes toward and interest in homeless patients. Therefore, increasing the opportunities to provide direct patient care to the homeless might improve the attitudes of health-care professionals toward this group.
The factor analysis suggested that we do have potential subscales in the instrument that may offer meaningful information regarding the general attitudes of health-care professionals who work with the homeless. The three subscales appear to represent: personal advocacy, which contains the items that reflect a personal commitment to work with the homeless; social advocacy, which consists of the items that reflect society's responsibility to care for the homeless population; and cynicism, which encompasses the items that reflect a negative attitude and a sense of futility in working with the homeless.
A limitation of this study is that only 160 health-care professionals participated in the online administration of the HPATHI. This small sample size limits the effectiveness of the factor analysis. Moreover, our sample focused only on the medical profession as represented by medical students and primary care residents and physicians. The participation of a larger number of health-care professionals from other medical specialties would strengthen the study.
Conclusions
The development of the HPATHI has many implications for and applications to future research on homeless health care, although its most valuable use may be to assess the attitudes toward homeless persons of medical students, residents, and practicing physicians. Throughout its various iterations, the instrument demonstrated strong internal consistency reliability for the total scale and satisfactory test-retest reliability. The scales identified by the factor analysis also showed satisfactory internal consistency reliability. The information collected from the expert panel and the literature search on attitudes toward the homeless and their health-care status proved to be an excellent framework for determining which statements to retain for the final instrument. The inter-item correlations and the correlations among the subscales indicate that the items are measuring similar underlying constructs within an overall theme – the attitudes of health-care professionals toward the homeless.
Despite the limited sampling, the research process has demonstrated that the HPATHI is a reliable and valid instrument that has the ability to assess the attitudes of health-care professionals toward the homeless population. We believe that the instrument may also be used in the future within the academic framework of medical schools to determine if attitudinal changes are affected by training experiences occurring with the homeless. To determine whether this is the case, we intend to administer the HPATHI as a pre/post-test survey to students who enroll in the homeless health-care track. Moreover, over the next year, we plan to keep on assessing the attitudes of health-care professionals toward the homeless by having new groups respond simultaneously to the ATHI and the HPATHI, as was done in Phase 3 of the original study. Additionally, we intend to include participants from other medical schools in the United States and to expand our sample to other health-care professionals who traditionally work with the homeless, to further test the instrument's overall validity.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
DB contributed to the conception, design, and acquisition of data, and drafted and revised the article. FMM contributed to the conception, design, and acquisition of data, helped draft the article, and carried out the data analysis. SK contributed to the conception, design, and acquisition of data, helped draft the article, and carried out the data analysis. DR contributed to the draft of the manuscript and critically revised it for important content. DLC contributed to the conception, design, and acquisition of data. AM contributed to the conception, design, and acquisition of data. RJV contributed to the interpretation of the data and critically revised it for important content. All authors read and approved the final manuscript
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This work was partially supported by an Advanced Research Training Grant awarded to David S. Buck, M.D., M.P.H., by the American Academy of Family Physicians and by funding from the Jewish Institute for Medical Research. We thank Pamela Paradis Tice, ELS(D), for her invaluable contribution in preparing the manuscript; L. Todd Weiss, for his indispensable assistance with data analysis; and Ellen Tseng, E.D.D., for creating the web interface that made the HPATHI accessible to other educational institutions. All three are with the Department of Family and Community Medicine at Baylor College of Medicine. We would also like to thank the members of the expert panel for their significant contributions during the initial phase of this research.
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| 15642125 | PMC545068 | CC BY | 2021-01-04 16:30:55 | no | BMC Med Educ. 2005 Jan 10; 5:2 | utf-8 | BMC Med Educ | 2,005 | 10.1186/1472-6920-5-2 | oa_comm |
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BMC Med Inform Decis MakBMC Medical Informatics and Decision Making1472-6947BioMed Central London 1472-6947-5-11563894010.1186/1472-6947-5-1Research ArticleComputer-aided DSM-IV-diagnostics – acceptance, use and perceived usefulness in relation to users' learning styles Bergman Lars G [email protected] Uno GH [email protected] Dept. of Learning, Informatics, Management and Ethics (LIME), Karolinska Institutet, Berzelius way 3, S-171 77 Stockholm, Sweden2005 7 1 2005 5 1 1 9 7 2004 7 1 2005 Copyright © 2005 Bergman and Fors; licensee BioMed Central Ltd.2005Bergman and Fors; 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
CDSS (computerized decision support system) for medical diagnostics have been studied for long. This study was undertaken to investigate how different preferences of Learning Styles (LS) of psychiatrists might affect acceptance, use and perceived usefulness of a CDSS for diagnostics in psychiatry.
Methods
49 psychiatrists (specialists and non-specialists) from 3 different clinics volunteered to participate in this study and to use the CDSS to diagnose a paper-based case (based on a real patient). LS, attitudes to CDSS and complementary data were obtained via questionnaires and interviews. To facilitate the study, a special version of the CDSS was created, which automatically could log interaction details.
Results
The LS preferences (according to Kolb) of the 49 physicians turned out as follows: 37% were Assimilating, 31% Converging, 27% Accommodating and 6% Diverging.
The CDSS under study seemed to favor psychiatrists with abstract conceptualization information perceiving mode (Assimilating and Converging learning styles).
A correlation between learning styles preferences and computer skill was found. Positive attitude to computer-aided diagnostics and learning styles preferences was also found to correlate.
Using the CDSS, the specialists produced only 1 correct diagnosis and the non-specialists 2 correct diagnoses (median values) as compared to the three predetermined correct diagnoses of the actual case. Only 10% had all three diagnoses correct, 41 % two correct, 47 % one correct and 2 % had no correct diagnose at all.
Conclusion
Our results indicate that the use of CDSS does not guarantee correct diagnosis and that LS might influence the results. Future research should focus on the possibility to create systems open to individuals with different LS preferences and possibility to create CDSS adapted to the level of expertise of the user.
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Background
Different types of decision support (DS) methods have been used in medicine for long. Computerized decision support systems (CDSS) including so-called "expert systems" can be used in for example interpretation of medical images, medical diagnostics or other areas. Examples are MYCIN for antibiotic treatments [1] and deDombal's system for acute stomach pain [2]. In Psychiatry, the DIAGNO system by Spitzer and Endicott was described 1968 as a computer program that simulated a DSM-1 diagnosis based on data from the psychiatric status schedule [3]. The CATEGO and DETOX systems are other examples [4,5].
For a more general reading on CDSS adoption in medical practice, see Fieschi et al [6] and for different approaches used to implement computers as diagnostic aids in medical decision making see for example Engle Jr. [7] and Miller [8]. Another study has been published by Berner and colleagues [9] who compared performance scores between four different diagnostic decision support systems.
Various models, or mode of actions, of DS exists including textual guidelines based on if-then-else strategies that forces the decision maker to make decisions in a logical and sequential manner; more advanced systems using fuzzy logic; neural networks; and systems using so called Artificial Intelligence. Many DS systems have been developed with aims to enable more accurate, consistent diagnosis and faster diagnostic procedures. CDSS have been applied in many medical disciplines, but have also been discussed in terms of e.g. reliability, usefulness, and user-acceptance. For example, Lu et al [10] found that the willingness to use CDSS rely heavily on preferences and perceived usefulness. Another example is Ridderikhoff and van Herk [11] who stated that although physicians indicate a need for diagnostic support, medical diagnostic support systems are not in widespread use. Miller [8] pointed out that it is misleading regarding the state of the art of these systems to just focus on the lack of widespread use. Miller's bibliography of systems from 1954 to 1993 convinced him that diagnostic systems nowadays can be seen as ubiquitous and "The prospects for adoption of large-scale diagnostic systems are better now than ever before, due to enthusiasm for implementation of the electronic medical record in academic, commercial, and primary care settings." Friedman et al [12] indicated that CDSS should be able to improve healthcare quality by providing accurate, useful and timely diagnostic information to clinicians and that most studies have emphasized the accuracy of the computer system alone without placing clinicians in the role as direct users. In exploring the extent to which CDSS might improve the diagnostic capability of clinicians, the success rate varied between different groups with different training levels. Larger improvements were observed for students than for residents and faculty. They concluded that "hands on" use of CDSS might influence diagnostic reasoning of clinicians. Regarding the decision procedure of a real expert, there are a number of theories. Many of these points out that there are differences between the decision process of an expert and a beginner (novice) [13,14].
It is unclear if the perceived usefulness of CDSS only is due to the DS model itself, or if the design and management of the computerized system is as important. Furthermore, there have been discussions about the design of computer systems, and how they might suit different users with different user-characteristics. Allen [15] argues that individual differences between users of information systems might influence search performance. Different types of cognitive resources such as topic knowledge, search skills, cognitive abilities, cognitive styles and learning styles have been shown to be related to a variety of search tactics and to tendencies to use certain information system features [16].
Learning styles
The concept of learning style (LS) might be regarded as an important characteristic and independent variable when individual differences in perceiving and processing information are investigated. A deep understanding of the user, his tasks and his environment is required to design a well-accepted (useful) computer program. Learning styles and its importance for users of computer systems has been demonstrated in various areas, for example in Internet use [17], web-based teaching [18], interactive multimedia environment [19], efficacy of computer training methods [20], hypertext environments [21], and in interactive learning systems [22].
There are a number of learning style models and inventories [23-27]. Claxton and Murrell [28] systematized the various learning style models based on Curry's [29] previous work on learning style constructs. The Kolb LS model [25], classified by Claxton and Murrell [28] as an information-processing model, has recently been further developed. The Kolb LS model has been widely applied during the years [30] and the latest version of the Kolb Learning Style Inventory instrument, is version 3 (LSI 3) [31,32].
This is a model of experience-based learning where all processes of the model are vital for the learning result. According to this model, the user/learner moves around the four modes in a circular direction (Figure 1). First there is an actual concrete learning experience. Second, the learner reflects on this experience. Third, the learner conceptualizes his/her observations and/or reflections into abstract theories or ideas. Fourth, the learner tests the theories or ideas by active experimentation. In this model, there are two processes for perceiving information: concrete experience mode and abstract conceptualization mode and two processes for processing experience into learning: active experimentation mode and reflective observation mode.
Figure 1 Structural Dimensions Underlying Learning Styles (After Kolb 1984)
These four processes combine into four learning styles: Converging (abstract conceptualization mode and active experimentation mode), Accommodating (concrete experience mode and active experimentation mode), Diverging (concrete experience mode and reflective observation mode) and Assimilating (abstract conceptualization mode and reflective observation mode).
Diverging learning style is associated with value generating skills: building relationships, helping others and sense making (reasoning). Assimilating learning style is associated with thinking skills: information gathering, information analysis and theory building. Converging learning style is associated with decision skills: quantitative analysis, technical device use and formulation of goals. Accommodating learning style is associated with action skills: leadership, initiative and action [25,33]. Learning styles of the Kolb model are not only associated with skill, but also with adaptivity and flexibility concerning management of different situations. Curry [34] points out that a learning style is different from ability, strategy and tactic. Styles might be observed across content domains, abilities, personalities and interpersonal behaviors and they are measured in terms of typical performance. According to Curry, learning style is spontaneously demonstrated without conscious awareness or choice across a wide variety of situations with similar requirements. Strategies, in contrast are the result of conscious decisions and tactics are specific observable activities in specific performance situations [34].
Diagnostics in psychiatry
In Psychiatry, the diagnostic process is mainly based on medical history, which is not often to be confirmed by lab tests or physical examinations. This leaves it more open for inter-personal differences in diagnostics, which might be a serious problem.
To facilitate the diagnostic procedures, and increase certainty and consistency in Psychiatry, a system called DSM has been created [35]. The DSM (Diagnostic Statistical Manual) system is a kind of information management system, an instrument that sort symptoms and handles them according to what the user judges as important symptoms. DSM is designed to assist the user by making the system criteria-based and by multi-axial descriptions, create a conflict-free base and thereby increase the reliability of the diagnosis. Currently, DSM version 4 (DSM-IV) is most used. The multi-axial DSM-system is based on the following reasoning:
1. Which symptoms have currently forced the patient to seek help?
2. How does the patient's overall pattern of experiences and behavior, compared with what is generally expected in the patients socio-cultural milieu, look like?
3. Are there any somatic diseases, which have to be attended to?
4. Have there occurred any stressful events in the patients' life along with the initial symptoms?
5. How serious are the problems just now, how is the patient functioning?
However, even DSM is not easily implemented in all situations, and therefore SCID, Structured Clinical Interview for DSM-diagnoses was created to further increase the reliability in psychiatric diagnostics [36]. SCID is a semi-structured interview instrument for DSM-IV-diagnoses, and is widely used in psychiatry internationally.
DSM training for physicians has been going on since 20 years during the Psychiatric course (9th semester) at Karolinska Institutet. The DSM-training is integrated in the course and the amount of time spent on DSM is approximately 4 hours. To our knowledge, there is no SCID-training in the Psychiatric course. However, in the training to become a specialist in Psychiatry at Karolinska Institutet, the DSM-framework is always used in teaching diagnostics and the amount of formal SCID-training is about 8 hours. Complementary to this, physicians becoming specialists in psychiatry are further trained in how to use SCID1 (axis 1 in DSM-IV) during their clinical training.
During a SCID interview, "jigsaw puzzle bits" are gathered, where DSM functions as a method to sort and put together these puzzle bits to known clinical syndromes. DSM is criteria based. For each criteria the users have to consider if the patient's symptom reach clinical significance so that the criteria can be regarded as fulfilled or not. There is some help for the interviewer in the DSM system in the form of a "decision tree" for axis 1 diagnosis.
Computer support for SCID
CB-SCID1 (Computer-Based SCID for axis 1 diagnostics) is a software program that is reported to have advantages as compared to the ordinary SCID-interview (according to the CB-SCID1-manual) [37]. The program handles most administration tasks for e.g. summing up of fulfilled criteria and also presents an overview of noted diagnoses. The order of questions and some control of possible conflicting diagnoses are also taken care of by the program. The "decision tree" mentioned above is integrated in the software.
Objectives
This study was undertaken to investigate and describe how different learning style preferences among psychiatrists might affect acceptance, use and perceived usefulness of the CDSS CB-SCID1 for DSM-IV-diagnostics.
Methods
Subjects
A number of practicing Psychiatrists, working at three different clinics, with different degree of expertise were asked to participate in this study. A fourth clinic was invited to participate in the study but could not do this due to lack of time. Out of 67 invited physicians, 49 volunteered to participate in the study. Out of these, 31 were experts at a senior level (being registered as specialists in psychiatry), and 18 were non-specialists (physicians with a position in psychiatry but without a specialist exam in psychiatry). In this study the groups are called "specialist group" (experts) and "non-specialist group" (not experts), respectively. They were all asked to complete a questionnaire regarding learning style preferences and to use the CB-SCID1 computer program for diagnostics of a real patient case (described in text) collected from the DSM-IV Case Book [38].
To be able to relate the Learning Styles of the 49 physicians in the study to the general situation in Sweden, a random sample of 250 (out of 1900 practicing psychiatrists in Sweden) were asked to fill in the same Learning Style inventories as mentioned above. This part of the study was done by sending out a letter including details of the study, which was followed up by a second letter as a reminder some weeks after the first one. All data were kept unidentified.
Survey instrument for LS
The learning style preferences for all participating physicians were measured according to the Kolb model [25] using the Kolb Learning Style Inventory instrument, version 3 (LSI 3) [31,32]. This instrument (questionnaire) presents specific questions and statements, which the test person enters his personal views on. The responses entered are then used to categorize the LS preferences for the person under study [31,32].
CDSS under study
The standardized terms and concepts of the DSM-system are the fundamentals of the CB-SCID1 system. CB-SCID1 uses logical inference of logical data (true, false), symbolically representing connections and dependency between components in the psychiatric knowledge base and presents questions according to the "paper" SCID-manual. CB-SCID1 takes care of the administration (for instance ordering of questions), correction possibility in criteria judgment, suggestion of answers according to previous in-data, summing up of fulfilled criteria, and also presentation of noted diagnoses. The system also handles some conflict control upon diagnoses. The user is asked to determine if various criteria are fulfilled or not and the system chooses how to go on, based on the user's input. If the number of fulfilled criteria reaches a certain level (according to DSM-IV) the program is automatically suggesting the corresponding DSM-IV diagnosis. The program is designed in a way that little training should be needed. CB-SCID1 also has a built-in, context sensitive help function in the consultation form, which put forward reminders and appropriate text information in tune with the decision problem at hand. The "assistance" from the system is based on the users input and also combined with the data driven rules derived from DSM-IV.
The physicians were instructed to use the CB-SCID-1 program as a tool to find the correct DSM-IV diagnoses of the paper case, and use the system as if the case had been a real one.
Data collection
A special version of CB-SCID1, CB-SCID1_Log, was created, with a logging function that automatically stored a number of data in a separate log file, while the physician was using the program trying to make DSM-IV diagnosis on the patient case. The data logged (outcome log-file variables) were:
• Total session time (total time spent in the CB-SCID1-program)
• Total decision time (total time used to decide about the different criteria used in CB-SCID1)
• Average decision time (mean time to decide about a criterion)
• Total "non decision" time (time spent in the program not making decisions)
• Total number of criteria judged (total number of criteria decided about)
• Total number of diagnoses (total number (correct and incorrect), of proposed diagnoses by CB-SCID1)
• Total number of correct diagnoses (total number of correct diagnosis according to the DSM-IV Case Book)
• Total number of incorrect diagnoses (all other diagnoses proposed by CB-SCID1 and not correct according to the DSM-IV Case Book)
• Ratio between correct diagnoses and proposed number of diagnoses
• Total number of regretted criteria-judgments (total number of times the user clicks on the Regret-button in the CB-SCID1 while deciding about a criterion)
• Total number of criteria judged unclear (total number of times using the Unclear-button in the CB-SCID1 while deciding about a criterion)
• Sum of numbers of regretted criteria-judgments and unclear criteria judgments
The CB-SCID1_Log system (identical to CB-SCID1 for the user) was installed at the office computers of the clinicians and a short oral introduction of the system functionality was given, explaining both the use of the system, its online help system, and the aim of the study.
Paper case used
The case used was picked from the DSM-IV Case Book [38]. The cases in this book are real, but unidentified, patients. These have been collected from a large number of clinicians (experts in particular areas of diagnosis and treatment). According to the Case Book, the recommended use of the cases is for example for researchers to assess the level of diagnostic expertise and the reliability with which members of their staff can make diagnostic assessments. A senior psychiatrist who is very experienced in DSM-IV and SCID training picked the actual case to be used. This specific case was chosen because it reflects multi-axial assessment (especially Axis 1 diagnoses), which was considered to be well suited for the CD-SCID1 program (which is aimed for Axis 1 diagnostics). The chosen case (called "Sickly" in the DSM-IV Case Book) is rather complex including three different diagnoses forcing the user to use the CB-SCID1 in full, taking decisions in problem areas like somatic problems, psychiatric problems and addiction problems.
The correct Axis 1 diagnoses (that is the assessment of Clinical Disorders and Other conditions that may be a focus of clinical attention) were in the actual case:
• Major Depressive Disorder, recurrent, mild
• Somatization Disorder
• Alcohol Dependence, in sustained full remission
To create a more realistic situation, the correct diagnoses were not revealed to the participating 49 psychiatrists, who all were given the same case with the aim to study the variation amongst the physicians using the CB-SCID1. They were not told what kind of case it was, neither the name of it ("Sickly"), nor where it came from.
Investigation procedure
The investigation procedure was performed in four steps as follows:
1. General information: The project leader gave oral information at each clinic on a regular meeting for psychiatrists about the study
2. Individual information and questionnaire: Each participating physician was given further oral information about the study and was asked to fill in a form about gender, age, professional training, DSM/SCID training, computer skill and attitude towards computer-aided diagnostics. This was followed by filling in the Kolb Learning Style Inventory questionnaire LSI 3.
3. CB-SCID1 test: The physicians received oral instructions on how to use the computer program and that it was more or less self-instructive compared to normal "paper" SCID-training, before using CB-SCID1. They were then instructed to diagnose the patient case with the help of the CB-SCID1-system. Ten minutes were offered reading the patient case before starting the CDSS-system
4. Follow-up interview and questionnaire: A follow-up interview within a week from the first interview was done. There were open-ended questions about the pros and cons about the CB-SCID1 (pro and con categories were built on the basis of the answers content). Also structured questions using a four-graded scale were given about clinical interviewing skill (without computer aid), perceived usefulness of CB-SCID1 and computer anxiety, which all were graded using a four-graded scale.
Statistical methods and analysis
Structured questions, constructed by the authors, and graded on a four-grade scale were given in the pre-assessment survey about computer skill and attitude to computer-aided diagnostics. In the post-assessment survey, structured questions were given about computer anxiety, clinical interviewing skill (without computer aid) and perceived usefulness of CB-SCID1. The questions were put in a clear statement which they could agree to/not agree to in an ordered categorical scale (Strongly disagree = 1, Disagree = 2, Agree = 3, Strongly agree = 4). The subject areas asked about were well defined and familiar to the users, why standardized attitude scales were not used. An open-ended question about the pros and cons about the CB-SCID1 were also given in the post-assessment survey. The answers to this question were grouped into several categories.
An analysis of correlations between the dimensions of LSI and the outcome log-file variables, as well as a comparison between specialists and non-specialists in this respect was also performed. Results were calculated as mean, standard deviation, median and lower – and upper quartile, where appropriate. Comparison between the two independent groups (specialists and non-specialists) was performed by the Mann-Whitney U Test and comparison between more than two independent groups (LS groups) was performed by the Kruskal-Wallis ANOVA by Ranks Test. Association between variables was calculated by Spearman Rank Order Correlations.
Ethical approval
All parts of this study have been approved by the ethical committee of Karolinska Institutet.
Results
General results
All of the 49 psychiatrists volunteering to participate in the original study group fulfilled all phases of the study. The randomly sampled 250 psychiatrists had a response rate of 42% (95). Only 226 questionnaires could actually be sent out because 24 of the randomly chosen 250 psychiatrists could not be reached due to for example that they had moved abroad or retired. One of the 95 actual responses was incomplete. Demographic data of the study groups can be seen in Table 1.
Table 1 Demographic variables in study groups
Variables Original study group (n = 49) Original study group – specialists (n = 31) Original study group – non-specialists (n = 18) Random sample group – specialists (Valid n = 93)
Age (mean ± SD) Male 48 ± 9 (n = 27) Male 53 ± 7 (n = 14) Male 43 ± 8 (n = 13) Male 52 ± 8 (n = 46)
Female 47 ± 11 (n = 22) Female 52 ± 7 (n = 17) Female 32 ± 6 (n = 5) Female 50 ± 8 (n = 47)
DSM/SCID-training (hours, mean) Male 7 Male 10 Male 3 Male 13
Female 9 Female 11 Female 3 Female 10
The 49 physicians in the original study group were reporting a value of 3 for general computer skill and a value of 1 for computer anxiety, both median values on a four-graded scale (where 1 is very negative or very low and 4 is very positive or very high). There were no differences in computer skill between specialists and non-specialists. Computer skill median was 3 in the random sample group.
The physicians were reporting to have a good clinical interviewing skill (without computer-aid), the median was 4 for specialists and 3 for non-specialists, which would predict high expected values on correct diagnosis and low values on incorrect diagnoses.
In the original study group, the attitude to computer-aided diagnostics presented as medians and lower – and upper quartile were for, male specialists 3 (2–4), female specialists 3 (2–3) and male non-specialists 3 (3-3), female non-specialists 3 (3-3). In the random sample group, where all were specialists, the results were for males 3 (2–4) and females 3 (2–3).
Other general variables like Gender, Age, Level of professional training, Computer skill, Attitude to Computer-aided diagnostics, DSM-IV/SCID-training were not found to be statistically correlated to the dependent log file variables (Total session time, Total decision time, Average Decision Time, Total "non decision" time, Total Number of Criteria judged, Total Number of Diagnoses, Total Number of Correct Diagnoses, Total Number of Incorrect Diagnoses, Correct Diagnoses ratio, Total Number of regretted criteria-judgments, Total Number of Criteria judged Unclear and Sum of numbers of regretted criteria-judgments and unclear criteria judgments).
Learning styles
The Learning Styles of the 49 physicians (tested by the LSI for learning style preferences) are shown in Table 2, where it is seen that the most common LS was Assimilating, followed by the Converging style. No major differences in Learning Style preferences were found between males (27) and females (22).
Table 2 Learning style preferences in the original study group and the random sample group
Group Assimilating Accommodating Converging Diverging Row Totals
Original study group 18 (37 %) 13 (27 %) 15 (31 %) 3 (6 %) 49
Specialists 10 (32 %) 6 (19 %) 12 (39 %) 3 (10 %) 31
Non-specialists 8 (44 %) 7 (39 %) 3 (17 %) 0 (0 %) 18
Random sample group 34 (36 %) 23 (24 %) 17 (18 %) 21 (22 %) 95
All groups 52 36 32 24 144
In comparison, the random sample of the psychiatrists in Sweden (also tested by the LSI for learning style preferences) turned out as indicated in Table 2. Here was also the most common LS Assimilating, followed by Accommodating style. No significant differences were found among the genders.
Among the 49 psychiatrists, it was found a correlation (p < .01) between learning styles and reported computer skill. The persons with highest score on computer skill were Converging, followed by Accommodating and Assimilating. Diverging styles were found to have the lowest computer skill.
A positive attitude to computer-aided diagnostics and learning styles were also found to correlate (p = .04). Most positive were Assimilating, followed by Converging and Accommodating. Diverging had the lowest figures in terms of attitude to computer-aided diagnostics.
Finally, it was also found that the distribution of learning styles and the Number of Criteria judged in the system significantly correlated (p < .01). The Accommodating group used the highest number of criteria, followed by the Converging, Diverging and Assimilating groups in that order.
Diagnostic results
Interestingly, the 49 physicians gave a rather low percentage of correct diagnoses. The correct diagnoses Major Depressive Disorder was found by only 27%, Somatization Disorder by 55% and Alcohol Dependence by 78%. Only 10% had all three diagnoses correct. 41% two correct diagnoses, 47% one correct diagnose and 2% no correct diagnose at all. Median values of proposed, correct, incorrect and ratio correct/proposed diagnoses for specialist and non-specialists are shown in Table 3.
Table 3 Median values of proposed, correct, incorrect and ratio correct/proposed diagnoses
Group Proposed diagnoses Correct diagnoses Incorrect diagnoses Ratio correct/proposed
Original study group (n = 49) 4 2 3 40 %
Specialists (n = 31) 4 1 3 33%
Non-specialists (n = 18) 4 2 3 50 %
As seen in Table 3, the non-specialist group seems to produce slightly better results (although not significant) than the specialist group. Also visible in the table, the median values for the number of proposed diagnoses (as a result from CB-SCID1 use) was as high as 4 for both specialists and non-specialists compared to the three correct diagnoses, which makes the above findings even more interesting and indicates a risk of over-diagnosing.
Acceptance
The indicator of acceptance, user's attitude to computer-aided diagnostics, was found to be 3 on a four-graded scale for both specialists and non-specialists in the original study group as well as for the random sample. No difference was found among genders. When correlating this to computer skill and computer anxiety it was found an overall negative significant correlation (-.48, p < .001) between positive attitude to computer-aided diagnostics and computer anxiety. Within the Accommodating group there was an even higher negative significant correlation (-.80, p < .001) between positive attitude to computer-aided diagnostics and computer anxiety.
There was only a tendency to a correlation between computer skill and a positive attitude toward computer-aided diagnostics (.27, p = .06) in the original study group. However, within the Accommodating group, there was a positive significant correlation (.62, p = .02). In the random sample group, a significant correlation between computer skill and positive attitude to computer-aided diagnostics (.43, p < .001) was found.
Finally, there was an overall negative significant correlation between computer skill and computer anxiety (-.51, p < .001).
Use
The use of CB-SCID1 was found to be varying among the 49 psychiatrists. For example, a positive significant correlation (.31, p = .03) was found between the active experimentation information processing mode (Converging and Accommodating) and the Total Number of Criteria judged in the program. No other significant correlations between the active experimentation-reflective observation dimension and the other 11 outcome log-file variables were found.
The total number of criteria judged for the patient case used varied from 44 to 119.
There were no significant correlations between the abstract conceptualization-concrete experience dimension and the 12 outcome log-file variables.
The comparison between the specialist group and non-specialist group concerning the 12 log-file variables revealed no significant differences of medians (See Table 4).
Table 4 Comparison of medians, lower – and upper quartile between specialists and non-specialists in outcome log-file variables
Variable Original study group (n = 49) Specialist group (n = 31) Non-specialist group (n = 18) p-value
Tot time (seconds) 1895 (1476–2405) 1710 (1476–2132) 2064 (1457–2532) 0.32
Decision time (seconds) 1193 (870–1559) 1035 (844–1486) 1269 (1107–1758) 0.20
Average decision time (seconds) 17 (13–22) 15 (12–20) 19.5 (14–22) 0.18
"Non decision time" (seconds) 661 (495–800) 650 (495–772) 677 (454–900) 0.83
Criteria judged (number) 68 (61–78) 69 (61–78) 67.5 (61–78) 0.74
Proposed diagnoses (number) 4 (3–5) 4 (3–5) 4 (4–5) 0.88
Correct diagnoses (number) 2 (1–2) 1 (1–2) 2 (1–2) 0.50
Incorrect diagnoses (number) 3 (2–4) 3 (2–4) 2.5 (2–3) 0.57
Ratio correct/prop diagnoses 40 (25–50) 33.3 (25–50) 50 (25–50) 0.24
Regretted judgments (number) 3 (1–8) 2 (1–8) 3.5 (2–10) 0.32
Unclear judgments (number) 7 (3–15) 7 (3–15) 8.5 (3–15) 0.80
Sum regretted unclear judgments (number) 14 (8–23) 14 (6–22) 13 (8–29) 0.56
Perceived usefulness
When analyzing the follow-up interview, (after trying CB-SCID1) a significant correlation (-.32) between perceived future usefulness and the Abstract Conceptualization – Concrete Experience dimension (p = .02) was found. The Abstract Conceptualizations orientated group had more Pros while the Concrete Experience orientated had more Cons on perceived future usefulness. This indicates that the Assimilating and Converging learning styles, which perceive information by abstract conceptualizations, are favored by the CB-SCID1-system.
According to the interviews, the perceived usefulness of the CB-SCID1-system was more negative than positive. 27 (55%) of the psychiatrists noted more Cons than Pros, 22 noted more Pros than Cons (45%).
The responses were also categorized according to their general content. 6 positive and 7 more negative general categories were found, see Table 5.
Table 5 Pro and Con categories of Perceived usefulness of CB-SCID1
Pros (6 different categories) Cons (7 different categories)
• Structure "there is a structure to hold on to in the program" • Appropriateness "not suited for the diagnostic interview situation"
• Accurate and reliable diagnoses "contradicts diagnoses by feeling", "more exact diagnose" • Empathy and intuition "risk of missing emotional and non-verbal information"
• Feedback "what works in treatment or not" • Conflict "managing the patient contact and the CB-SCID1-system at the same time"
• Help "help in diagnostic thinking while working with the program" • Underestimation "risk of underestimating your own skill, risk of getting dependent of the program"
• Correction "you will be noticed if you are on the wrong track" • Routine questioning "promotes exhaustion effects and lack of initiative"
• Timesaving "the program runs all administration and presents the diagnostic results" • Dialogue "breaking up of dialogue, missing emotional states and risk of irrelevant questions"
• General picture "risk of losing the overall picture"
Discussion
General results
Given the reported high clinical interviewing skill, high computer skill and relatively positive attitude to computer-aided diagnostics for the group, the low number of correct diagnoses and high number of incorrect diagnoses is very interesting. The DSM-IV-diagnosis is a symptom-diagnose derived from a deliberately limited amount of relevant diagnostic information, pattern of symptoms and development within predefined limits. This is not intended to be compared with the clinical diagnosis, which is building on patterns of symptoms, complete development, actual circumstances, anamnestic data, etiological discussion, laboratory tests, psychological tests etc. The symptom-diagnose and clinical diagnose perspective might be complementary to each other and it is usually recommended that a clinical interview always should be done before the SCID or CB-SCID1 interview. One possible explanation of our findings is that diagnosis in psychiatry is so complex that neither the DSM-system nor the "paper-SCID" or the CB-SCID1 systems might help. Another possible explanation is that CDSS have their limitations. This is in accordance with for example Dreyfus and Dreyfus who argued that computers could be good competent manipulators of symbols according to prepackaged algorithms, but they lack the type of intuition that real experts have [39].
There are alternatives to the SCID and CB-SCID1 to make DSM-IV-diagnosis. For example, the SCAN (Schedules for Clinical Assessment in Neuropsychiatry) and the computerized version, the SCAN 2.1 system, are systems developed by the WHO (World Health Organization) [40,41]. The computerized SCAN system is more based on "facts" from the patients' answers transformed by algorithms to DSM-IV diagnosis compared to the CB-SCID1 where the interviewer's judgment of each criterion is of importance. If a system like SCAN would have given other results, remains to be investigated.
The fact that this study did not diagnose living patients and that it was done with the help of a computer program may affect the results in that tacit knowing could not come into play as much as in a real situation. Polanyi [42] mentions that we can notice and do things without being able to tell how we recognize something or tell exactly what we do. Maybe the results of the experts, number of correct diagnoses for instance, are most affected by this in their intuitive way of functioning due to extensive personal experience, exercise and experience of former master-trainee relationship that is, "tacit knowing knowledge".
We also have to keep in mind that the 49 physicians tried the CB-SCID1 for the first time and with limited training in the program (even if the system is said to be possible to be used with little training). When trying to evaluate the results, we have to consider the different needs, habits and working style of experts and non-experts of different levels. For example, any expert, with a possible intuitive way of thinking, might be confused when they use a program addressed to non-experts that emphasize rule-following and logical step-by-step working procedures.
But our interpretation is still that the explanation is that CDSS do not suit all clinicians. This is in accordance with for example Ridderikhoff and van Herk [11] who found that despite need for diagnostic help, computer-aided support systems was ranked lower than other computer-aids and the use of a diagnostic computer-aided support system.
Limitations of the study
A limitation of this study is that only one case was used. The main reason for only using one single case was that the 49 physicians participating were very busy and reported that they had little time to spend for research projects like this. Furthermore, most of the 18 physicians not accepting to participate in the study reported lack of time as the main reason; asking the participants to use more cases, would most probably have resulted in more dropouts. The fact that physicians decided not to participate due to time constraints, could also raise a concern about selection bias since they might actually be less prone to use computer technology, differ in learning style or else. However, as the real case used is judged as being rather standardized and has been used successfully in training, we consider the risk of incorrect results being rather low.
Another limitation is that the computer skill was self-reported, and that no standardized skill test was used.
Finally, one limitation is that some of the psychiatrists participating in this study might have seen the used case before. However, since the DSM Case Book covers more than 200 cases, the chance that those psychiatrists remembered both the actual case and its three specific diagnoses is considered to be very low.
LS and psychiatry
It has been reported that domain specialists might have different LS preferences, for example Baker III, et al [43] using the Kolb model found that there is an identifiable surgical learning style: Converging (46 %). The other styles were Accommodating (26%), Assimilating (20%) and Diverging (8 %). This is in line with the Plovnick [44,45] results, which suggests surgeons as Converging in Medicine. Our results concerning learning styles did not confirm the Plovnick results that psychiatrists should be Diverging. This could depend on various reasons. The role for the psychiatrist today is not the same as it was 30 years ago. The "Diverging" aspect of relational skill is less vital compared to diagnostic methods of today and various modern treatment methods.
Another possible explanation is, according to the Kolb theory, Diverging-preference persons with their Concrete perceiving and Reflective observation are predicted to be the least interested in computer work, and thus that there is a possibility that the result is due to selection bias and low response rate. Theoretically the nearly 60 % of the 226 Swedish psychiatrists who did not answer the questionnaire, might have a Diverging preference. If so psychiatrists would have a Diverging preference, and the CB-SCID1 perceived usefulness values would probably be even more negative. Divergers also had the lowest figures in terms of attitude to computer-aided diagnostics and computer skill. Could it be that the low number of Diverging preferences, 6% in the original study group is the result of "selection bias"? After all, they were volunteers to participate in the study. They were asked to try the CB-SCID1 and may have hesitated to participate due to lack of computer skill? The random sample group was not asked to try the CB-SCID1 and Diverging preferences in that group were 22%. The higher numbers with Converging preference (high computer skill) in the Original study group compared to the Random sample group might have been explained by the same reason.
The fact that there were no significant correlations between the general variables and the dependent log-file variables indicate that further analyses of for example attitudes, use, perceived usefulness and learning style must be made in the future. This is supported by our findings that individual values regarding computer skill, computer anxiety and attitude to computer-aided diagnostics are related to learning styles and acceptance, use and perceived usefulness.
Acceptance
Computer skill, computer anxiety and the attitude to computer-aided diagnostics are interpreted to be important variables in the acceptance of the CB-SCID1-system. Accommodating psychiatrists with computer anxiety have a very low value on the indicator of acceptance. This means that this group has difficulties accepting the CB-SCID1. Again within the Accommodating group, those with computer skill are positive to the indicator of acceptance.
This highlights the importance of training as a means to increase computer skill and ease the acceptance of systems like CB-SCID1. Maybe the acceptance would increase if only targeting beginners or persons on "medium level" of training. As shown, a tendency for a more positive regard to computer-aided diagnostics exists in the non-expert group. The specialists, as proficient and on expert level, might not have a need of such computer-aid, or at least a different need in accordance with their way of working as experts with an intuitive frame of reference compared to the step by step, rule-following work of the novice.
Use
A possible explanation for the low number of significant results between LS and the outcome log-file variables might be that learning style is used with more flexibility in a real situation (using CB-SCID1) compared to more "attitude-like" variables like acceptance and perceived usefulness.
It might be so that LS must be linked to learning skill and adaptivity/flexibility in different specific situations to give significant results against the log-file variables. The LSI is measuring learning style preferences not associated to specific situations.
Perceived usefulness
Our findings that the program seems to be more attractive to psychiatrists with learning styles which prefer Abstract Conceptualizations (Assimilating and Converging learning styles) and in the majority negative to the Concrete Experience (Accommodating and Diverging learning styles) is in accordance with Lu et al [10] who found that the willingness to use CDSS rely heavily on preferences and perceived usefulness.
The Cons in future perceived usefulness against the CB-SCID1 about the appropriateness of such a program, the conflict perspective about using it and the breaking up of dialogue contain an all or none view. However, the Cons arguments have much in similar with the Dreyfus and Dreyfus standpoint regarding the limitations of computer use and seems to be reflecting the expert view, for instance lack of appropriateness, empathy and intuition and underestimation of your own skill. The Pros arguments seem to meet the non-expert view of learning to diagnose, for instance the program as a structure, help and correction facility. How to use, by whom and when, in which situations the CB-SCID1 is to be used must be elaborated upon. Non-experts with Diverging preferences might increase their flexibility using learning styles as a result of training and maybe a more positive regard to computer-aided diagnostics. That is, going around the "Kolb circle" in a more flexible and balanced way, using all learning styles, and give up their strong Diverging preferences.
Overall results from acceptance, use and perceived usefulness
Our results indicate that computer skill is of importance, and computer anxiety of negative impact on the attitude to computer-aided diagnostics. The highest computer skills were found within the Converging and Accommodating groups, which use Active Experimentation as information processing mode. Most positive to computer-aided diagnostics were the Assimilating and Converging groups, which in perceiving information are using Abstract Conceptualization.
The results also indicate that the Active Experimentation information-processing mode (Accommodating and Converging learning styles) is significantly correlated to number of criteria judged in the program.
Furthermore, the results also indicates, that although the 49 psychiatrists reported a positive attitude to computer-aided diagnostics, physicians with computer anxiety are less positive. Moreover, the CB-SCID1 CDSS seemingly invites the Accommodating and Converging learning styles to significantly adopt an Active Experimentation information-processing mode with a high number of criteria judged. However, this experimental mode may increase the number of incorrect diagnoses. The Assimilating and Converging learning styles also seems to be favored by the computer program concerning the Abstract Conceptualization mode in perceiving information.
Conclusions
The results of this study suggests that a CDSS is no guarantee of improved diagnostic procedures in Psychiatry and that even a clinically experienced user might end up with several incorrect diagnoses using such a system. The results also indicate that the use of CDSS-tools seems to favor users with learning style preferences using abstract conceptualization information perceiving mode.
Furthermore, our results indicate that future research on CDSS should focus on the possibility to create systems open to individuals with different LS preferences. Future research should also focus on the importance of computer training and different professional levels to optimize the usefulness of CDSS. The relationship between learning style preferences and working style habits at different professional levels might also be elaborated upon as well as the importance of learning style flexibility and decision modes in various diagnostic situations.
List of abbreviations used
CDSS Computerized decision support system
DS Decision support
DSM Diagnostic Statistical Manual, "DSM-system"
DSM-IV Diagnostic Statistical Manual, version 4
LS Learning Style
SCID Structured Clinical Interview for DSM-diagnoses
CB-SCID1 Computer-Based Structured Clinical Interview for DSM-diagnoses (axis 1)
Competing interests
Competing interests exists between the commercial CB-SCID1 system from Pilgrim Press and the SCAN-system developed by WHO, World Health Organization. However, the authors have no interests in any of these competitors.
Authors' contributions
UF has supervised LB in the planning, carrying out and analysis of this research work. LB has carried out all experiments. The manuscript has been written on close collaboration between LB and UF.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
Thanks to the Psychiatrists who volunteered to participate, despite heavy workload, to participate in this study. Special thanks to Jörgen Herlofson, Pilgrim Press, for letting us use the CB-SCID1 system.
Many thanks to Elisabeth Berg, specialist in medical statistics at LIME, Karolinska Institutet.
This study was supported by the Karolinska Institutet and the Stockholm County Council. (Stockholms Läns Landsting)
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| 15638940 | PMC545069 | CC BY | 2021-01-04 23:52:12 | no | BMC Med Inform Decis Mak. 2005 Jan 7; 5:1 | utf-8 | BMC Med Inform Decis Mak | 2,005 | 10.1186/1472-6947-5-1 | oa_comm |
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J Transl MedJournal of Translational Medicine1479-5876BioMed Central London 1479-5876-2-451561323110.1186/1479-5876-2-45ResearchCD28 expression in sentinel node biopsies from breast cancer patients in comparison with CD3-ζ chain expression Schüle Jana M [email protected] Leif [email protected]åkansson Leif [email protected] Bertil [email protected]åkansson Annika [email protected] Department of Surgery, Central Hospital, SE-72189 Västerås, Sweden2 Center for Clinical Research of Uppsala University, SE-72189 Västerås, Sweden3 Department of Oncology, University Hospital of Linköping, SE-58185 Linköping, Sweden4 Department of Pathology and Cytology, University Hospital of Linköping, SE-58185 Linköping, Sweden2004 21 12 2004 2 45 45 5 10 2004 21 12 2004 Copyright © 2004 Schüle et al; licensee BioMed Central Ltd.2004Schüle 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
Immunosuppression is documented in several malignant diseases, including breast cancer. Subsequently, future therapeutic concepts might include immunological approaches. However, detailed knowledge about tumor immunogenicity and host immunoreactivity, and how to assess these adequately, is still limited. We studied CD28 and CD3-ζ expression in sentinel node biopsies (SNB) from breast cancer patients to analyze tumor-related changes in T cell activity.
Method
25 women underwent surgery for primary breast cancer, including SNB. Frozen sections from 21 sentinel nodes could be analyzed with a double-staining technique. CD28 expression was studied in CD4+ and CD8+ T-lymphocyte subsets and compared with CD3-ζ expression in three specified nodal regions.
Results
The degree of CD28 expression varied between the different lymph node areas. The lowest degree of CD28 expression was observed in CD4+ T-lymphocytes in the paracortex and germinal centers. Here, a good agreement with CD3-ζ expression was found. A higher CD28 expression was noted in CD4+ T-cells in the primary follicles, where concordance with CD3-ζ expression was weaker. The CD8+ T-lymphocyte subset displayed generally a higher degree of CD28 expression than the CD4+ subset.
Conclusion
Sentinel lymph nodes from breast cancer patients displayed local immunosuppression of varying extent. In the areas with the lowest degree of CD28 expression an accordingly low CD3-ζ expression was found. The SNB might prove an important diagnostic tool for the evaluation of interactions between tumor and the host immune system, helping to select patients who might benefit from adjuvant immunotherapy.
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Background
Numerous studies [1] portray a decreased anti-tumor immunoreactivity in patients with malignancies, including breast cancer [2-4], and its correlation with disease progression and survival [5,6]. Antigen presentation and subsequent T-cell activation play a major role in initiating and maintaining an adequate anti-tumor response. However, the complex signaling cascades engaged in this process are not yet fully understood, rendering it difficult to be successfully addressed in therapeutical approaches. Better knowledge of these mechanisms is therefore essential for further development of immunological treatment strategies.
The CD28 surface receptor is normally expressed on 95% of CD4+ T-cells and approximately 50% of CD8+ T-cells in human peripheral blood [7]. Its natural ligands, the B7 molecules, are found on various antigen-presenting cells [8]. CD28 expression increases in activated T-cells [9]. Ligation of CD28 possesses major importance as a second, co-stimulatory signal during antigen/MHC complex presentation [10], hereby leading to a lower T-cell activation threshold and a longer duration of the proliferative response [11]. However, activation via the T-cell receptor alone induces transient T-cell proliferation [12], T-cell anergy, or deletion [13].
Decreased CD28 expression is described in dysfunctional peripheral T-lymphocytes from patients with hairy cell leukemia [14] and chronic lymphocytic leukemia [15]. In colorectal cancer, tumor-infiltrating lymphocytes (TIL) lack CD28 in contrast to those in normal colon interstitium [16]. This is consistent with findings in TIL from primary melanoma patients [17]. In melanoma metastases, CD28 down-regulation is more pronounced in areas of tumor regression [18,19]. Compared to healthy controls, breast cancer patients display significantly lower percentages of CD28+ T cells in peripheral blood [20]. To our knowledge, no studies as to the expression of CD28 in sentinel node biopsies from breast cancer patients have yet been published.
The expression of the zeta chain of the T-cell receptor (CD3-ζ) is decreased in sentinel node biopsies from breast cancer patients [21]. This down-regulation is most pronounced in the paracortex, the main T-cell activation area. In the present study, CD28 expression was analyzed in the same material and subsequently compared with CD3-ζ expression in parallel sections.
Methods
Study population
The study comprised 25 patients who underwent surgery for primary breast cancer, using the sentinel node biopsy technique. Inclusion criteria for enrolment in the study protocol were informed patient consent and a newly diagnosed palpable invasive breast cancer. Exclusion criteria were palpable axillary metastases, multifocality of the cancer, ongoing pregnancy or preoperative cytotoxic treatment.
In two cases, the sentinel node could not be immunologically analyzed due to lack of technical quality. In two other cases, nodal tumor growth was too abundant and remaining lymphoid tissue too little to analyze the sections. Thus, the remaining study population comprised 21 patients. Patient and tumor characteristics are presented in Table 1.
Table 1 Tumor and other selected characteristics of 21 women operated on for primary breast cancer.
Median (range) n (%)
Age 60 (36–86)
Menopausal status
premenopausal 5 (24)
postmenopausal 16 (76)
Nodal status
negative SNB 12 (57)
positive SNB 9 (43)
only positive SNB 7 (33)
3–4 positive lymph nodes 2 (10)
Tumor stage
I 7 (33)
IIa 7 (33)
IIb 7 (33)
Histological type
ductal invasive 17 (81)
lobular 3 (14)
mucinous 1 (5)
Tumor size (mm) 21 (1–60)
Estrogen receptor status
positive 19 (90)
negative 2 (10)
DNA ploidy status
euploid 9 (43)
aneuploid 12 (57)
S phase
high 3 (14)
low 18 (86)
(Elston) histological grade
I 2 (10)
II 11 (52)
III 7 (33)
Not measured 1 (5)
SNB = sentinel node biopsy
The study protocol was approved by the ethics committees at the University of Uppsala and the University Hospital of Linköping.
Identification of sentinel node
Sentinel nodes were identified by injection of a radioactive tracer (Tc-99-nanocolloid) close to the tumor site, preoperative lymphoscintigrams, and injection of Patent blue dye (Guerbet, Paris, France). After biopsy, the sentinel nodes were sent fresh to the pathology department. Specimens were snap-frozen and tissue sections (6–7 μm thick) were obtained for immediate diagnostic analysis regarding the occurrence of metastasis in the sentinel node. Additional frozen sections were wrapped in parafilm and stored at -70°C until processed further at University Hospital of Linköping. Routine diagnostic studies of both tumor tissue and lymph nodes were performed at the pathology department of Central Hospital, Västerås and comprised all parameters listed in Table 1.
Monoclonal antibodies
The monoclonal antibodies used were as follows: CD4 (Clone SK3, Becton-Dickinson, Stockholm, Sweden), CD8 (Clone SK1, Becton-Dickinson, Stockholm, Sweden), CD28 (Clone L293, Becton-Dickinson, Stockholm, Sweden), CD3 (Clone UCHT1, DAKO Stockholm, Sweden), TCR-zeta (Clone 2H2D9 (TIA-2), Immunotech, Stockholm, Sweden).
Preparation of node biopsies and immunological staining of tissue sections
Tissue sections obtained as described above were fixed with 4% paraformaldehyde (pH 7.4) (Riedel-de Haen AG, Seelze, Germany), supplemented with 5.4 g/L of glucose for 5 min and soon afterward washed three times in Hanks' balanced salt solution (BSS; Gibco, Paisley, United Kingdom) supplemented with 0.01 M HEPES solution. To avoid unspecific binding, sections were blocked with normal rabbit serum before the first staining and subsequently incubated with the primary antibodies CD3 (1/40), CD4 (1/25) and CD8 (1/50) for 30 minutes. After the slides had been washed in BSS/saponin, biotinylated rabbit anti-mouse immunoglobulin was added at a 1/100 dilution in BSS/saponin. Mouse IgG (Sigma, Stockholm, Sweden) was used as a negative control. The slides were then incubated with peroxidase-labeled streptavidine (P0397; Dako, Stockholm, Sweden) at a 1/100 dilution in BSS/saponin for 30 minutes. DAB (3,3'-diaminobenzidine, D-5637, Sigma, Stockholm, Sweden) was used as a substrate, which resulted in a brown color. The expression of the zeta chain or CD28 was identified using mouse monoclonal antibodies to these substances. The sections were first blocked by incubation with normal goat serum and subsequently incubated with the primary antibodies. Sections stained for the zeta chain (1/2.5) were incubated for 30 minutes while sections stained for CD28 (1/25) were incubated over night. The slides were again washed in BSS/saponin and incubated with goat anti-mouse immunoglobulin for 30 minutes and then with the alkaline phosphatase-anti-alkaline phosphatase (APAAP) mouse monoclonal antibody (Dakopatts D 651) at a dilution of 1/25 in BSS/saponin. After washes in BSS/saponin and Tris-buffered saline (TBS) and incubation with the alkaline phosphatase substrate (Naphthol AS-MX), 2 mg phosphate (Sigma N4875), 0.2 ml dimethylformamide, 9.8 ml 0.1 M Tris buffer pH 8.2, 50 μl 1 M levamisole (Sigma L-9756) and 10 mg Fast-Red TR salt (Sigma F 1500) for 20 minutes, the sections were again washed in TBS. Thereafter, the sections were counterstained in Mayer's haematoxylin for 15 minutes and mounted in Glycergel (Dakopatts, Sweden). All antibody solutions contained 2% normal blood donor AB serum and all incubations were performed in a moist chamber. The APAAP technique resulted in bright red staining for CD3-ζ and CD28. Double-stained cells appeared as red-brown; those with down-regulated CD3-ζ or CD28 were dominated by a brown color.
Evaluation of occurrence and distribution of CD28
Two independent investigators (A.H., B.G.) analyzed whole sections of the sentinel nodes. The degree of CD28 expression was evaluated in both CD4+ and CD8+ T-cell subsets within three lymph node areas: primary follicles, secondary follicles (germinal centers) and paracortex. The number of CD4+ and CD8+ T-lymphocytes expressing CD28 was semiquantitatively scored as "high" (>75% CD28+), "moderate" (50–75% CD28+) and "low" (<50% CD28+). The regional scores were then put into correlation with our earlier data on expression of CD3-ζ in parallel sections of the same patients where the same scoring system was applied [21]. There was good agreement between the scores of the two investigators (80%). The few discrepancies between scores were discussed and the reasons for the difference in the scores could be identified. Thus, a consensus regarding the proper evaluation could always be reached.
Statistical analysis
For testing correlations between the patient data depicted in Table 1 and expression of CD28 and CD3-ζ, the Mann-Whitney U test was used. The Kruskal-Wallis test was applied for comparing the three analyzed lymph node regions regarding the expression of the respective markers and for the comparison of CD28 expression in the T-cell subgroups (CD4+ and CD8+) within the same lymph node region. A p-value less than 0.05 was considered significant. For testing the agreement between the expression of CD28 (in the two separate T-cell subsets) and the expression of CD3-ζ for every nodal area separately, the Prevalence-Adjusted Bias-Adjusted Kappa (PABAK) was used. It has the same interpretation as Cohen's Kappa [22]. A value of >0.80 indicates excellent agreement, 0.61–0.80 good agreement, 0.41–0.60 moderate agreement, 0.21–0.40 fair and 0.00–0.20 poor agreement. Values less than zero suggest that the agreement is worse than expected by chance.
Results
Expression of CD28
The degree of CD28 expression varied considerably, both between individual patients and between different regions of the sentinel lymph nodes. The lowest degree of CD28 expression was seen in CD4+ T-lymphocytes located in the paracortex and the secondary follicles. CD8+ T-lymphocytes displayed a significantly higher degree of CD28 expression than CD4+ T-cells in both paracortex (p < 0.001) and primary follicles (p < 0.001). There were statistically significant differences in CD28 expression in CD4+ T-cells comparing the three nodal regions (p < 0.01), with CD28 expression being higher in primary follicles than in secondary follicles and paracortex. No such differences were found analyzing CD28 expression in CD8+ T-lymphocytes. No significant correlation could be found between the degree of CD28 expression and tumor size, histological type, hormone receptor status, DNA ploidy status, Elston histological grade, S-phase, patient age, nodal status or tumor stage following the TNM classification of malignant tumors, neither were there any significant correlation detected regarding clinical data and the degree of CD3-ζ expression in our previously published material [21].
Primary follicles
Primary follicles are cortical lymph node areas housing predominantly B-lymphocytes. The analysis could be performed in 20 cases. In the CD4+ subset (Table 2), 7 of 20 patients displayed a high degree of CD28 expression (Figure 1a), 5 of 20 a moderate and 8 of 20 a low degree of CD28 expression (Figure 1b).
Table 2 Degree of CD28 expression in the CD4+ T-lymphocyte subset in different areas of the sentinel nodes from 21 breast cancer patients (numbers are frequencies).
Expression of CD28 on CD4+
High Moderate Low
Primary follicles 7 5 8
Secondary follicles 1 3 12
Paracortex 0 5 15
Figure 1 Double staining of a sentinel node for CD4 (brown staining) and CD28 (red staining).1a). Primary follicle showing a high expression of CD28.1b). Primary follicle showing a low expression of CD28. 1c). Germinal center showing a moderate expression of CD28. 1d). Germinal center showing a low expression of CD28. 1e). Paracortical area showing a moderate expression of CD28. 1f). Paracortical area showing a low expression of CD28. 1g). Double staining of a sentinel node for CD3 (brown staining) and the zeta chain (red staining). Paracortical area showing a low expression of the zeta chain. 1h). Double staining of a sentinel node for CD8 (brown staining) and CD28 (red staining). Corresponding paracortical area showing a high expression of CD28.
In the CD8+ subset of T-lymphocytes (Table 3), a high degree of CD28 expression was found in all cases except two, which displayed a moderate degree.
Table 3 Degree of CD28 expression in the CD8+ T-lymphocyte subset in different areas of the sentinel nodes from 21 breast cancer patients (numbers are frequencies).
Expression of CD28 on CD8+
High Moderate Low
Primary follicles 19 2 0
Paracortex 15 5 1
Germinal centers
These B-cell areas, which develop as secondary follicles upon antigenic stimulation, could be analyzed in 16 patients. In the remaining five cases, no or very few germinal centers were found. In the CD4+ subset (Table 2), 4 of 16 patients exhibited a high or moderate degree of CD28 expression (Figure 1c), and 12 of 16 a low degree (Figure 1d). The small numbers of CD8+ T-lymphocytes in this compartment did not permit immunological analysis in any of the cases.
Paracortex
A markedly low expression of CD28 was observed in the paracortex, the principal T-cell activation area. Sections of 20 sentinel nodes could be analyzed. In the CD4+ subset (Table 2), none of the cases established a high CD28 expression, while it was moderate in 5 of 20 patients (Figure 1e), and low in 15 (Figure 1f). In contrast, 15 of 21 cases displayed a high CD28 expression in the CD8+ subset, 5 of 21 cases a moderate, and only one case a low CD28 expression (Table 3).
Comparison between CD28 and CD3-ζ expression
As described in a previous paper of our group [21], a low expression of CD3-ζ was seen in the primary follicles in 6 of 24 patients, in the germinal centers in 13 of 18 patients and in the paracortex in 19 of 24 patients. In the present study, the expression of CD28 was analyzed in parallel sections from the same patients.
The expression of CD28 and CD3-ζ was always compared in the same area of the same lymph node. Degrees of expression were only considered corresponding if a patient displayed exactly the same degree of expression (i.e. low, moderate or high) of both markers in that area.
In primary follicles, corresponding degrees of expression were found in 12 of 20 patients analyzing the CD4+ subset (Table 4), with a PABAK value of 0.40. Nevertheless, some sentinel node biopsies with a high CD3-ζ expression indicated a low (n = 3) or moderate (n = 3) CD28 expression on CD4+ T-cells. This discrepancy is hardly due to the presence of CD8+ T-cells as these cells presented a low or moderate CD28 expression in only one of these six biopsies. In the CD8+ subset, only 10 of 21 patients showed corresponding degrees (Table 7), resulting in a PABAK value of 0.21.
Table 4 Degree of CD28 expression on CD4+ T-lymphocytes in comparison with CD3-ζ expression in primary follicles. The table comprises 20 cases, where both analyses could be done in the same sentinel node biopsies (numbers are frequencies).
Expression of CD3-ζ Expression of CD28 on CD4+
High Moderate Low
High 5 3 3
Moderate 1 2 0
Low 1 0 5
Table 7 Degree of CD28 expression on CD8+ T-lymphocytes in comparison with CD3-ζ expression in the primary follicles. Parallel sections of all 21 sentinel node biopsies were analyzed (numbers are frequencies).
Expression of CD3-ζ Expression of CD28 on CD8+
High Moderate Low
High 10 1 0
Moderate 4 0 0
Low 5 1 0
In secondary follicles, 11 of 15 patients had the same degrees of CD28 and CD3-ζ in the CD4+ subset (Table 5), corresponding a PABAK value of 0.60. As earlier mentioned, the CD8+ subset was excluded from immunological analysis in this area due to the small numbers of CD8+ T-cells present.
Table 5 Degree of CD28 expression on CD4+ T-lymphocytes in comparison with CD3-ζ expression in secondary follicles (germinal centers). The table comprises 15 cases, where both analyses could be done in the same sentinel node biopsies (numbers are frequencies).
Expression of CD3-ζ Expression of CD28 on CD4+
High Moderate Low
High 0 1 1
Moderate 0 1 1
Low 0 1 10
In the paracortex, the degree of CD28 and CD3-ζ expression was corresponding in 15 of 20 cases in the CD4+ subset (Table 6). Here, the highest PABAK was observed, reaching a value of 0.62. As the vast majority of T-lymphocytes in the paracortex belonged to the CD4+ subset, the expression of the zeta chain by CD3+ T-cells actually represented the expression by CD4+ lymphocytes. In the CD8+ subset, only 2 of 21 patients displayed corresponding degrees (Table 8), leading to a PABAK value of -0.36. Instead, 11 patients with a low degree of CD3-ζ expression (figure 1g) showed a high CD28 expression in parallel sections (figure 1h).
Table 6 Degree of CD28 expression on CD4+ T-lymphocytes in comparison with CD3-ζ expression in the paracortex. The table comprises 20 cases, where both analyses could be done in the same sentinel node biopsies (numbers are frequencies).
Expression of CD3-ζ Expression of CD28 on CD4+
High Moderate Low
High 0 0 1
Moderate 0 2 1
Low 0 3 13
Table 8 Degree of CD28 expression on CD8+ T-lymphocytes in comparison with CD3-ζ expression in the paracortex. Parallel sections of all 21 sentinel node biopsies were analyzed (numbers are frequencies).
Expression of CD3-ζ Expression of CD28 on CD8+
High Moderate Low
High 1 0 0
Moderate 3 0 0
Low 11 5 1
Discussion
CD28 expression was studied in CD4+ and CD8+ T-lymphocyte subsets and compared with CD3-ζ expression in sentinel node biopsies from breast cancer patients. The lowest expression of CD28 was found in the CD4+ subset in the paracortex and germinal centers. In these areas, a moderate to good agreement between expression of CD28 on CD4+ T-cells and expression of CD3-ζ was observed. In the primary follicles, where CD28 expression on CD4+ T-cells was higher, the concordance between the two markers was less pronounced.
The CD8+ subset of T-lymphocytes displayed generally a higher degree of CD28 expression than the CD4+ subset. This applied for all analyzed regions of the lymph nodes. Moreover, the congruence with CD3-ζ expression was weaker than in the CD4+ subset.
The different functions of CD4+ and CD8+ T lymphocyte subsets are well documented. Some investigators suggest that CD8+ T-lymphocytes be closer connected to the CD28/B7 pathway than CD4+ T-cells [23]. Moreover, higher levels of B7 seem to be necessary to evoke an activation of CD8+ T-cells equivalent to CD4+ T-cells [24]. CD8+ T-cells from HIV positive individuals illustrate a more pronounced CD28 down-regulation than CD4+ T-cells, and their weakened proliferative response is marked by decreased Ca influx [25]. The presented data show a higher CD28 expression in CD8+ than in CD4+ T-cells, which stands in contrast to findings described in peripheral blood from healthy donors [7]. Thus, our results might suggest altered functions of CD4+ and CD8+ T-lymphocytes in the context of anti-tumor immunoreactivity.
It was proposed that T-lymphocytes from secondary lymphoid organs, rather than from peripheral blood, should be used to assess host immunoreactivity [26]. In bone marrow-derived T-cells from breast cancer patients, immunological changes are more pronounced than in peripheral blood lymphocytes [27,28]. Several studies document decreased CD28 and CD3-ζ expression in peripheral blood from breast cancer patients [2,4,20] in comparison to healthy individuals. However, there are no immunological data from axillary sentinel lymph nodes from normal controls. Even if such data would be most valuable in the interpretation of the present findings, it seems ethically unjustifiable to obtain such material. Still, the sentinel node biopsy might be the method of choice for assessing early immunological alterations in that the host immune response against tumors is initiated here. To confirm its distinct key position, future studies should include the comparative analysis of non-sentinel axillary lymph nodes in a distance-dependent fashion. In the present study, the down-regulation of CD28 and CD3-ζ clearly seems to be more accentuated in sentinel node biopsies than otherwise described in breast cancer patients.
Induction of T-cell anergy is one of the major tumor escape mechanisms [1], and its abrogation is therefore a main focus in the development of immunological anti-cancer strategies. Co-stimulation via the CD28/B7 pathway can prevent the induction of anergy in T-cell clones [29], but it is assumed that this might not be sufficient to reverse T-cell anergy once it is established [30]. Addition of exogenous IL-2 promotes reversal of T-cell anergy in this situation [31]. It was even suggested that decreased CD28 expression might be the result of continuous antigenic stimulation, aiming at reconstituting the non-responsiveness of T-cells [32]. The high numbers of CD4+/CD28- T-cells found in patients with rheumatoid arthritis are consistent with this theory [33]. Thus, the functional significance of decreased CD28 expression remains complex to interpret.
The functions of CD28 and CD3-ζ might be closely linked together. Engineered T-cells co-delivering CD28 activation in addition to the T-cell receptor (TCR) subunit CD3-ζ are more effective to activate an anti-tumor response in vivo than T-cells with TCR-CD3-ζ only [34]. Without co-stimulatory signaling via CD28/B7, tyrosine phosphorylation of the zeta chain of the T-cell receptor is inhibited [35]. Moreover, signaling via CD3-ζ could activate protein-tyrosine kinases that subsequently augment signal transduction via CD28/B7 [36]. The present data document a good agreement in the expression of the two markers in areas of pronounced down-regulation.
Several studies point towards a role for immunological treatment approaches in breast cancer [37-39]. The present data on decreased expression of CD28 and CD3-ζ in sentinel lymph nodes confirm that breast cancer has an impact on local immunoreactivity. However, the occurrence of varying individual patterns could be explained by the existence of both non-immunogenic and immunogenic, if not immunotoxic tumor types. Tumors with little inherent immunogenicity might not be as susceptible to immunotherapy as immunogenic tumor types [40]. Therefore, the decision whether to include immunotherapy in a treatment could be facilitated by analyzing the individual patient's immunological anti-tumor response. In this context, the sentinel node biopsy offers a unique opportunity to study early indicators of host-tumor interaction, and contributes valuable information for further conception and development of immunological treatment strategies.
Conclusions
The axillary lymph node status is of utmost importance for breast cancer prognosis and the choice of adequate adjuvant treatment. Today, the sentinel node biopsy technique is widely used as a diagnostic tool to gain that valuable information while limiting the operative procedure to a necessary minimum. However, it is also in the sentinel node that the immunoreactivity against the tumor is initiated.
In this study, a varying extent of immunosuppression was observed, represented by a decreased expression of CD28 and CD3-ζ in certain areas of the lymph node. The concordance between both markers was highest in the areas of most pronounced down-regulation, namely the paracortical region and the germinal centers. As immunological reactivity was not distributed evenly over the different areas of the lymph node, this might illustrate the dynamic nature of the immunological response to malignant disease within the complex functional anatomy of the lymph node. Thus, the immunohistochemical analysis of the sentinel node biopsy with respect to its preserved architecture might prove an important instrument for the evaluation of interactions between the tumor and the host immune system, helping to select patients who might benefit from adjuvant immunotherapy.
Authors' contributions
All authors participated in the study design and contributed with the collection of material and data. The microscopic analysis was carried out by AH and BG. All authors read and approved the manuscript.
Acknowledgements
The authors thank Karin Hellander and Catharina Tranaeus-Röckert at the Department of Pathology and Cytology, University Hospital of Linköping, for excellent technical help in performing the immunohistochemical staining. We also express great gratitude to Ulrika Hansson, Lena Scheibenpflug and their staff at the Department of Pathology, Central Hospital, Västerås, for preparing and providing the frozen sections. We would like to thank John Öhrvik from Centre for Clinical Research, Västerås, for statistical support. This work was supported by the County Council of Östergötland, Sweden.
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| 15613231 | PMC545070 | CC BY | 2021-01-04 16:39:24 | no | J Transl Med. 2004 Dec 21; 2:45 | utf-8 | J Transl Med | 2,004 | 10.1186/1479-5876-2-45 | oa_comm |
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BMC Mol BiolBMC Molecular Biology1471-2199BioMed Central 1471-2199-5-221559601110.1186/1471-2199-5-22Research ArticleChromosomal duplications and cointegrates generated by the bacteriophage lamdba Red system in Escherichia coli K-12 Poteete Anthony R [email protected] Anita C [email protected] Ashwini [email protected] Dept. of Molecular Genetics & Microbiology, University of Massachusetts Medical School, Worcester, MA 01655, USA2004 13 12 2004 5 22 22 20 9 2004 13 12 2004 Copyright © 2004 Poteete et al; licensee BioMed Central Ltd.2004Poteete 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 Escherichia coli strain in which RecBCD has been genetically replaced by the bacteriophage λ Red system engages in efficient recombination between its chromosome and linear double-stranded DNA species sharing sequences with the chromosome. Previous studies of this experimental system have focused on a gene replacement-type event, in which a 3.5 kbp dsDNA consisting of the cat gene and flanking lac operon sequences recombines with the E. coli chromosome to generate a chloramphenicol-resistant Lac- recombinant. The dsDNA was delivered into the cell as part of the chromosome of a non-replicating λ vector, from which it was released by the action of a restriction endonuclease in the infected cell. This study characterizes the genetic requirements and outcomes of a variety of additional Red-promoted homologous recombination events producing Lac+ recombinants.
Results
A number of observations concerning recombination events between the chromosome and linear DNAs were made: (1) Formation of Lac+ and Lac- recombinants depended upon the same recombination functions. (2) High multiplicity and high chromosome copy number favored Lac+ recombinant formation. (3) The Lac+ recombinants were unstable, segregating Lac- progeny. (4) A tetracycline-resistance marker in a site of the phage chromosome distant from cat was not frequently co-inherited with cat. (5) Recombination between phage sequences in the linear DNA and cryptic prophages in the chromosome was responsible for most of the observed Lac+ recombinants. In addition, observations were made concerning recombination events between the chromosome and circular DNAs: (6) Formation of recombinants depended upon both RecA and, to a lesser extent, Red. (7) The linked tetracycline-resistance marker was frequently co-inherited in this case.
Conclusions
The Lac+ recombinants arise from events in which homologous recombination between the incoming linear DNA and both lac and cryptic prophage sequences in the chromosome generates a partial duplication of the bacterial chromosome. When the incoming DNA species is circular rather than linear, cointegrates are the most frequent type of recombinant.
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Background
The Red recombination system of bacteriophage λ promotes efficient double strand break repair/recombination. An Escherichia coli strain in which RecBCD has been genetically replaced by Red exhibits greatly elevated levels of recombination between its chromosome and short linear double-stranded DNA species sharing sequences with the chromosome [1].
In previous studies, we have characterized a recombination event, pictured in Figure 1A, in which a 3.5 kbp dsDNA consisting of the cat gene and flanking lac operon sequences recombines with the E. coli chromosome to generate a chloramphenicol-resistant Lac- recombinant [2-4]. The dsDNA was delivered into the cell as part of the chromosome of a non-replicating λ vector, from which it was released by the action of the PaeR7 restriction endonuclease in the infected cell. Formation of Lac- recombinants was found to depend upon red and the bacterial recombination genes recA, recF, recO, recR, recQ, ruvAB, and ruvC. Large numbers of chloramphenicol-resistant Lac+ recombinants were generated in these crosses as well. In this study, we characterize the Lac+ recombinants and the processes which generate them. Most appear to arise from events in which homologous recombination between the incoming DNA and the chromosome generates a partial duplication of the bacterial chromosome.
Figure 1 λ lac::cat variants A. Recombination between a 3.6 kbp linear dsDNA fragment and lac genes in the bacterial chromosome. A non-functional lacZ::cat allele is generated by the recombination event pictured. The linear DNA is released from the chromosome of a non-replicating λ phage, by the action of PaeR7 restriction endonuclease in the infected cell. The lac genes are shown as oriented in the conventional E. coli map. Their transcription is from right to left; replication forks travel through them from left to right. B. Structures of the cat substitutions in the λ phages used in this study. White bars represent cat and adjacent sequences from Tn9 (1 kbp). Colored bars represent lac sequences (1.3 kbp or 40 bp). PaeR7 sites are represented by P. All the substitutions replace the same λ sequences, bp 23,135–33,498, with the indicated sequences and an additional 1.9 kbp of sequence from phage P22 gene 9 (not shown in the diagrams). λ sequences replaced by the substitutions include the attachment site, int, xis, exo, bet, gam, and cIII. The substitutions result in net deletions of 5.0–7.6 kbp from the λ chromosome.
Results and discussion
Genetic requirements for Lac+ recombinant formation
Data presented in Table 1 show that formation of Lac+ recombinants depends upon the same recombination functions as the Lac- recombinants: recA, recF, recO, recR, recQ, ruvAB, and ruvC. Deletion of recG increased the production of both Lac+ and Lac- recombinants. Lac+ recombinants accounted for 10–60% of the total chloramphenicol-resistant offspring of the crosses. These results strongly suggest that formation of the Lac+ recombinants takes place via homologous recombination, though they do not rule out the possibility that non-homologous end joining, or other varieties of "illegitimate" recombination, might be involved as well.
Table 1 Genetic requirements of Lac+ recombinant formation
Strain Relevant genotype Recombination % Lac+
recG+ background
507 wild type 1.00 ± 0.16 41
527 recA 0.01 ± 0.01 42
638 recQ 0.05 ± 0.02 37
540 ruvAB 0.34 ± 0.06 30
523 ruvC 0.22 ± 0.08 29
606 sulA 1.00 ± 0.31 40
608 sulA lexA 0.51 ± 0.01 44
615 sulA recF 0.12 ± 0.09 51
614 sulA recO 0.06 ± 0.00 42
625 sulA recR 0.03 ± 0.02 61
recGΔ background
554 wild type 1.00 ± 0.13 28
532 recA 0.01 ± 0.01 37
639 recQ 0.05 ± 0.03 46
559 ruvAB 0.34 ± 0.25 41
555 ruvC 0.08 ± 0.02 10
607 sulA 1.00 ± 0.14 37
609 sulA lexA 0.29 ± 0.12 22
628 sulA recF 0.03 ± 0.03 58
626 sulA recO 0.01 ± 0.01 21
627 sulA recR 0.02 ± 0.01 34
Bacterial strains were grown to log phase and infected at a multiplicity of 10 with λ lac::cat819 nin5. Infected cells were aerated for 1 hour at 37°C, then plated on LB agar and LB agar supplemented with chloramphenicol, isopropylthiogalactopyranoside, and X-Gal. Recombination frequencies represent the ratio of chloramphenicol-resistant recombinants to total cell titer, normalized to the ratio of the strain at the top of each group of strains. Means and standard errors for at least three measurements are shown. Actual frequencies were 507, 2.49%; 606, 2.50%, 554, 7.76%; 607, 7.81%. The frequencies of Lac- recombinants in these crosses were published previously (Poteete and Fenton, 2000).
The reason for considering mechanisms other than homologous recombination in the formation of the Lac+ recombinants is because of the expectation that some of the phage-borne lac::cat sequences would be attached to phage sequences. Heitman et al. [5] showed that double strand breaks generated by a restriction endonuclease in vivo are rapidly repaired by DNA ligase. The results of previous physical studies with other substituted λ phages cut in vivo by PaeR7 lead to the expectation that the λ lac::cat819 chromosome would be uncut, or only singly cut, much of the time in the infected cell [2,6].
Preliminary characterization of the Lac+ recombinants formed in cells which were wild-type for all the recombination functions revealed that most, possibly all, were unstable. When streaked on plates containing chloramphenicol and X-Gal, they segregated Lac- (colorless) progeny at variable frequencies. Southern gel analysis of chromosomal DNA from unstable Lac+ recombinants revealed that some of them were multiploid for lacZ (not shown). When the lac::cat dsDNA was delivered into the cell by a λ vector bearing a tetracycline resistance determinant (Δ nin::tet859 – described below) neither the Lac+ nor Lac- recombinants acquired tetracycline resistance. These findings suggest that the process which formed the recombinants did not involve the entire phage chromosome.
A baseline level of Lac+ recombinants was to be expected in these crosses. The recombination event pictured in Figure 1A, occurring in a cell with a pre-existing duplication of the lac locus, will produce a Lac+, chloramphenicol-resistant recombinant. Data presented below, in which simpler lac::cat recombining substrates generate Lac+ recombinants at a frequency approximately 1000-fold lower than Lac-, suggest the frequency of spontaneous lac duplications in Red+ but otherwise wild type E. coli is approximately 10-3, consistent with estimates of spontaneous duplication frequency in Salmonella made by Roth et al. [7]. The Lac+ recombinants formed in the crosses summarized in Table 1 occurred at a much higher frequency – approaching 5% of the infected cells – suggesting that pre-existing chromosomal duplications were not involved in the generation of the majority of the Lac+ recombinants. Apparently, normal Red-mediated homologous recombination between the phage and bacterial chromosomes duplicated or amplified sequences in the bacterial chromosome.
Multiplicity effects
The crosses described above were done by infecting log phase host cells grown in rich medium. Such cells contain multiple copies of their chromosome. Complications might arise if the lac::cat segment were to recombine with more than one chromosome at a time. Further complicating interpretation of the experiment, the cells were infected at a multiplicity of 10 phages per cell.
To reduce the complexity of the system, we switched to a cross procedure involving low multiplicity infection (0.1 phage per cell) of stationary phase cells. Presumably, under these conditions, most of the events producing chloramphenicol-resistant recombinants take place between single copies of the lac::cat segment and single copies of the bacterial chromosome. As shown in Table 2, the low copy infections produced substantial numbers of Lac+ recombinants, though fewer than the high copy infections (4–7% of the chloramphenicol-resistant progeny versus 28–40%). The Lac+ colonies produced in this way were still unstable, segregating Lac- progeny (colorless colonies) when restreaked, or suspended and plated, on medium containing chloramphenicol and X-Gal (not shown).
Table 2 Recombinant formation by λ lac::cat variants
phage description host genotypea % recombinant % Lac+
181 λ lac::cat819 cut both sides 507 wild 0.54 ± 0.06 7
197 λ lac::cat930 cut right 507 wild 0.92 ± 0.04 10
198 λ lac::cat931 cut left 507 wild 2.8 ± 0.07 0.2
196 λ lac::cat929 cut neither side 507 wild 0.055 ± 0.001 48
196 λ lac::cat929 cut neither side 839 hsdR 0.039 ± 0.002 29
181 λ lac::cat819 cut both sides 554 recG 0.93 ± 0.08 7
197 λ lac::cat930 cut right 554 recG 3.7 ± 0.04 13
198 λ lac::cat931 cut left 554 recG 4.6 ± 0.2 0.2
196 λ lac::cat929 cut neither side 554 recG 0.10 ± 0.03 47
186 λ lac::cat819 nintet 507 wild 0.28 ± 0.04 4
186 λ lac::cat819 nintet 849 pae 0.024 ± 0.003 45
186 λ lac::cat819 nintet 842 pae hsdR 0.027 ± 0.005 49
186 λ lac::cat819 nintet 850 pae recA 0.001 ± 0.002 95
186 λ lac::cat819 nintet 856 pae red 0.008 ± 0.008 52
208 λ cat988 nintet right flank only 849 pae 0.008 ± 0.003 58
209 λ cat989 nintet left flank only 849 pae 0.003 ± 0.0002 100
211 λ cat995 nintet no flank 849 pae 0.004 ± 0.0002 100
195 λ lac::cat921 short flank 507 wild 0.002 ± 0.001 30
195 λ lac::cat921 short flank 554 recG 0.002 ± 0.0002 50
Bacterial strains were grown to stationary phase without active aeration, and infected at a multiplicity of 0.1 with the indicated phages. Recombination frequencies represent the percentages of infected cells which became chloramphenicol-resistant recombinants; % Lac+ refers to the percentage of chloramphenicol-resistant recombinants which were Lac+. Means and standard errors for at least three measurements are shown. a. All strains have the Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 substitution, except as noted.
Recombinant formation by single-cut variants of λ lac::cat
To examine the dependence of Lac- and Lac+ recombinant formation on the structure of the DNA substrate, we constructed variants of λ lac::cat, which are diagrammed in Figure 1. λ lac::cat819, the progenitor of this series, has two PaeR7 sites. Upon infecting a host cell bearing the Δ recBCD::Ptac-gam-bet-exo-pae-cI substitution, it injects its chromosome, which circularizes. Expression of its lytic genes, including those necessary for phage DNA replication, is blocked by the action of cI repressor. Cutting of its chromosome by the PaeR7 restriction endonuclease in the infected cell releases a 3.5 kbp linear dsDNA, consisting of the cat gene and 1.3 kbp flanks of lac sequences on the right and left. (In this and subsequent descriptions, "right" refers to the upstream, or 5' end of the lac operon). The ends of the flanking lac sequences match precisely their counterparts in the chromosome.
λ lac::cat930 and λ lac::cat931 have only single PaeR7 sites, on the right and left, respectively. Cutting by PaeR7 in the infected cell should produce large linear dsDNAs related to the lac::cat819 fragment, but with long tails of non-homologous DNA extending from the left and right sides, respectively. Interestingly, both of these single-cut phages produce recombinants more efficiently than the double-cut λ lac::cat819, in both recG+ and Δ recG backgrounds (Table 2). The right-cut λ lac::cat930 produces relatively more Lac+ recombinants, whereas the left-cut λ lac::cat931 produces fewer. The frequency of Lac+ λ lac::cat931 recombinants, 0.2%, is roughly consistent with the expected frequency of pre-existing lac duplications in the population of infected cells, suggesting that λ lac::cat931 does not generate duplications, but λ lac::cat930 does. We cannot rule out the possibility that some of the 0.2% Lac+ recombinants made by λ lac::cat931 are cointegrates. Cointegrate formation is discussed further in the next section.
Recombinant formation by circular λ lac::cat
λ lac::cat929 has no PaeR7 site. Its chromosome should stay circular in the infected Δ recBCD::Ptac-gam-bet-exo-pae-cI cell. Surprisingly, it produces chloramphenicol-resistant recombinants only 10-fold less efficiently than λ lac::cat819. Unlike the PaeR7-cuttable λ lac::cat variants, λ lac::cat929 also efficiently produces weakly chloramphenicol-resistant microcolonies (not counted in the data presented in Table 2). The provenance of the microcolonies is readily understandable. The uncuttable λ lac::cat929 chromosome cannot replicate; it also is not destroyed, and should produce chloramphenicol acetyl transferase in the cell in which it resides. The infected cell, and perhaps its descendants for one or two generations, might be able to divide in the presence of low-concentration chloramphenicol.
Approximately half of the strongly chloramphenicol-resistant recombinants generated by the uncuttable phage λ lac::cat929 are Lac- (Table 2). This observation suggests that they are "legitimate" recombinants, in which the single chromosomal copy of lacZ is replaced by lacZ::cat. These recombinants were unexpected. All Red-mediated recombination events are thought to involve at least one linear partner [6]. How, then, can circular λ lac::cat929 recombine with the circular E. coli chromosome? Three possible explanations were considered. (1) Some of the λ lac::cat929 chromosomes might be cut by the EcoK restriction endonuclease. The infected cells contained a functional EcoK restriction-modification system. λ lac::cat929 was grown in a similarly EcoK+ host, and therefore was presumably EcoK-modified. However, if the modification were not complete, there might be residual EcoK restriction activity, resulting in 10% of the phage chromosomes being cut. (The phage chromosome contains five EcoK sites. If each site were 98% methylated, then approximately one in ten phage chromosomes would have a single unmethylated site). (2) The production of the recombinants might not be Red-mediated. (3) The linear partner might be a broken bacterial chromosome.
To test the first explanation, we replaced the gene encoding the EcoK restriction endonuclease, hsdR, with a tetracycline resistance determinant. This replacement had little effect on the formation of recombinants by λ lac::cat929 (Table 2).
To explore further the origins of the recombinants formed between circular phage chromosomes and the bacterial chromosome, we constructed a series of bacterial strains bearing variants of the Δ recBCD::Ptac-gam-bet-exo-pae-cI substitution lacking pae. In these hosts, no cutting takes place at PaeR7 sites, and so all of the lac::cat-substituted phages should remain circular. The results of crosses in these strains are shown in Table 2. As in the Pae+ crosses, elimination of hsdR in the Pae- background did not reduce the yield of recombinants. In the Pae- background, elimination of recA reduced the yield of recombinants twenty-fold. The great majority of the residual recombinants were Lac+, suggesting that their formation was by a process not necessarily involving homologous recombination. Elimination of red reduced the yield of recombinants three-fold, showing that most, but not all of the recombination events were Red-mediated. As in the Red+ cross, approximately half of the recombinants were Lac-, indicating that formation of both Lac- and Lac+ recombinants was Red-mediated in approximately the same proportions. Thus, the second hypothesis mentioned above, that the recombinants are formed by a Red-independent process, is not supported. The third hypothesis, that the linear partner might be a broken bacterial chromosome, is thus favored. It additionally seems reasonable, given that spontaneous double-strand breaks are frequent in E. coli [8].
We sought to detemine what proportion of the circular phage-by-chromosome recombinants were cointegrates, by testing for co-inheritance of cat and a tetracycline resistance-conferring element present at a remote location in the phage chromosome. Among the Lac+ recombinants produced in the Pae+ host TP507 (Table 2), only 2 out of 39 tested were found to be tetracycline-resistant; none of the 40 Lac- recombinants we tested was tetracycline-resistant. This result was consistent with our earlier finding that the great majority of recombinants formed in the high mulitiplicity infections of log phase cells acquired only the homology-flanked cat segment of the infecting λ chromosome. In contrast, in the Pae- host, 27 of 55 Lac- recombinants, and 23 of 25 Lac+ recombinants, were additionally tetracycline-resistant. These observations indicate that the most frequent Red-generated product of recombination between the uncut phage chromosome and the bacterial chromosome is a cointegrate.
Cointegrates could in principle be formed by four different single reciprocal recombination events between the circular λ lac::cat and bacterial chromosomes. These events could involve the right side lac sequences or the left side lac sequences, as diagrammed in Figure 2. A third event, not shown, involves recombination between the cI genes borne by both the phage and bacterial chromosomes (the latter at the substituted recBCD locus). A fourth event, also not shown, involves recombination between λ genes and homologues in cryptic prophages in the E. coli chromosome (discussed below). As suggested in the figure, cointegrates formed by recombination in the right-side lac flank are phenotypically Lac-, while recombination in the left-side lac flank (or other loci) forms Lac+ recombinants. To test this idea, we constructed variants of λ lac::cat missing either or both lac flanks; they are diagrammed in Figure 1. As shown in Table 2, in the non-cutting Pae- host, only the right flank-containing phage chromosome forms Lac- recombinants. The left-flank and no-flank phages form only Lac+ recombinants.
Figure 2 Cointegrates formed by recombination between circular λ cat and the bacterial chromosome. A. The cointegrate formed by recombination between the chromosome and λ cat989, which bears only the left-side lac flank, leaves the chromosomal lacZ gene intact. B. The cointegrate formed by recombination between the chromosome and λ cat988, which bears only the right-side lac flank, disrupts the chromosomal lacZ gene.
Short-flank recombination
The ability of the λ Red system to promote recombination events involving short sequence homologies makes it particularly useful for genetic engineering. Yu et al. [9] reported that a linear cat cassette with 1000 bp homologous flanks was only 10-fold more efficient than one with 40 bp flanks. Their experiments were done with cells which had been subjected to heat shock and electroporation. To test the efficiency of short-flank recombination under less extreme conditions, we constructed a short-flank λ lac::cat variant.
The fragment released by PaeR7 from the chromosome of λ lac::cat921 consists of the cat gene flanked by 40 bp sequences corresponding exactly to the terminal 40 bp at each end of the 1.3 kbp flanks of λ lac::cat819. As shown in Table 2, λ lac::cat921 exhibits a several hundred-fold lower efficiency of recombinant formation than its long-flank counterpart, in both recG+ and Δ recG backgrounds. λ lac::cat921 was similarly inefficient in log phase cells (data not shown). These observations suggest that short-flank recombination may not be a significant activity of the Red system in nature. However, they do not speak to the question of whether long flanks work better because they provide a larger homology target for synapsis, or because they provide non sequence-specific protection, perhaps delaying exonucleolytic degradation of the recombining sequences long enough to permit recombination to take place.
Recombination with inverted partners
The observation that Lac+ recombinants are formed efficiently by the right side-cut λ lac::cat930, but not by the left side-cut λ lac::cat931 (Table 2, discussed above) raised questions concerning the sequence determinants of this directionality. To explore these questions, we constructed locally inverted variants of the bacterial chromosome and the phages, as diagrammed in Figure 3.
Figure 3 Construction of inversions. A. A plasmid containing the lac operon and some flanking sequences was constructed. The SphI site was converted to a BsrG1 site, the large BsrG1 lac segment was inverted, and the inverted operon was crossed into the bacterial chromosome. Sequence segments between certain boundaries – the inversion endpoints, the ends of the lac flanks, and the 12 bp of lacZ replaced by the cat gene – are given letter designations to simplify representations of parent and recombinant chromosomes in crosses described below. B. The structure of λ lac::cat930 is represented as a linear map. T designates the left side flanking λ sequences shared by plasmids and phages, consisting of λ bp 22346–23134. 9 designates phage P22 bp 17725–15955. R is the right side λ flank, bp 33502–34504. P is the rest of the phage λ chromosome. The lac::cat segment was inverted relative to the flanking sequences in plasmids pTP930 and 931 (generating pTP1032 and 1033), and the inverted elements were crossed into λ. The structure of λ lac::cat1033 is illustrated.
The strategy for inverting the chromosomal lac operon consisted of five steps (detailed in the Methods section and Table 4): (1) deletion of the entire operon from the chromosome, replacing it with the cat gene; (2) cloning the cat gene from the deletion mutant, along with flanking chromosomal sequences; (3) using the cloned, plasmid-borne flanking sequences, and Red-mediated gap repair, to clone the lac operon in a plasmid; (4) inverting the lac operon, relative to its flanking sequences, in the plasmid; (5) replacing the Δ lac::cat chromosomal allele with the plasmid-borne lac-inv allele.
Table 4 Bacterial strains used in this study
Strain Relevant Genotype Source, reference, or construction
AB1157 Backgrounda
KM22 Δ(recC-ptr-recB-recD)::Plac-gam-bet-exo-kan Murphy, 1998
KM32 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-cat Poteete et al., 1999
TP507 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 "
TP523 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 ruvC53 eda::Tn 10 Poteete and Fenton, 2000
TP527 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 Δ(srl-recA)306::Tn10 "
TP532 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 recG258::kan Δ(srl-recA)306::Tn 10 "
TP540 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 Δ ruvAB6203::tet "
TP554 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 Δ recG6202 "
TP555 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 Δ recG6202 ruvC53 eda::Tn10 "
TP559 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 Δ recG6202 Δ ruvAB6203::tet "
TP606 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 Δ sulA6209::tet "
TP607 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 Δ recG6202 Δ sulA6209::tet "
TP608 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 Δ sulA6209::tet lexA71::Tn5 "
TP609 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 Δ recG6202 Δ sulA6209::tet lexA71::Tn5 "
TP614 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 Δ sulA6209::tet recO1504::Tn5 "
TP615 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 Δ sulA6209::tet recF400::Tn5 "
TP625 Δ (recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 Δ sulA6209::tet recR252::Tn10–9kan "
TP626 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 Δ recG6202 Δ sulA6209::tet recO1504::Tn5 "
TP627 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 Δ recG6202 Δ sulA6209::tet recR252::Tn10–9kan "
TP628 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 Δ recG6202 Δ sulA6209::tet recF400::Tn5 "
TP638 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 Δ recQ6216::tet "
TP639 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 Δ recG6202 Δ recQ6216::tet "
TP839 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 Δ hsdR::tet TP507 × PCR productb
TP842 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-cI978 Δ hsdR::tet TP849 × P1(TP839)
TP849 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-cI978 KM22 × pTP978 linearb
TP850 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-cI978 Δ recA6207::tet TP849 × P1(TP796)
TP856 Δ(recC-ptr-recB-recD)::Ptac-gam-cI996 KM32 × pTP996 linearb
MG1655 Background
TP798 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-cat MG1655 × P1(KM32)
TP829 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 TP798 × pTP822 linearb
TP832 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-bla979 TP798 × pTP979 linearb
TP872 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-bla979 Δ lac::cat TP832 × PCR productb
TP890 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 Δ lac::cat TP829 × P1(TP872)
TP894 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 lac-inv TP890 × pTP1034 linearb
TP896 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-bla979 lac-inv TP872 × pTP1034 linearb
MDS12 Backgroundc
TP750 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 Poteete, 2004
TP796 Δ(recC-ptr-recB-recD)::Ptac-gam-bet-exo-pae-cI822 Δ recA6207::tet "
a. These strains are presumed to bear all the other genetic markers of AB1157 (Bachmann BJ: Derivations and genotypes of some mutant derivatives of Escherichia coli K-12. In: Escherichia coli and Salmonella: cellular and molecular biology. Edited by Neidhardt FC, III RC, Ingraham JL, Lin ECC, Low KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE. Washington, D.C.: ASM Press; 1996: 2460–2488).
b. Plasmids and PCR products used in the construction of some of the strains are described in the Methods section.
c. MDS12 is a derivative of wild type E. coli strain MG1655 bearing 12 large deletions (Kolisnychenko et al., 2002).
The single-cut lac::cat930 and lac::cat931 alleles were constructed by replacing the left-side and right-side PaeR7 sites, respectively, of pTP819, with XbaI sites. To produce inverted derivatives of these two alleles, the plasmids pTP930 and pTP931 were digested with XbaI and XhoI (a PaeR7 isoschizomer); lac::cat inserts and bla-ori backbone fragments from the two plasmids were exchanged. The inverted alleles were then crossed into phage λ, as described in the Methods section.
In constructing the chromosomal inversion, we switched genetic backgrounds, from the AB1157-derived strains with which most recombination studies have been done, to MG1655, the sequenced wild type E. coli K-12 [10]. In the MG1655 background, the same directionality of Lac+ recombinant formation was observed. The efficiencies of recombination of the MG1655 derivatives with both λ lac::cat930 and λ lac::cat931, and the efficiency of Lac+ recombinant formation by λ lac::cat930, were slightly elevated relative to those in the corresponding AB1157-derived strain.
The eight combinations of normal and inverted phages and bacteria (left- and right-cut phages, and their inverted counterparts, in normal and lac-inverted E. coli) were tested for Lac+ recombinant formation. The results are shown in Figure 4. Only two of the eight combinations produced large numbers of Lac+ recombinants: lac::cat930 (right-cut, normal orientation) in lac-wild type, and lac::cat1033 (left-cut, inverted orientation) in lac-inv.
Figure 4 Normal and inverted phage-by-chromosome crosses. Phages bearing the indicated lac::cat alleles were crossed with wild type (TP829) and lac-inv (TP894) bacteria. Sequence segments are designated as shown in Figure 3. Bacterial sequences are colored blue, phage sequences yellow, and the cat gene white. The percentages of Lac+ bacteria among the chloramphenicol-resistant recombinant progeny are indicated, as the means and standard errors from three measurements. Crosses were done by low multiplicity infection of stationary phase cells. The total yields of chloramphenicol-resistant recombinants ranged from .02 to .04 per infected cell.
Recombination with electroporated linear DNA species
The linear DNA produced by cutting of the λ lac::cat phages at a single site is large and complex relative to the lac::cat segment itself. To reduce the complexity, we generated linear DNA species which correspond to shortened versions of the single-cut phage chromosomes. The DNA species were generated by transferring, into a conditionally-replicating vector, parts of the plasmids previously used to introduce the lac::cat substitutions into λ. The vector can replicate only in a host which supplies the plasmid R6K Pir protein [11]. Details of the plasmid constructions are given in the Methods section. Plasmids bearing the cloned lac::cat and flanking sequences from λ were digested with restriction enzymes, and the DNA fragments were introduced into bacteria by electroporation. The results of some of these crosses are shown in Figure 5. The electroporated DNAs faithfully mimicked their single-cut phage counterparts: only the two crosses corresponding to the high Lac+ producer crosses of Figure 4 generated high proportions of Lac+ recombinants.
Figure 5 Normal and inverted linear DNA-by-chromosome crosses. Plasmids bearing the indicated lac::cat alleles (corresponding to the phages in Figure 4; the numbers of the plasmids bearing these alleles are given in the Methods section) were digested with BamHI and XhoI (PaeR7 isoschizomer), and electroporated into wild type and lac-inv bacteria (strains TP832 and TP896). Sequence segments are labeled as in Figure 3. The percentages of Lac+ bacteria among the chloramphenicol-resistant recombinant progeny are indicated, as the means and standard errors from two measurements. The total yields of chloramphenicol-resistant recombinants ranged from 20,000 to 200,000 per pmol.
The observation that only DNAs with sequences designated "T-9" on the left side generated many Lac+ recombinants (Figure 5) led us to try a linear species in which T was detached by digestion with a restriction enzyme (KpnI). Removal of the T segment greatly reduced the ability of the linear DNA to generate Lac+ recombinants (Table 3). A BLAST search of the E. coli genome for sequences related to T revealed four loci in MG1655 with close matches to T's leftmost 200 bp, which constitute the C-terminal third of the λ tfa gene. The four bacterial sequence segments are located in cryptic prophages: ybcX and an unnamed gene fragment in prophage DLP12, tfaR in prophage Rac, and tfaQ in prophage Qin. The locations and orientations of the first three of these are such as to permit a linear DNA species to recombine both with them and with the lac locus. Such an event is diagrammed in Figure 6, in which the three recombining bacterial loci are designated I, II, and III; closely related events have been extensively documented in other studies [7]. In the pictured cross, recombination between the linear DNA and the bacterial chromosome at lac and I generates a recombinant bearing intact lac, lac::cat, a duplication of all sequences between lac and I, a smaller duplication of the left lac flank, and an insertion of λ sequences unrelated to the cryptic prophages (see Figure 6c).
Table 3 Recombinant formation by variant linear DNA species
Plasmid Restriction digest Recombining linear species % Lac+
1019 Bam + Xho T9ACB 27 ± 2
1019 Bam + Xho + Kpn 9ACB 0.6 ± 0.2
1047 Bam + Xho T9ACB(Δχ) 27 ± 2
1052 Bam + Xho TACB 14 ± 0.1
1055 Sph + Xho GCB 95 ± 3
1056 BsrG + Xho ACF 99 ± 0.5
1057 Bam + Xho TCB 96 ± 2
1018 Bam + Xho ACB 0.2 ± 0.04
The indicated DNAs were introduced into TP832 by electroporation. DNA segments are labeled as in Figures 3–5. B(Δχ) is a variant of the 1.3 kbp right-side lac flank from which the 396 bp immediately adjacent to the cat segment (C) have been deleted, eliminating a Chi site. Figures indicate the means and standard errors from two measurements. Numbers in the first row are from Figure 5, top left, and are repeated here for comparison.
Figure 6 Model for Lac+ recombinant formation in linear DNA-by-chromosome crosses. (a) Recombination between the B sequence segments generates two broken chromosome arms. In the diagram, which is not to scale, I, II, and III designate the cryptic prophage tfa homologues ybcX in the DLP12 prophage remnant at 0.58 Mbp in the MG1655 chromosome, a closely linked unnamed pseudogene in the same element, and tfaR in the Rac prophage at 1.43 Mbp, respectively. The lac operon is at 0.36 Mbp. (b) The T segment-ended arm can recombine with any of the three T segments in an unbroken copy of the chromosome, generating a Lac+ chloramphenicol-resistant recombinant. Short arrows indicate the primers used to demonstrate the unique junction formed in the type I recombinant. (c) The recombinant contains two repeated sequence segments, a short one consisting only of the A segment (1.3 kbp), and a long one (0.2 or 1.1 Mbp) consisting of bacterial sequences from B through T.
The hypothesis that the high-frequency Lac+ chloramphenicol-resistant recombinants are generated by homologous recombination with cryptic prophage sequences in the chromosome predicts that such recombinants would not be generated in an E. coli strain in which the cryptic prophages are deleted. To test this prediction, we electroporated linear lac::cat930 DNA into TP750, a Red+ derivative of MDS12, the reduced-genome E. coli strain constructed by Kolisnychenko et al. [12]. In this cross, Lac+ recombinants constituted only 0.06% (average of 6 measurements) of the total chloramphenicol-resistant progeny, a frequency no higher than expected for spontaneous pre-existing duplications in the bacterial chromosome.
The hypothetical Lac+ chloramphenicol-resistant recombinant pictured in Figure 6 has some predicted properties which were confirmed experimentally. First, it is predicted to segregate Lac- recombinants at low frequency, and chloramphenicol-sensitive recombinants at high frequency, the results of recombination between the short and long duplicated segments, respectively. Overnight cultures of three Lac+ recombinants grown in the absence of selection were found to include Lac- chloramphenicol-resistant clones at an average frequency of 0.03%, and Lac+ chloramphenicol-sensitives at 30%. Second, it should be possible to demonstrate specific duplication junctions in each of the three types of recombinants by the use of PCR (see Figure 6c). Primers were designed to this end, and employed in colony PCR with a collection of 12 Lac+ recombinants. Each of the 12 was found to have one of the three predicted junctions: seven were type I, two were type II, and three were type III. Examples are shown in Figure 8.
Figure 8 PCR products indicating specific duplication junctions. Colonies of Lac+ recombinants were tested by PCR as described in the Methods section. Lanes labeled S are standards (1 kb ladder, Invitrogen). Lanes 1, 2, and 3 are type I, II, and III recombinants, respectively, formed by electroporation of cells with the lac::cat930 DNA species diagrammed in Figure 5. The sizes of the expected products are: type I-1272 bp, II-1026 bp, III-1872 bp. Lane 4 is a recombinant formed by electroporation of cells with the GCB DNA species diagrammed in Figure 7. The size of the expected product is 3032 bp. Lane 5 is a recombinant formed by electroporation of cells with the ACF DNA species diagrammed in Figure 7. The size of the expected product is 2765 bp. Lane 6 is a recombinant formed by infection of cells with λ lac::cat819. The expected size of the product is 3653 bp. Lanes 7–10 are wild type cells used as template in control PCRs with the primers used in lanes 1–3, 4, 5, and 6, respectively.
We constructed several other linear DNA substrates to test the generality of the model in Figure 6. The abilities of these substrates to generate Lac+ recombinants are shown in Table 3. The first of these was a derivative of lac::cat930 lacking its χ site. The χ site is located in the right lac flank, which is labeled "B" in Figures 3–6, close to the cat gene. The orientation of this χ site is such that it would be expected to interact productively with RecBCD enzyme approaching from the PaeR7- or Xho-generated right end of lac::cat930. While no activity of χ in these crosses was expected, as the bacteria lack RecBCD, the directionality of Lac+ recombinant formation was reminiscent of the directionality of χ-RecBCD interaction (see [13] for a review). The Δχ substrate was just as active as the χ + version, showing that χ does not contribute significantly to Lac+ recombinant formation. Similarly, a lac::cat930 derivative (pTP1052) lacking its sequences derived from phage P22 was equally proficient at generating Lac+ recombinants. A derivative missing the left lac flank made Lac+ recombinants almost exclusively; the small number of Lac- recombinants in this case probably represent cointegrates made by uncut plasmid DNA in the fragment preparation.
Plasmids pTP1055 and 1056 were constructed to test whether duplications of the type pictured in Figure 6 could be generated by Red-mediated recombination involving arbitrarily chosen sequences on either side of lac in the chromosome. As indicated in Table 3, linear DNAs from these plasmids also generated Lac+ recombinants almost exclusively. The structures of the plasmids, chromosome, and expected recombinants are diagrammed in Figure 7. That the expected duplications were in fact generated was demonstrated by the production of duplication-spanning products in PCR using the primers Ddi and Edi, described in the Methods section and represented schematically in Figure 7. Examples are shown in Figure 8.
Figure 7 Red-generated duplications to the left and right of lac. Recombination between electroporated linear dsDNA species ACF (left) and GCB (right) generate duplications of chromosomal sequences to the left and right of lac, respectively, through a sequence of recombination events as pictured in Figure 6. (a) In the diagram, the first events are crossovers between the A or B lac segments, with crossovers between the associated F or G flanks pictured as occurring second (b). The same recombinants would also be formed if the orders were reversed, for example, if F recombined before A (not shown). Short arrows indicate the primers initiating DNA synthesis divergently from the D and E segments. (c) In the recombinants, the primers generate PCR products spanning the duplications, of 2765 and 3032 bp, respectively.
Structures of the λ lac::cat Lac+ recombinants
The major class of Lac+ chloramphenicol-resistant recombinants formed in crosses involving infection by λ lac::cat819 (cut left and right) and λ lac::cat930 (cut right) behave like their counterparts generated by electroporation of linear dsDNAs into Red+ cells: they segregate Lac- chloramphenicol-resistant clones at low frequency, and Lac+ chloramphenicol-sensitives at high frequency (data not shown). In addition, their formation also depends upon the presence of cryptic prophages in the chromosome: λ lac::cat819 was found to produce Lac+ chloramphenicol-resistant recombinants as only 0.13% (average of six measurements) of the total chloramphenicol-resistant progeny in crosses with TP750. These properties suggested both kinds of recombinant might have the same structures as well, but PCR tests of 50 of the phage-generated recombinants with the primers used to demonstrate type I, II, and III recombinants (Figure 8) were negative (data not shown).
Further computer analysis revealed a cryptic prophage sequence which could recombine with the phages, but not with the shorter, plasmid-derived linear DNAs, producing recombinants by the mechanism drawn in Figure 6: a 3.5 kbp patch of DLP12 closely matching sequences in the vicinity of the λ cos site. This site is located immediately to the left of the tfaD locus in the chromosome. Recombinants formed by crossing over at this site and at the right lac flank would be expected to contain duplications of bacterial sequences identical to those of type I recombinants (pictured in Figure 6), but to contain a larger part of the phage chromosome as well. PCR tests of six λ lac::cat819 and six λ lac::cat930 recombinants with primers designed to demonstrate cos recombinant junctions showed that all twelve had them. An example is shown in Figure 8.
The predominance of cos recombinants over tfa recombinants among the phage-generated Lac+ recombinants is to be expected. First, λ Exo, traveling in from the end of the long left-side non-homologous tail of λ lac::cat930 encounters cos before tfa; cos therefore presumably has a kinetic advantage. Second, the bacterial cos homology patch is significantly larger than the tfa homologies. A third possible advantage of cos is that, at least in some parts of the phage lytic cycle, it is the site of a double-strand break for the DNA encapsulation step of phage assembly. We expected that cos would be unbroken almost all the time in our crosses. The λ chromosome is linear at the time of injection, but is rapidly circularized by annealing and ligation [14]. The other parts of the phage lytic cycle are inhibited by the presence of cI repressor in the infected cells. Even so, the hypothesis that Red sometimes manages to gain access to cos ends in these crosses remains plausible; particularly so because Red is present in the cell at all times, whereas, in a normal infection, Red is not present until its genes are expressed from the phage chromosome. An unexpectedly significant presence of cos ends in the infected cells could also help explain the otherwise surprisingly high frequency of chloramphenicol-resistant recombinants seen in crosses involving phage chromosomes not cut by restriction endonucleases, described above.
One aspect of Lac+ recombinant formation by λ lac::cat819 is not explained by the model of Figure 6. The λ lac::cat819 contains two PaeR7 sites. Cutting by PaeR7 should release a linear dsDNA with lac flanks and no attached non-lac DNA. This DNA species did not generate Lac+ recombinants (above the background of pre-existing duplications) when electroporated directly into cells (Table 3). How does it apparently do so in the infected cells? As discussed above, it is expected that the λ lac::cat819 chromosome would be uncut, or only singly cut, much of the time in the infected cell. If it recombined at a time at which it was cut only on the right, the type of recombinant pictured in Figure 6 could be formed. In this recombinant, an uncut and unmodified PaeR7 site would sit in the chromosome. Presumably, the presence of this site would make the recombinant unstable, initially; but the PaeR7 site might eventually become modified, that is, escape restriction. The pae-expressing strains employed in these experiments restrict plaque formation by single PaeR7 site-bearing λ phages only approximately 5-fold (unpublished data).
Conclusions
The bacteriophage λ Red recombination system is of general interest for two main reasons. First, it is an intensively characterized and relatively simple system, which serves as a model for studies of homologous recombination [15-17]. Second, it has emerged as a powerful tool for genetic engineering in gram-negative enteric bacteria [1,9,18-24]. It was therefore of interest to elucidate the structures of the previously observed but unexplained Lac+ recombinants formed by recombination between lac::cat-bearing λ phages and the bacterial chromosome, as well as the mechanism of their formation. Our studies uncovered a diversity of recombinant structures, including complex duplications and cointegrates. Recombination between λ and cryptic prophage sequences in the bacterial chromosome was found to be the most significant mechanism generating Lac+ recombinants. All of the Lac+ recombinants could be generated by homologous recombination events of types which have been previously described. In particular, there was no evidence for end-joining or other non-homologous recombination events. Perhaps the most surprising finding was the high frequency with which large duplications in the bacterial chromosome – up to 1 Mbp – could be generated by the Red system.
Methods
Bacteria
E. coli strain DH5α (λ pir) [11] was used for propagation of plasmids bearing the R6K origin. Other strains used in this study are described in Table 4. The Δ hsdR::tet allele in TP842 was constructed by using a Tn 10-containing E. coli strain as template in PCR with primers hsdRut (5'-TTGGACAGGCCCGCACAGCAATGGATTAATAACAATGATGCTCGACATCTTGGTTACCGT-3') and hsdRdt (5'-GCTGAATTTGCCCAGCAGGGTATCGAGATTATCGTCAAAGCGCGGAATAACATCATTTGG-3'). The Δ lac::cat allele in TP872 was constructed by using a Tn 9-containing E. coli strain as template in PCR with primers cat15 (5'-TCTGGTGGCCGGAAGGCGAAGCGGCATGCATTTACGTTGAATGAGACGTTGATCGGCACG-3') and cat16 (5'-AGAGTACATCTCGCCGTTTTTTCTCAATTCATGGTGTACAATTCAGGCGTAGCACCAGGC-3').
Plasmids
Δ nin::tet
An EcoR1 fragment of λ cI857 Sam7 containing the nin region genes was cloned into the EcoR1 site of pBR322. In the resulting plasmid, pnin, the nin genes are read clockwise in the conventional map of pBR322. pTP772 was constructed by deleting sequences between the two HindIII sites of pnin. pTP859 was constructed by cutting pTP772 with SacII and ClaI, blunting the ends with T4 DNA polymerase, ligating with NotI linkers (5'-AGCGGCCGCT-3'), cutting with NotI, and ligating with a NotI fragment of pTP857 [25] containing the tetR and tetA genes of transposon Tn10.
lac::cat PaeR7 site variants
pTP819, in which the cat gene is flanked on both sides by lac sequences, PaeR7 sites, and λ sequences (for crossing into the phage), has been described [2]. pTP828 and pTP829 were described previously (without names) as intermediate plasmids in the construction of pTP819 [2]; each bears the cat gene with a single lac flank and PaeR7 site. pTP922 was constructed by deleting the lac and cat sequences between the two PaeR7 sites in pTP819. (In this and other plasmid constructions, PaeR7 sites were cut with XhoI, an isoschizomer). pTP926, pTP927, and pTP928 were constructed by ligating the oligonucleotide 5'-TCGACAGTCTAGACTG-3' into the PaeR7 sites of pTP828, pTP829, and pTP922, respectively, eliminating the PaeR7 sites and replacing them with XbaI sites. pTP929 was constructed by ligating the XbaI-NcoI fragment of pTP926 containing the N-terminal coding sequences of cat and the NcoI-XbaI fragment of pTP927 containing the C-terminal coding sequence of cat into the XbaI site of pTP928. The orientation of the reconstructed cat gene is the same as in pTP819. pTP930 was constructed by ligating together the large ApaI-SacII fragment of pTP929 and the small ApaI-SacII fragment of pTP819. pTP931 was constructed by ligating together the small ApaI-SacII fragment of pTP929 and the large ApaI-SacII fragment of pTP819.
pTP921 (short-flank lac::cat)
pTP921 was constructed by ligating together two XhoI-digested DNAs: pTP922 and a PCR product made by amplifying the cat gene from a Tn9-containing E. coli strain with primers 5'-GACGCACTCGAGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGCCGGCCACTGGAGCACCTCAAAAACACCA-3' and 5'-GACGCACTCGAGGCACACAGCGCCCAGCCAACACAGCCAAACATCCGCGCGGGCCCGACCGGGTCGAATTTGCTTTCGAA-3'. The presence of the expected sequences from the synthetic oligonucleotides in pTP921 DNA was verified by automated sequencing (data not shown).
lac flank variants
pTP988 (left lac flank only) was constructed by replacing the XbaI-ApaI lacZY segment of pTP930 with a mixture of two oligonucleotides, 5'-CTAGTTGCAAGCTTGGGCC-3' and 5'-CAAGCTTGCAA-3'. pTP989 (right lac flank only) was constructed by replacing the NgoMI-XhoI lacZ segment of pTP930 with a mixture of two oligonucleotides, 5'-CCGGCAAGCTTGCTGGTGGGCAA-3' and 5'-TCGATTGCCACCAGCAAGCTTG-3'. pTP995 (no lac flank) was constructed by ligating together the small NcoI fragment of pTP988 and the large, ori-containing NcoI fragment of pTP989.
Inverted lac::cat
pTP1032 was constructed by ligating together the XbaI- and PaeR7-ended cat-containing fragment of pTP930 and the XbaI- and PaeR7-ended ori-containing fragment of pTP931. pTP1033 was constructed by ligating together the XbaI- and PaeR7-ended cat-containing fragment of pTP931 and the XbaI- and PaeR7-ended ori-containing fragment of pTP930.
Inverted lac
pTP1016 was constructed by ligating into the NotI site of pTP809 [25] the NotI-digested PCR product made by amplifying the cat gene and flanking sequences from strain TP872 (Δ lac::cat) with primers 5'-CATCATCACGCGGCCGCGACGTTTGCCGCTTCTGAA-3' and 5'-ATCATCCACGCGGCCGCTGCGTTTTGCACCAGTACG-3'. In pTP1016, the cat gene is flanked closely by unique SphI and BsrGI sites. pTP1027 was constructed by electroporating SphI- and BsrGI-digested pTP1016 into strain TP829 (lac+); a plasmid formed by gap repair was isolated. pTP1028 was constructed by ligating SphI-digested pTP1027 with the oligonucleotide 5'-GTTGTACAACCATG-3', converting the SphI site into a BsrGI site. pTP1034 was constructed by cutting pTP1028 with BsrGI, ligating the two fragments back together, and screening for plasmids in which the lac genes were inverted relative to their flanking sequences.
R6K oriγ lac::cat plasmids
pTP1029, a tetracycline resistance bearing vector capable of replicating only in cells expressing R6K pir function, was constructed by ligating together two AatII- and Bam-digested DNA species: a PCR product made by amplifying a Tn10-containing E. coli strain with primers 5'-TCAACGTAAATGCATGGACGTCCTCGACATCTTGGTTACCGT-3' and 5'-TGTACACCATGAATTGGATCCCGCGGAATAACATCATTTGG-3'; and the ori-containing fragment of plasmid pLD54 [26]. pTP1018, 1019, 1020, 1044, and 1045 were constructed by ligating the BamHI lac::cat-containing fragments of pTP819, 930, 931, 1032, and 1033, respectively, into the BamHI site of pTP1029.
Rearranged lac::cat variants (Table 3)
pTP1047 was constructed by ligating the complementary oligonucleotides 5'-TCGTCTAGAGT-3' and 5'-CCGGACTCTAGACGAAGCT-3' between the SacI and NgoMIV sites of pTP1019. pTP1051 was constructed by ligating the complementary oligonucleotides 5'-CAGCATGCAT-3' and 5'-CTAGATGCATGCTGGTAC-3' between the KpnI and XbaI sites of pTP930. pTP1052 was constructed by ligating the complementary oligonucleotides 5'-CAGCATGCAGGGCC-3' and 5'-CTGCATGCTGGTAC-3' between the KpnI and ApaI sites of pTP1019. pTP1053 was constructed by ligating the complementary oligonucleotides 5'-GGCATGCAGGTTCTTTGAGTCCTTTGGGCGGCCGCGGGCC-3' and 5'-CGCGGCCGCCCAAAGGACTCAAAGAACCTGCATGCCGC-3' between the SacII and ApaI sites of pTP1019. pTP1054 was constructed by ligating the complementary oligonucleotides 5'-CCGGCGCGGCCGCAGGTTCTTTGAGTCCTTTGGTGTACAGAGCT-3' and 5'-CTGTACACCAAAGGACTCAAAGAACCTGCGGCCGCG-3' between the NgoMIV and SacI sites of pTP1020. pTP1055 was constructed by ligating the small SphI-NotI fragment of pTP1016 between the SphI and NotI sites of pTP1053. pTP1056 was constructed by ligating the small NotI-BsrGI fragment of pTP1016 between the NotI and BsrGI sites of pTP1054. pTP1057 was constructed by ligating the BamHI cat-containing fragment of pTP1051 into the BamHI site of pTP1029.
Δ recBCD::Ptac-gam-bet-exo-pae-cI variants
pTP822, which bears a synthetic Ptac-gam-bet-exo-pae-cI operon flanked by sequences upstream from recC on one side and sequences internal to recD on the other, has been described [2]. pTP978 was constructed by ligating together two NcoI- and XbaI-digested DNA species: pTP822, and a PCR product made by amplifying the cI gene from E. coli strain TP507 with the same primers used in the construction of pTPP822. This construction has the effect of simply deleting the pae genes from pTP822. pTP996 was constructed by deleting the C-terminal coding sequences of bet and the N-terminal coding sequences of exo between the two HpaI sites of pTP978. pTP979 was constructed by ligating together two NcoI- and XbaI-digested DNAs: pTP822 and a PCR product made by amplifying the bla gene from pBR322 with primers 5'-CCACCAATCATCCATGGCGCGGAACCCCTATTTGTTT-3' and 5'-TTGTTGGACGATCTAGAGGTCTGACAGTTACCAATGC-3'. This construction has the effect of replacing pae and cI with bla.
Phages
λ lac::cat819 and λ lac::cat819 nin5 have been described [2]. λΔ nin::tet859 was made by crossing λ wild type with plasmid pTP859, infecting a tetracycline-sensitive strain with the resulting lysate, and selecting a tetracycline-resistant lysogen. The Δ nin::tet859 substitution replaces λ bp 40388–43825 with the tetR and tetA genes of transposon Tn 10.
The lac::cat921, 929, 930, 931, 988, 989, 995, 1032, and 1033 substitutions were crossed into λ wild type and/or λ Δ nin::tet859. The parent phages were crossed with the cat substitution-bearing plasmids. Phages which had acquired the substitution were either selected by plating on a strain lysogenic for phage P2 (Spi- phenotype), or identified by their clear-plaque morphologies (all the cat substitutions replace the cIII gene). The structures of the substituted phages were all verified by their ability to generate specific products when used as templates in PCR (not shown).
Crosses
Phages were crossed with plasmids by spotting enough phage to make a confluent zone of lysis on a lawn of a sensitive bacterial strain bearing the parent plasmid. After overnight incubation at 37°C, material from the zone was collected in TM (10 mM Tris-HCl pH 7.5, 10 mM MgSO4), and shaken with chloroform. Two methods for crosses monitoring recombination between phage-injected DNA and the bacterial chromosome, both previously described, were employed: high-multiplicity infection of log phase cultures [27], and low-multiplicity infection of cells grown to stationary phase by standing overnight incubation [4].
Crosses involving recombination between bacterial chromosomes and electroporated DNA fragments were carried out as described by Murphy and Campellone [23]. The DNA fragments were generated by digesting various plasmids with restriction endonucleases. Amounts of DNA used varied among experiments, corresponding to 250–500 ng of the 3595-bp lac::cat Xho fragment; within an experiment, equimolar amounts of different recombining linear species were used. DNA was quantitated by running samples in an agarose gel, staining with ethidium bromide, and measuring fluorescence of the bands by the use of a Kodak Gel-Logic 200 system with 1-D software.
PCR
The structures of various Red-generated duplications in the E. coli were verified by PCR. Colonies were picked and added to 40 μl mixtures containing 1 unit Taq polymerase, 63 μM dNTPs, 1.88 mM MgCl2, 5% (v/v) dimethylsulfoxide, 20 mM Tris-HCl pH 8.4, 50 mM KCl, and primers at 0.7 μM. Samples were heated to 95C for 5 min, then put through 30 cycles of 94C for 1 min, 55C for 1 min, 72C for 2–4 min, depending upon the length of the expected products. Primers for demonstrating the type I, II, and III tfa junctions were combined in a single mixture of 4 oligonucleotides: Jsp1 (GTTGAATGGGCGGATGCTAA), Jsp2 (TCTTCCACCAGAAAGCTACC), JncA (TGCCGTGTGAACGGTTTAC), and Jnc2 (TTCTAGCCCCATCATCTGTG). Primers for spanning the ACF-generated duplication (see Figure 7) were Ddi1 (CTCTTTCCGTTACGGGACAC) and Ddi2 (TGGTGAACATGATGCCGACA). Primers for spanning the GCB-generated duplication were Edi1 (CGCCGAAATCCCGAATCTCT) and Edi2 (ACCGGCATACTCTGCGACAT). Primers for demonstrating the DLP12 cos junction were Csj1 (GATTGAGCGTGAAGTCTGTTTGTG) and Csj2 (CGAATAGTCGGCTCAACGTGGGTT).
Abbreviations
PCR: polymerase chain reaction. X-Gal: 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside. IPTG: isopropyl β-D-thiogalactopyranoside. bp: base pairs.
Authors' contributions
ACF carried out the experiments summarized in Table 1. AN and ARP carried out the experiments summarized in Table 2. ARP carried out the other experiments and wrote the paper. ARP, ACF, and AN constructed the plasmids, phages, and bacterial strains.
Acknowledgements
Yanxia Guo and Daniel Aiello provided excellent technical assistance. ARP thanks Kenan Murphy for helpful advice and discussions. This research was supported by grant MCB-0234991 from the National Science Foundation and grant GM51609 from the National Institutes of Health.
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| 15596011 | PMC545071 | CC BY | 2021-01-04 16:48:00 | no | BMC Mol Biol. 2004 Dec 13; 5:22 | utf-8 | BMC Mol Biol | 2,004 | 10.1186/1471-2199-5-22 | oa_comm |
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BMC PharmacolBMC Pharmacology1471-2210BioMed Central London 1471-2210-4-311557595510.1186/1471-2210-4-31Research ArticleCharacterisation of cytotoxicity and DNA damage induced by the topoisomerase II-directed bisdioxopiperazine anti-cancer agent ICRF-187 (dexrazoxane) in yeast and mammalian cells Jensen Lars H [email protected] Marielle [email protected] Lasse T [email protected] Morten [email protected] Peter B [email protected] Maxwell [email protected] Department of Pathology, Diagnostic Centre, Rigshospitalet 5444, Frederik V's Vej 11, DK-2100 Copenhagen, Denmark2 Institute of Molecular Pathology, University of Copenhagen, Frederik V's Vej 11, DK-2100, Copenhagen, Denmark3 Laboratory of Experimental Medical Oncology, Finsen Centre, Rigshospitalet 5074, Blegdamsvej 9, DK-2100 Copenhagen, Denmark2004 2 12 2004 4 31 31 21 7 2004 2 12 2004 Copyright © 2004 Jensen et al; licensee BioMed Central Ltd.2004Jensen 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
Bisdioxopiperazine anti-cancer agents are inhibitors of eukaryotic DNA topoisomerase II, sequestering this protein as a non-covalent protein clamp on DNA. It has been suggested that such complexes on DNA represents a novel form of DNA damage to cells. In this report, we characterise the cytotoxicity and DNA damage induced by the bisdioxopiperazine ICRF-187 by a combination of genetic and molecular approaches. In addition, the well-established topoisomerase II poison m-AMSA is used for comparison.
Results
By utilizing a panel of Saccharomyces cerevisiae single-gene deletion strains, homologous recombination was identified as the most important DNA repair pathway determining the sensitivity towards ICRF-187. However, sensitivity towards m-AMSA depended much more on this pathway. In contrast, disrupting the post replication repair pathway only affected sensitivity towards m-AMSA. Homologous recombination (HR) defective irs1SF chinese hamster ovary (CHO) cells showed increased sensitivity towards ICRF-187, while their sensitivity towards m-AMSA was increased even more. Furthermore, complementation of the XRCC3 deficiency in irs1SF cells fully abrogated hypersensitivity towards both drugs. DNA-PKcs deficient V3-3 CHO cells having reduced levels of non-homologous end joining (NHEJ) showed slightly increased sensitivity to both drugs. While exposure of human small cell lung cancer (SCLC) OC-NYH cells to m-AMSA strongly induced γH2AX, exposure to ICRF-187 resulted in much less induction, showing that ICRF-187 generates fewer DNA double strand breaks than m-AMSA. Accordingly, when yeast cells were exposed to equitoxic concentrations of ICRF-187 and m-AMSA, the expression of DNA damage-inducible genes showed higher levels of induction after exposure to m-AMSA as compared to ICRF-187. Most importantly, ICRF-187 stimulated homologous recombination in SPD8 hamster lung fibroblast cells to lower levels than m-AMSA at all cytotoxicity levels tested, showing that the mechanism of action of bisdioxopiperazines differs from that of classical topoisomerase II poisons in mammalian cells.
Conclusion
Our results point to important differences in the mechanism of cytotoxicity induced by bisdioxopiperazines and topoisomerase II poisons, and suggest that bisdioxopiperazines kill cells by a combination of DNA break-related and DNA break-unrelated mechanisms.
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Background
Type II topoisomerases are essential nuclear enzymes found in all living organisms [1]. Their basic role in cells is to catalyse the transport of one DNA double helix through a transient double strand break in another DNA molecule [2]. This activity helps relieve tensions built up in DNA during various DNA metabolic processes such as DNA replication, chromosome condensation and de-condensation, chromosome segregation and transcription [3].
Topoisomerase II is also a major drug target in human cancer therapy, where a number of clinically active drugs such as the epipodophyllotoxins VP-16 and VM-26, the aminoacridine m-AMSA, and antracyclines such as doxorubicin, daunorubicin and epirubicin are widely used. These drugs have collectively been called topoisomerase II poisons due to their mechanism of action on topoisomerase II. Rather than inhibiting the basic catalytic activity of the enzyme, these drugs perturb the topoisomerase II catalytic cycle resulting in an increase in the level of a transient reaction intermediate, where DNA is cleaved and covalently attached to DNA [4].
Catalytic inhibitors of topoisomerase II have a different mode of action. These drugs exemplified by merbarone, aclarubicin, F11782 and the bisdioxopiperazines work by inhibiting topoisomerase II at other stages in the reaction cycle where DNA is not cleaved as reviewed in [5,6]. Amongst these, the bisdioxopiperazines have gained much attention due to their distinct and well-characterised mode of action. These compounds exemplified by ICRF-187, ICRF-159 and ICRF-154 inhibit the DNA strand passage reaction of topoisomerase II by sequestering this protein as a salt-stable closed clamp on DNA whose formation depends on the presence of ATP [7-9]. This closed clamp complex has retained the capability to hydrolyse ATP, although at a reduced level [10].
Several studies indicate that the closed clamp complex on DNA represents a novel form of DNA lesion to cells, – and that inhibition of topoisomerase II catalytic activity (DNA strand passage activity) is not responsible for bisdioxopiperazine-induced cell kill: (i) Expression of bisdioxopiperazine-sensitive topoisomerase II in cells also expressing bisdioxopiperazine-resistant topoisomerase II confers dominant sensitivity to these drugs [7,11] – a modality reminiscent of that of topoisomerase II poisons. (ii) Mouse embryonic stem cells [12] and chicken lymphoma DT40 cells [13] having one topoisomerase II α allele knocked out with concomitant reduced levels of topoisomerase II, are resistant to both ICRF-193 and the topoisomerase II poison etoposide, – while the opposite result is to be expected if ICRF-193 kill cells by depriving them of essential topoisomerase II catalytic activity. (iii) Killing of yeast cells by exposure to ICRF-193 occurs more rapidly and to a higher level than killing of yeast cells induced by the depletion of endogenous topoisomerase II catalytic activity [7]. (iv) The ICRF-193-induced topoisomerase II closed clamp complexes on DNA work as a "road block" signalling selective degradation of topoisomerase II β as well as p53 activation in a transcription dependent fashion [14].
Some studies have recorded elevated levels of DNA breaks in cells after exposure to the bisdioxopiperazine analog ICRF-193. In one study, ICRF-193 was found to increase the level of topoisomerase II-DNA covalent complexes in vitro and in vivo [15]. However, in this study efficient trapping of this covalent intermediate was only evident when guanidine was used to denature topoisomerase II attracted to DNA, while the agent normally used to trap the topoisomerase II-DNA cleavage complex, SDS, was not effective. In another study, the comet assay and pulsed field gel electrophoresis were used to demonstrate elevated levels of DNA breaks in mammalian cells after exposure to ICRF-193 [16]. In this study, inhibiting DNA replication with aphidicolin reduced the level of DNA breaks induced by the topoisomerase II poison m-AMSA, but had no effect on DNA breaks induced by ICRF-193. These results point towards bisdioxopiperazines poisoning DNA topoisomerase II in cells by a mechanism different from that of the classical topoisomerase II poisons such as etoposide and m-AMSA. In a recent paper, it was directly demonstrated that m-AMSA-induced dominant cytotoxicity only required the DNA cleavage activity of topoisomerase II, while dominant cytotoxicity towards ICRF-193 depended strictly on the DNA strand passage reaction of the enzyme[17].
Based on these observations, the present study aims to further elucidate the mechanism of cytotoxicity induced by the bisdioxopiperazines. We here characterise the effect of the clinically approved analog ICRF-187 (dexrazoxane) by using a number of different cell-based pharmacological assays, taking advantage of genetically modified yeast and mammalian cells.
Results
ICRF-187 sensitivity of yeast cells depends on their homologous recombination status, albeit to a lesser extent than for m-AMSA sensitivity
To pinpoint the mechanism of cytotoxicity of ICRF-187 versus m-AMSA, we employed a panel of human topoisomerase II α-transformed haploid single-gene knockout yeast strains, defective in various aspects of DNA repair, checkpoint control, membrane transport and protein degradation. All yeast strains are depicted in table 1. We used doses of these two drugs equitoxic to wild-type cells having no mutations. Clonogenic survival of all yeast strains is depicted in additional file 1, and the degree of drug resistance / hypersensitivity is also listed in table 2.
Table 1 Yeast strains used in the study
BY4741 + pMJ1 BY4741Δrad9 + pMJ1
BY4741Δrad51 + pMJ1 BY4741Δtel1 + pMJ1
BY4741Δrad52 + pMJ1 BY4741Δchk1 + pMJ1
BY4741Δrad54 + pMJ1 BY4741Δmhl1 + pMJ1
BY4741Δrad55 + pMJ1 BY4741Δpms1 + pMJ1
BY4741Δrad57 + pMJ1 BY4741Δmsh2 + pMJ1
BY4741Δrad59 + pMJ1 BY4741Δmsh3 + pMJ1
BY4741Δdcm1 + pMJ1 BY4741Δatr1 + pMJ1
BY4741Δsae2 + pMJ1 BY4741Δpdr5 + pMJ1
BY4741Δrad50 + pMJ1 BY4741Δyor1 + pMJ1
BY4741Δmre11 + pMJ1 BY4741Δubc4 + pMJ1
BY4741Δxrs2 + pMJ1 BY4741Δubc13 + pMJ1
BY4741Δrad6 + pMJ1 BY4741Δdoa4 + pMJ1
BY4741Δrad18 + pMJ1 BY4741Δqri8 + pMJ1
BY4741Δrev1 + pMJ1 BY4741Δrnr3 + pMJ1
BY4741Δrev3 + pMJ1 BY4741Δsml1 + pMJ1
BY4741Δrad1 + pMJ1 BY4741 + PYX112
BY4741Δrad14 + pMJ1 BY4741Δrad6 + pYX112
BY4741Δapn1 + pMJ1 BY4741Δrad50 + pYX112
BY4741Δyku70 + pMJ1 BY4741Δrad52 + pYX112
BY4741Δyku80 + pMJ1 BY4741Δsae2 + pYX112
BY4741Δmec3 + pMJ1 BY4741Δyku70 + pYX112
BY4741Δdcc1 + pMJ1
BY4741Δrad17 + pMJ1 JN362At2-4+ pMJ1
Table 2 Hypersensitivity (or resistance) scoring of pMJ1-transformed BY4741 deletion strains towards ICRF-187 and m-AMSA as determined in clonogenic assay using 22.5 hours drug exposure.
Drug ICRF-187 m-AMSA
Gene deleted
WT 0 0
Nucleotide Excision Repair (NER)
Single Strand Annealing (SSA) Recombination
Anti-Recombination
Δrad1 R 0
Δrad14 0 0
Mismatch Repair (MMR)
Anti-Recombination
Δmsh2 R 0
Δmsh3 0 0
Δmhl1 R 0
Δpms1 R 0
Base Excision Repair (BER)
Δapn1 0 0
Post Replication Repair (PRR)
Δrev1 0 0
Δrev3 0 0
Δrad18 0 +
Δrad6 0 ++
Homologous Recombination (HR)
Non-Homologous End Joining (NHEJ)
Δrad50 + ++
Δmre11 + ++
Δxrs2 + ++
Homologous Recombination (HR)
Δrad52 + ++
Δrad51 + +
Δrad54 + ++
Δrad57 + ++
Δrad55 + ++
Δrad59 0 0
Δdmc1 0 0
Δsae2 + +
Non-Homologous End Joining (NHEJ)
Δyku70 + 0
Δyku80 0 0
DNA Damage Checkpoints
Δtel1 0 0
Δrad9 0 0
Δmec3 0 0
Δddc1 0 0
Δrad17 0 0
Δchk1 0 0
ABC Transporters (Yeast MDR1 homologous)
Δatr1 0 0
Δpdr5 + 0
Δyor1 0 0
Ubiquitin conjugation / hydrolysis
Δubc4 0 0
Δubc13 0 0
Δdoa4 0 R
Δqri8 0 0
Ribonucleotide-reductase regulation
Δrnr3 0 0
Δsml1 0 0
R : Cells are more than a 1/2 log resistant at any drug concentration.
0 : Cells are no more than a 1/2 log resistant and no more than a 1/2 log hypersensitive at any drug concentration.
+ : Cells are at least a 1/2 log but no more than 2 log hypersensitive at any concentration.
++ : Cells are more than 2 log hypersensitive at any concentration.
Hypersensitivity (resistance) was graduated as follows
The products of the three genes RAD50, MRE11 and XRS2 together form the Rad50/Mre11/Xrs2 hetero-trimer protein complex that has catalytic and structural functions in many kinds of DNA metabolic processes including HR as reviewed in [18]. We observed that Δrad50, Δmre11, and Δxrs2 single knockout strains were extremely hypersensitive towards m-AMSA, while they displayed considerably less hypersensitivity towards ICRF-187 (additional file 1 and table 2).
We also tested the effect of deleting a number of genes exclusively involved in HR namely RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, DMC1 and SAE2 [18]. Deleting RAD51, RAD52, RAD54, RAD55, RAD57 and SAE2 had a profound effect on the sensitivity of the yeast cells towards m-AMSA while having a smaller, but significant, effect on the sensitivity of these cells towards ICRF-187 (additional file 1 and table 2), again pointing to the HR pathway as being most important for the repair of DNA damage caused by cleavage complex stabilising drugs. We found that deleting RAD59 had no effect on drug sensitivity, confirming reported data that RAD59 only becomes functionally important in the absence of functional Rad51 protein [19]. We also observed no effect of deleting DMC1 (additional file 1 and table 2). This may be explained by the fact that Dmc1p is primarily involved in meiotic recombination [20].
NHEJ represents another DNA repair-pathway. In yeast, this repair pathway is generally less important than HR for the repair of DNA breaks [21]. In accordance with this we observed no effect of deleting the NHEJ genes YKU70 and YKU80 on the sensitivity towards m-AMSA. We did however, observe some hypersensitivity of Δyku70 cells towards ICRF-187, while Δyku80 cells were not hypersensitive (additional file 1 and table 2). This is a surprising result, because Yku70p and Yku80p have been demonstrated to play equally important roles for NHEJ activity in yeast [21]. These results suggest that the effect of deleting YKU70 is unrelated to its DNA repair functions.
The DNA binding Rad18p forms a hetero-dimer with Rad6p that is involved in post replication repair (PRR) [22]. We found that although Δrad18 cells were clearly hypersensitive towards m-AMSA, Δrad6 cells were markedly more sensitive. Δrad6 cells were actually among the most sensitive towards m-AMSA (figure 1, additional file 1 and table 2). Interestingly, the sensitivity of Δrad6 and Δrad18 cells towards ICRF-187 is indistinguishable from that of wild-type cells (figure 1). The vast difference in the sensitivity of Δrad6 cells towards ICRF-187 and m-AMSA confirms the notion that the DNA lesions induced by these drugs are different in nature. The finding that Δrad6 cells are much more sensitive towards m-AMSA than Δrad18 cells is surprising, and may indicate that Rad6p functions unrelated to DNA repair affect cellular sensitivity towards m-AMSA. Rad6p has ubiquitin conjugating activity [22], and therefore such Rad18p-unrelated functions could involve protein degradation via the 26S proteasome pathway. To test this hypothesis, we analysed the drug sensitivity of four yeast strains with impaired protein degradation; Δubc4, Δubc13, Δdoa4 and Δqri8. These deletion strains were not hypersensitive towards m-AMSA (or ICRF-187) (additional file 1 and table 2), suggesting that Δrad6 cells are hypersensitive towards m-AMSA due to impaired PRR activity. The involvement of both HR repair and PRR in determining the sensitivity of yeast cells towards the topoisomerase II cleavage complex stabilising drugs mitoxantrone and idarubicin has previously been reported [23]. The observed lack of hypersensitivity of the Δrev1 and Δrev3 strains (additional file 1 and table 2) suggests that trans-lesion DNA synthesis plays no role in determining the sensitivity towards ICRF-187 or m-AMSA.
Figure 1 Clonogenic sensitivity of PRR defective Δrad6 and Δrad18 yeast cells towards equitoxic doses of ICRF-187 and m-AMSA. A Δrad52 strain is included for comparison. Error-bars represent SEM of at least 3 experiments.
We also analysed the effect of deleting genes belonging to the nucleotide excision repair (NER) pathway – RAD1 and RAD14, the base excision repair (BER) pathway – APN1, and the mismatch repair (MMR) pathway – MLH1, PMS1, MSH2 and MSH3. None of these deletions caused cells to become more sensitive towards ICRF-187 and m-AMSA, indicating that these pathways are not involved in repairing DNA damage induced by these drugs. Interestingly, deleting genes involved in the MMR and NER pathways caused cells to become somewhat resistant to ICRF-187, and to a lesser extent towards m-AMSA (additional file 1 and table 2). The products of these genes have been implicated to have anti-recombination activities [24]. Increased levels of recombination in these cells could therefore be responsible for the observed low-level resistance towards ICRF-187.
Deletion of DNA damage checkpoint genes has little effect on both ICRF-187 and m-AMSA sensitivity of yeast cells
While deleting genes involved in DNA repair caused cells to be hypersensitive towards both drugs tested, we observed little effect of deleting the DNA damage checkpoint genes MEC3, DDC1, RAD17, TEL1, RAD9 and CHK1 (additional file 1 and table 2). Our finding that checkpoint control regulation plays no important role for bisdioxopiperazine sensitivity supports earlier data showing that arresting yeast cells in G1 phase did not protect against ICRF-193 cytotoxicity [7]. The lack of importance of checkpoint function in determining sensitivity towards m-AMSA is also in accordance with published observations [23], where sensitivity towards the cleavage complex stabilising topoisomerase II poisons mitoxantrone, idarubicin, daunorubicin and doxorubicin were only marginally affected by inactivating the RAD9, RAD17, MEC1, and RAD53 genes, while the sensitivity of yeast cells towards the topoisomerase I poison camptothecin showed a strong dependency on these pathways.
ICRF-187 is a possible substrate for the Pdr5 ABC transporter in yeast
In mammalian cells resistance towards various structurally-unrelated anti-neoplastic agents is often associated with over-expression of ABC-type drug efflux transporters such as p-glycoprotein and multi-drug resistance protein (MRP) as reviewed in [25]. Among the three yeast ABC transporters assessed in our study, Pdr5 is by far the best characterised [26]. While deleting YOR1 and ATR1 had no effect on drug sensitivity, Δpdr5 cells were clearly hypersensitive towards ICRF-187 but not towards m-AMSA (additional file 1 and table 2), suggesting that ICRF-187 is a substrate for the Pdr5 pump in yeast. It has to be emphasized, that over-expression of drug efflux pumps has not been associated with resistance towards bisdioxopiperazines in mammalian cells.
Transcriptional profiling of yeast cells after exposure to equitoxic concentrations of ICRF-187 and m-AMSA
In order to assess the effect on global gene expression of the interaction between human topoisomerase II α and the two drugs in yeast cells, transcriptional profiling was performed using Affymetrix gene chip technology. We exposed pMJ1-transformed JN362At2–4 yeast cells expressing human topoisomerase II α as their sole active topoisomerase II to equitoxic doses of ICRF-187 and m-AMSA for two hours at 34°C. This treatment resulted in a 50 % reduction in clonogenic survival after exposure to both drugs (additional file 2). Genes whose average expression in two independent experiments was up- or down-regulated more than 1.5 fold by exposure to the drugs were filtered out. 138 transcripts were induced by exposure to ICRF-187 while the number was 90 for m-AMSA. 26 transcripts were repressed by exposure to ICRF-187 while the number was 16 for m-AMSA. Additional file 3 lists transcripts induced or repressed by ICRF-187 while additional file 4 lists transcripts induced or repressed by m-AMSA. The expression profile of selected genes is listed in Table 3 and discussed below.
Table 3 Selected genes induced or repressed by exposure of pMJ1-transformed yeast cells to equitoxic concentrations of ICRF-187 and m-AMSA for 2 hours
Transcriptional activation
Gene function Gene Name Effect of ICRF-187 Effect of m-AMSA
DNA damage RNR3 3.2 4.0
HUG1 3.1 5.0
RAD51 1.9 2.0
RAD54 1.7 1.6
RNR2 1.5 1.8
Membrane transport PDR12 2.4 1.0
PDR15 2.0 1.0
Stress response HSP12 2.8 1.6
HSP26 1.7 1.8
WSC4 1.6 1.3
XBP1 1.6 1.5
HSP42 1.5 1.5
Others SWI1 1.6 1.1
Transcriptional repression
Others SNZ1 0.6 1.1
PCL9 0.7 0.6
Genes induced by both drugs
Both compounds induced the expression of a number of genes known to be up-regulated by DNA damage. The expression of four well-established DNA-damage inducible genes; RNR2, RNR3 [27,28] and RAD51, RAD54 [29] was thus induced by both drugs. Both compounds also stimulated the expression of HUG1 recently shown to be up-regulated by DNA damage and replication arrest [30] (See Table 3, additional files 3 and 4). Furthermore, both drugs stimulated expression of the stress-inducible XBP1 gene whose protein product is a transcription factor. XBP1 expression is reportedly induced in response to heat shock, high osmolarity, oxidative stress, glucose starvation and DNA damage, and induces a slow-growth phenotype with lengthening of the G1 cell cycle phase [31]. The PCL9 gene product has cyclin-dependent protein kinase regulator activity suggesting a role for Pcl9p in cell cycle regulation [32]. Repression of PCL9 by exposure to both drugs (table 3, additional files 3 and 4) may thus be indicative of drug-induced cell cycle arrest in accordance with the XBP1 expression data. Finally, both drugs induced the expression of general stress-induced HSP genes as expected (table 3, additional files 3 and 4). Although expression of the RNR3 and HUG1 genes was up-regulated by both drugs, pMJ1-transformed cells having RNR3 or SML1 deleted (the latter is a functional non-inducible homolog of HUG1) have wild-type sensitivity towards both drugs (table 2, additional file 1), showing that although these genes are induced by both drugs, they are probably not involved in determining their cytotoxicity.
Genes specifically induced by ICRF-187
We found that ICRF-187 specifically induced the expression of two genes encoding the ABC efflux transporters Pdr12 and Pdr15, while m-AMSA had no effect on the expression of these genes (table 3, additional files 3 and 4). Transcription of the stress-inducible WSC4 gene was likewise enhanced by exposure to ICRF-187 (table 3, additional files 3 and 4). Knocking out WSC4 in yeast cells has been found to enhance their sensitivity towards various stresses including heat, ethanol and DNA damage [33]. Recently, the SWI/SNF complex was directly shown to repress transcription in S. cerevisiae cells [34]. We found that SWI1 was specifically induced by ICRF-187 (table 3, additional files 3 and 4). Finally, we found that ICRF-187 specifically repressed the expression of the stationary phase-induced SNZ1 gene [35] (table 3, additional files 3 and 4).
Exposure of yeast cells to ICRF-187 causes less transcriptional induction of DNA damage-inducible genes than exposure to m-AMSA at equitoxic drug concentrations
To verify the array data we performed real-time PCR to assess the expression of the RNR3, HUG1, RAD51 and RAD54 genes after exposure to the two drugs using the actin gene ACT1 as internal control (figure 2). Real-time PCR confirmed induction of these established DNA damage-inducible genes by both drugs assessed. Furthermore, exposure of the cells to m-AMSA resulted in a higher level of induction than did exposure to ICRF-187 for the four genes tested, especially for HUG1. These data suggest that when yeast cells are exposed to equitoxic concentrations of the two drugs, m-AMSA generates more extensive DNA damage than ICRF-187.
Figure 2 Analysis of gene expression by real time PCR. Real-time PCR was used to determine the expression of the DNA-damage inducible genes RNR3, HUG1, RAD51, and RAD54 by using the 2-ΔΔCt method. Gene expression was normalized to that of the actin gene ACT1. It can be seen that exposure of yeast cells to m-AMSA results in higher levels of induction of transcription of these four genes than exposure to ICRF-187 when the two drugs are applied at equitoxic concentrations. Error-bars represent SEM of two independent experiments each performed in duplicate.
ICRF-187 sensitivity of mammalian hamster cells depends on their homologous recombination status, albeit to a lesser extent than seen for m-AMSA sensitivity
The yeast clonogenic assays presented above point to an important role of HR in the repair of m-AMSA-induced DNA damage, while the importance of this pathway in the repair of ICRF-187-induced DNA damage is less so. Because HR is the major repair pathway in yeast [21], while both NHEJ and HR are important for the repair of DNA breaks in mammalian cells [36], we next turned to assess the importance of these pathways in mammalian cells having reduced levels of HR and NHEJ. In this analysis we used a panel of four hamster cell lines; AA8 cells (wild-type), irs1SF cells [37] (recombination defective caused by non-functional XRCC3), CXR3 cells [37] (recombination proficient due to ectopic expression of human XRCC3), and V3-3 cells [38] (reduced level of NHEJ due to non-functional DNA-PKcs).
We observed a strong dependence on HR for the sensitivity towards m-AMSA (figure 3A). Thus, only 1 % relative survival was seen for the irs1SF cells (recombination defective) at 6 nM of this drug, while wild-type AA8 cells were only slightly sensitive to15 nM m-AMSA. Furthermore, ectopic expression of human XRCC3 fully reversed the m-AMSA hypersensitivity as CXR3 cells were no more hypersensitive than AA8 wild-type cells, confirming the notion that HR plays a role in the repair of topoisomerase II-induced DNA breaks in mammalian cells. We also observed that irs1SF cells were hypersensitive towards ICRF-187 (figure 3B), but the degree of hypersensitivity was much less than observed for m-AMSA, as also seen for recombination deficient yeast cells. Again, ectopic expression of the human XRCC3 homolog reversed the hypersensitivity as CXR3 cells displayed near wild-type sensitivity towards ICRF-187.
Figure 3 Assessing the clonogenic sensitivity of HR and NHEJ deficient and proficient hamster cells towards ICRF-187 and m-AMSA. To determine the sensitivity of the four cell lines AA8 (wild-type), irs1SF (recombination defective caused by non-functional XRCC3), CXR3 (recombination proficient due to ectopic expression of human XRCC3), and V3-3 (defective in NHEJ due to non-functional DNA-PKcs) towards ICRF-187 and m-AMSA, a clonogenic assay with continuous drug exposure was used. Error-bars represent SEM of two independent experiments.
DNA-PKcs deficient hamster cells show slightly increased sensitivity towards both m-AMSA and ICRF-187
To assess the effect of NHEJ on drug sensitivity we also employed the V3-3 cell line (DNA-PKcs deficient, with concomitant reduced level of NHEJ). The results of these experiments are depicted in figure 3A and 3B. The V3-3 cells were slightly hypersensitive towards both drugs suggesting a role for NHEJ in the repair of DNA lesions induced by both drugs.
AA8, irs1SF, CXR3 and V3-3 cells have similar levels of topoisomerase II catalytic activity
The sensitivity of cells towards topoisomerase II directed drugs depends both on their levels of topoisomerase II catalytic activity, and on their capability to repair topoisomerase II-induced DNA damage. We therefore determined the level of topoisomerase II catalytic (DNA strand passage) activity in crude protein extracts isolated from the four cell lines used in clonogenic assays, by applying a radioactive decatenation assay. No significant difference in the level of topoisomerase II DNA strand passage activity was recorded between the four cell lines (additional file 5). This result rules out the possibility that varying levels of topoisomerase II catalytic activity in these cells is responsible for their differential drug sensitivity.
ICRF-187 induces lower levels of homologous recombination in hamster cells than m-AMSA at equitoxic concentrations
The hypersensitivity of the recombination defective irs1SF cells towards both drugs suggests that HR is involved in repairing DNA lesions induced by both drugs. To address this directly we applied a mammalian recombination assay to measure stimulation of HR by ICRF-187 and m-AMSA by using SPD8 hamster cells [39]. This assay measures the repair of a defective chromosomal hprt gene by the activity of HR. From figure 4A it is evident that both drugs stimulated the level of HR in a dose dependent manner. When recombination frequency is expressed as a function of surviving cells (figure 4C) it becomes evident that the recombination frequency increases with increasing cell mortality for both drugs tested. From figure 4C it is also evident that at equitoxic concentrations of the two drugs, m-AMSA stimulated HR to much higher levels than did ICRF-187. Thus, at 50 % survival, no induction of HR was seen with ICRF-187 (in three independent experiments), while m-AMSA caused an approximately 10-fold induction at equitoxic doses.
Figure 4 Assessing the effect of equitoxic concentrations of ICRF-187 and m-AMSA on the level of HR in SPD8 hamster cells. The SPD8 cell line has a defective hprt gene that can be repaired by HR. Panel A depicts the induction of HR induced by increasing concentrations of the two drugs. Panel B depicts the relative survival of cells exposed to similar concentrations of the two drugs. The data represented in Panel A and B was used to generate Panel C, where recombination frequency is plotted against the surviving fraction of cells. This data presentation allows a direct comparison of recombination levels at equitoxic concentrations of the two drugs. Representative data from one of three independent experiments is shown.
ICRF-187 induces only low levels of H2AX phosphorylation in human SCLC cells as compared to m-AMSA
Induction of γH2AX is a well-established marker for topoisomerase-induced DNA double strand breaks in mammalian cells [40-42]. We therefore assessed the effect of exposing human SCLC OC-NYH cells to 10 μM m-AMSA and 1 mM of ICRF-187 at increasing time points (figure 5A and 5B). Exposure to 10 μM m-AMSA quickly resulted in γH2AX induction. Thus, induction was evident after 30 min, and after 24 hours more than 10-fold induction was observed. In contrast, when cells were exposed to 1 mM ICRF-187, much less γH2AX induction was observed, and after 24 hours the level of induction was less that three-fold.
Figure 5 Assessing the effect of m-AMSA and ICRF-187 on γH2AX induction in human SCLC OC-NYH cells. To assess the level of DNA breaks in human cells after exposure to 10 μM m-AMSA and 1 mM ICRF-187 for increasing time points, total histones were isolated after incubation with the drugs. 10 μg of purified histones was then used in western blotting experiments. Panel A depicts a typical Western blot showing increased γH2AX induction with increasing drug incubation times. Panel B depicts fold γH2AX induction plotted against drug incubation time to analyse the kinetics of induction of DNA double strand breaks by the two drugs. Insert shows γH2AX induction from 0 to 2 hours for better resolution. Error-bars represent SEM of three independent experiments for ICRF-187 treatment and SEM of two independent experiments for m-AMSA treatment.
Discussion
We initiated the study by assessing the clonogenic sensitivity of yeast single-gene deletion mutants ectopically expressing human topoisomerase II α towards m-AMSA and ICRF-187. The results presented in table 2 and additional file 1 indicates that HR plays a role in the repair of ICRF-187-induced DNA damage. Previous studies addressing the bisdioxopiperazine sensitivity of yeast cells have generated different results. In one study rad52-cells had the same sensitivity towards ICRF-187 and ICRF-193 as did RAD52+ cells [7], while in other studies, HR deficient cells were found to be hypersensitive towards bisdioxopiperazines, although to a much lesser extent than towards topoisomerase II cleavage complex stabilising drugs [43,44]. Our present study involving numerous other genes involved in various aspects of HR clearly establishes this pathway as being a functional determinant for bisdioxopiperazine sensitivity in yeast cells. In a recent work by Simon and colleagues, where a panel of yeast deletion strains was also applied to pinpoint the mechanism of action of various anticancer drugs, a given drug was classified as selective if one single pathway was mainly involved in determining cellular sensitivity [23]. The selective involvement of the HR pathway in determining the sensitivity towards ICRF-187 classifies this drug as highly selective according to this definition. However, it is important to note that although HR clearly does play a role in protecting yeast cells from ICRF-187 cytotoxicity, the importance of this pathway on cell survival in the presence of m-AMSA is much greater (additional file 1, table 2) – in accordance with this drug being a topoisomerase II poison killing cells solely by the generation of topoisomerase II-mediated DNA breaks.
We find that the relative sensitivity of AA8, irs1SF, and CXR3 cells towards m-AMSA (figure 3) closely resembles their sensitivity towards etoposide [45], showing that the hypersensitivity of XRCC3 deficient irs1SF cells is general to topoisomerase II poisons, suggesting a role for HR in the repair of topoisomerase II-induced DNA breaks in these cells. The involvement of HR in the repair of DNA lesions induced by topoisomerase II poisons in higher eukaryotes is also supported by a recent work suggesting that RAD51 plays an important role in the repair of etoposide-induced DNA damage in human small cell lung cancer cells [46], and by work by Adachi and colleagues who recently found that knocking out RAD54 in chicken DT40 cells enhances their sensitivity towards the topoisomerase II poison etoposide [13].
We find that XRCC3 defective irs1SF cells are more sensitive towards ICRF-187 than the parental AA8 cells, although the XRCC3 defect has a much more pronounced effect on m-AMSA sensitivity (figure 3A versus figure 3B) – as seen with the yeast deletion mutant panel. Adachi and colleagues have found that knocking out RAD54 in DT40 chicken cells does not increase sensitivity towards ICRF-193 [13]. The reason for this discrepancy in not clear. The difference observed between the DT40 and irs1SFcells may relate to the fact that different DNA repair genes are deleted in the two cell lines possibly resulting in different processing of bisdioxopiperazine-induced DNA damage. In any case, this discrepancy does not challenge the overall finding that HR plays a more important role in protecting cells of various origin from cytotoxicity induced by topoisomerase II poisons as compared to cytotoxicity induced by bisdioxopiperazines.
To study the importance of NHEJ in determining the sensitivity towards m-AMSA and ICRF-187 we employed a DNA-PKcs defective hamster cell line, V3-3, which has reduced levels of NHEJ activity. We find that V3-3 cells are hypersensitive towards m-AMSA (figure 3A). This result is in accordance with a recent publication by Willmore and colleagues who found that a specific small-molecule inhibitor of DNA-PKcs NU7026 could potentate the sensitivity of human leukemic K562 cells towards various topoisomerase II poisons [47]. Our result is also in accordance with a recent report by Adachi and colleagues showing that DNA-PKcs knockout chicken DT40 cells are hypersensitive towards etoposide [48]. These result points towards an important role of DNA-PKcs in determining the sensitivity of higher vertebrate cells towards topoisomerase II poisons. Different studies have demonstrated a more pronounced effect of inactivating Ku as compared to DNA-PKcs on cellular sensitivity towards topoisomerase II poisons [48-50]. Consequently, the importance of NHEJ in determining the sensitivity towards topoisomerase II poisons in mammalian cells is likely to be underestimated from our V3-3 cell data. This notion is confirmed by early publications demonstrating that Ku deficient hamster cells are highly sensitive towards m-AMSA and etoposide [51,52].
Our finding that V3-3 cells are more sensitive towards ICRF-187 than AA8 cells (figure 3B) is also in accordance with observations by Adachi and colleagues who find that DNA-PKcs deficient chicken DT40 cells are hypersensitive towards another bisdioxopiperazine analog, ICRF-193. These authors found the effect of inactivating DNA-PKcs to be much more pronounced than seen in our present study. While the reason for this difference is not clear, it has to be mentioned that studies addressing the effect of DNA-PKcs on the sensitivity towards topoisomerase II targeting drugs and ionising radiation have produced varying results. Thus, in a study by Jin and colleagues, DNA-PKcs defective murine cells were much less sensitive towards etoposide than Ku70 and Ku80 deficient cells [50], while in a study by Gao and colleagues the importance of DNA-PKcs on the sensitivity towards ionising radiation was found to depend on cell type and/or cell cycle distribution [49]. Such variation could well explain the different importance of DNA-PKcs observed in our study and in the work by Adachi and colleagues.
In order to study directly the effect of exposing mammalian cells to ICRF-187 and m-AMSA on the levels of HR, we employed a mammalian recombination assay previously described [39]. In this assay, m-AMSA enhanced the level of recombination in SPD8 cells to higher levels than ICRF-187 at all cytotoxicity levels tested (figure 4C), demonstrating directly pronounced differences in the mechanism(s) by which topoisomerase II poisons and bisdioxopiperazines kill cells. This notion is further confirmed by our γH2AX induction experiments, where ICRF-187 causes much lower levels of induction (figure 5), demonstrating that ICRF-187 induces less DNA breaks in cells than m-AMSA. Our observation that ICRF-187 induces both HR and γH2AX induction in mammalian cells, is in agreement with a recent paper demonstrating by the use of comet assay and pulsed field gel electroforesis that ICRF-193 induces DNA breaks in mammalian cells [16]. This result is also in agreement with our real-time PCR results where ICRF-187 tended to induce the expression of established DNA damage-inducible genes.
The finding that ICRF-187 induces lower levels of HR than m-AMSA in SPD8 cells at equitoxic doses may be explained in at least two ways. Bisdioxopiperazine-induced DNA breaks could be more toxic to cells than breaks induced by topoisomerase II poisons, or the DNA breaks could be only partly responsible for killing the cells. Three lines of evidence support the latter possibility. (i) Functional ATR, but not ATM, is required for a cell cycle checkpoint arrest induced by ICRF-193 [53], suggesting that DNA breaks are not involved in triggering the checkpoint signal. (ii) Exposure of mammalian cells to the topoisomerase II poison etoposide induces degradation of the large subunit of RNA polymerase II indicative of DNA breaks, while this is not the case for ICRF-193 [14]. (iii) Our finding that cell survival in the presence of ICRF-187 depends less on HR than cell survival in the presence of m-AMSA suggests that ICRF-187-induced DNA breaks contribute less to overall cytotoxicity than m-AMSA-induced DNA breaks. If ICRF-187-induced DNA breaks were more toxic to cells than m-AMSA-induced DNA breaks, cell survival in the presence of ICRF-187 would be expected to depend at least as much on HR as cell survival in the presence of m-AMSA. This is not the case.
What mechanisms are then responsible for producing the DNA breaks induced by bisdioxopiperazines in cells? In a recent work by Oestergaard and colleagues).)[17], it is suggested that the toxic intermediate causing bisdioxopiperazine cytotoxicity is topoisomerase II stably bound to two DNA segments – a conformation they suggested would only be attainable if the DNA strand passage reaction of topoisomerase II is functioning. HR could then be required for the repair of DNA breaks generated by the collision of DNA tracking complexes with such four-way DNA junctions / topoisomerase II closed clamp complexes on DNA. It has recently been demonstrated by the use of pulsed field gel electrophoresis, that inhibiting DNA replication by aphidicolin does not reduce the level of DNA breaks generated by exposure of mammalian cells to ICRF193, while the level of m-AMSA-induced DNA breaks was reduced by aphidicolin treatment [16]. This result suggests that collision of the DNA replication complex with bisdioxopiperazine-induced topoisomerase II closed clamp complex on DNA is not involved in generating the DNA breaks.
In a recent study by Lundin and colleagues, it was demonstrated that inhibiting DNA replication by exposing cells to hydroxyurea resulted in the generation of DNA breaks [54]. Furthermore, in this work as well as in a subsequent work [45], HR was shown to be functionally involved in repairing such DNA breaks. The first of these two studies used the same four hamster cell lines that are also used in our present study. Remarkably, the relative sensitivity of these cell lines towards hydroxyurea exactly resembles their sensitivity towards ICRF-187 seen in our present work. This may suggest that replication arrest is involved in generating DNA breaks induced by bisdioxopiperazines in cells. Here, replication forks stalled at the bisdioxopiperazine-induced closed clamp complexes could be the source of DNA breaks in newly replicated DNA [54]. This would also explain the lack of effect of aphidicolin on the level of ICRF-193-induced DNA breaks observed by Hajji and colleagues [16]. If the DNA breaks result from arrested replications forks, and not from the collision of the DNA replication complex with the closed clamp complex on DNA, no effect of aphidicolin would be expected. This mechanism would also explain why yeast cells arrested in intra-S phase are not protected from ICRF-193 cytotoxicity [7]. We therefore suggest that this mechanism is responsible for generating DNA breaks induced by bisdioxopiperazines in cells.
Together our HR, γH2AX, and cytotoxicity data suggest that bisdioxopiperazines kill cells by a combination of DNA break-related and DNA break-unrelated mechanisms. This raises the question as to which mechanism(s) is / are involved in mediating the DNA break-unrelated part of bisdioxopiperazine cytotoxicity. Exposure of mammalian cells to ICRF-193 represses global transcription and mediates selective degradation of topoisomerase II β via a transcription dependent mechanism [14]. Inhibition of the RNA polymerase II – transcription complex by bisdioxopiperazine-induced topoisomerase II complexes on DNA could therefore be involved in mediating the DNA break-unrelated component of bisdioxopiperazine cytotoxicity.
Treatment of mammalian cells with high doses of ICRF-187 for one hour is capable of antagonising DNA breaks and the cytotoxicity of topoisomerase II poisons [55,56], and this antagonism can be extended to animal models, where ICRF-187 can antagonise etoposide toxicity [57,58] and bone marrow depression (unpublished results). How are bisdioxopiperazines capable of antagonising the effects of topoisomerase II while at the same time producing DNA breaks? Two independent studies assessing the dose- and schedule-dependency of combinations of bisdioxopiperazines and topoisomerase II poisons on cytotoxicity in mammalian cells may provide important clues. One study investigated the effect of combinations of ICRF-193 and etoposide [59]. Here, continuous administration of low doses of both drugs resulted in synergistic cell kill, while treatment with high concentrations of ICRF-193 for one hour efficiently antagonised etoposide-mediated cytotoxicity. A similar effect of schedule and concentration on cytotoxicity has also been observed for combinations of ICRF-187 and daunorubicin [60], but here long time exposure of the cells to both drugs resulted in an additive effect on cell kill. We have previously shown that exposure of mammalian cells to high concentrations of ICRF-187 (500 – 1000 μM) alone for 60 min is non-toxic, and that this treatment efficiently antagonises etoposide-induced DNA breaks and cytotoxicity [61,62]. In these studies, exposure of cells to 200 μM ICRF-187 was found to trap most cellular topoisomerase II α and β as non-extractable complexes on DNA. The inability of topoisomerase II poisons to act on bisdioxopiperazine-stabilised closed clamp complexes on DNA could therefore explain the antagonistic effect of high concentrations of bisdioxopiperazines generally observed in one-hour drug exposure experiments [59-62]. When a low concentration of bisdioxopiperazine is administered, it is most likely that only a small fraction of the topoisomerase II molecules in the cell is trapped as closed clamp complexes on DNA, leaving some or most topoisomerase II molecules available for the action of topoisomerase II poisons. Therefore, after long-time exposure of cells to low concentrations of bisdioxopiperazine and a topoisomerase II poison, covalent and non-covalent complexes of topoisomerase II on DNA could both contribute to cytotoxicity by generating DNA breaks via different mechanisms, thus explaining the additive or synergistic effect on cell kill observed under these circumstances.
To summarise, our data are consistent with a model where bisdioxopiperazine-induced cytotoxicity results from a combination of DNA break-related and -unrelated mechanisms, where the DNA-break unrelated mechanism is clearly not mediated by the inhibition of catalytic topoisomerase II activity in the cells.
Conclusion
Since the discovery by Andoh and colleagues in 1991, that the bisdioxopiperazines target eukaryotic topoisomerase II [63,64], their mode of cytotoxicity has been the cause of debate. While early publications tended to classify these compounds as "pure" catalytic inhibitors of topoisomerase II, expected to kill cells by depriving them of essential topoisomerase II catalytic activity, numerous recent reports present data that are not consistent with this view [7,11-14,16,17]. In the present report we have characterised bisdioxopiperazine (ICRF-187) induced cytotoxicity in yeast and mammalian cells by using a combination of genetic and molecular approaches. Our results are consistent with a model where bisdioxopiperazines cause cytotoxicity by stabilising a topoisomerase II reaction intermediate / complex on DNA inducing DNA breaks in cells which are repaired by HR and NHEJ. We propose that cells exposed to bisdioxopiperazines die by a combination of DNA break-related and-DNA break-unrelated mechanisms. Our study clearly establishes that bisdioxopiperazines do not kill cells solely by depriving them of topoisomerase II catalytic activity.
Methods
Drugs
ICRF-187 (Cardioxane, Chiron group) was dissolved in sterile water at 20 mg/ml and kept at – 80°C. To avoid hydrolysis of the drug, fresh aliquots were used for each experiment. m-AMSA (Pfizer) was diluted in DMSO and stored at – 80°C at 1 mg/ml. L-azaserine and thymidine (both from Sigma) were added directly to tissue culture medium. 6-thioguanine and hypoxanthine (both from Sigma) were dissolved in 5 M NaOH and immediately added to the tissue culture medium.
Yeast strains and constructs
BY4741 haploid Saccharomyces cerevisiae cells (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and a panel of single-gene deletion derivatives hereof (table 1) were purchased from EUROSCARF, Institute of Microbiology, Johann Wolfgang Goethe University Frankfurt, Germany. The construction of BY4741 and its deletion derivatives have been described [65]. JN362At2–4 cells with the relevant genotype (MATa ura3–52 leu2 trp1 his7 ade1–2 ISE2 top2-4) were kindly provided by Dr. John L. Nitiss, St. Jude Children's Research Hospital, Memphis TN, USA. This strain and the construct for functional expression of human topoisomerase II α in yeast pMJ1 (URA3) have been described previously [66]. All yeast strains were transformed with pMJ1 to functionally express human topoisomerase II α in a cell cycle independent fashion. BY4741 wild-type and Δrad6, Δrad50, Δrad52, Δsae2 and Δyku70 cells were also transformed with an empty URA3 vector (pYX112). The ICRF-187 and m-AMSA sensitivity of pYX112-transformed cells was assessed to assure that the drug sensitivity of the pMJ1-transformed cells (Table 2, S1) is related to the ectopic expression of human topoisomerase II α in the cells, which was the case. Transformation and selection was carried out according to standard procedures using lithium acetate cell wall permeabilisation and PEG-mediated DNA uptake by using single-stranded DNA as carrier as described [67]. Selection was done on SC-URA plates. Three independent pMJ1-transformed yeast clones were selected and propagated for each transformation. All strains were propagated at 30°C, to be subsequently used in clonogenic assays at 34°C.
Yeast clonogenic assay
The clonogenic sensitivity of the yeast cells towards ICRF-187 and m-AMSA was determined using a clonogenic assay essentially performed as described in [7]. Briefly, overnight cultures of the strains were grown in SC-URA medium at 34°C at 200 rpm. Cells in log phase were diluted to 2 × 106 cells/ml in pre-warmed YPD medium, and 3 ml cultures were exposed to different concentrations of drug at 34°C for 22.5 hours. After drug exposure the samples were diluted up to 105 times (depending on the combination of strain and drug used) in distilled sterile water. Yeast cells that were not diluted before plating were spun down by brief centrifugation, and re-suspended in the same volume of sterile water. Next, 200 μl of diluted cells were transferred to SC-URA plates, which were incubated for 5 days at 30°C before counting. 200 to 600 colonies were typically counted for each drug concentration in each single experiment. Finally, the relative survival at the different drug concentrations as compared to the no drug sample was calculated to generate dose-response curves. For each combination of yeast strain and drug, at least three dose-response curves were generated using pMJ1-transformed cells from at least two independent clones (mostly from three).
Yeast microarray gene expression analysis
Microarray experiments were performed with yeast strain JN362t2–4 transformed with pMJ1 to functionally express human topoisomerase II α. Fresh colonies were inoculated into YPD medium and grown overnight at 34°C, 180 rpm. The cultures were then diluted to obtain an OD600 of 0.2. Cultures of 50 ml in YPD medium were first grown for two hours to assure exponential growth of the cells. 1 mg/ml ICRF-187 or 50 μg/ml m-AMSA (equitoxic concentrations) were then added to the cell cultures (a no-drug sample was also included), and the cells were grown for an additional two hours. Each treatment was performed in duplicate. The used concentration of both drugs resulted in a reduction in the clonogenecity of the cells of 50 %. After treatment cells were harvested by centrifugation. Total RNA was isolated by the hot acidic phenol method [69]. All the steps for cDNA synthesis, cRNA synthesis, biotin labeling and array hybridization to Affymetrix S98 yeast arrays were performed as described in the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix), and performed at the microarray core facility at Rigshospitalet, Copenhagen Denmark. Briefly, cDNA was synthesized from 5 μg RNA using a (dT)24 primer containing a T7 RNA polymerase promoter sequence and SuperScript II reverse transcriptase (Invitrogen) for 1 h at 42°C followed by second-strand synthesis using DNA polymerase I and RNase H digestion followed by isolation of cDNA using GeneChip Sample Cleanup Module (Affymetrix). The cDNA was used as template for synthesis of biotin-labeled cRNA by incubation with biotin-labeled ribonucleotides and T7 RNA polymerase for 5 h at 37°C. Biotin-labeled cRNA was purified using GeneChip Sample Cleanup Module. Biotinylated cRNA was fragmented and 15 μg used for hybridization to Affymetrix Yeast Genome S98 arrays at 45°C for 16 h as described in the Affymetrix users' manual. Washing and array staining with streptavidin-phytoerythrin were performed using the GeneChip Fluidics Station 400 and scanning was performed with a Gene Array Scanner G25 (Agilent technology). Data was analyzed using the DNA-Chip Analyzer (dChip) software [70].
Real-time PCR analysis
The RNA preparations used for microarray analyses were also used for real-time PCR. This analysis was performed on an ABIPrism 7900HT (Applied Biosystems). RNA samples were DNase treated using the DNA-free™ DNase treatment and removal kit (Ambion), and RNA concentrations were measured before conversion to cDNA using the TaqMan RT kit (Applied Bioscience). Priming was performed by random hexamers converting 2 μg RNA pr 100 μl reaction volume, to make 20 ng/μl cDNA. Primers were designed for coding sequences from the Saccharomyces genome database using the Primer 3 input program . All primers were purchased at DNA Technology A/S, with melting temperatures close to 60°C. Reaction mixtures containing the following components at the indicated end-concentrations were prepared. To make a total of 40 μl in sterile water, 20 μl 1x SYBR® green PCR master mix (Applied Biosystems), 250 nM forward primer, 250 nM reverse primer, and 5 ng template was mixed. Cycling conditions: 95°C for 10 min, followed by 40–45 cycles of 95°C for 15 s and 60°C for 60 s. Relative values of gene expression were calculated with untreated samples as calibrator, and normalized to levels of actin, according to the 2-ΔΔCt method [71] and (User bulletin #2, AbiPrism 7700 Sequence Detection System, Applied Biosystems) after primer optimisation and target efficiency evaluation. The following primers were used:
HUG1-forward, AGGCCTTAACCCAAAGCAAT;
HUG1-reverse, TCTTGTTGACACGGTTGCTC;
RNR3-forvard, ATGCATCTCCAGTTCCATCC;
RNR3-reverse, GGGGCAACACTATCTTCCAA;
RAD51-forward, GTGGCGGTGAAGGTAAGTGT;
RAD51-reverse, GTCTAATCCGAACCGCTGAG;
RAD54-forward, CTAAAGCAGGTGGGTGTGGT;
RAD54-reverse, CTTGTTGATCAGCAGCAGGA;
ACT1-forward, CGGTGATGGTGTTACTCACG;
ACT1-reverse, GGCCAAATCGATTCTCAAAA.
Mammalian cells
The CHO cell lines AA8, irs1SF, CXR3, V3-3, and the hamster lung fibroblast cell line SPD8 were kindly provided by Dr Thomas Helleday, University of Sheffield, UK. AA8 is a wild-type cell line. The AA8-derived irs1SF cell line is XRCC3-defective and has reduced levels of HR [37]. CXR3 is a human-XRCC3-cosmid complemented strain of irs1SF, which is proficient in HR [37]. The V3-3 cell line is DNA-PKcs-deficient and consequently deficient in NHEJ [38]. The SPD8 cells carry a non-functional hprt gene that can be repaired by HR [39]. HPRT+ cells can then be selected on HAsT medium containing hypoxanthine, L-azaserine and thymidine. When SPD8 cells were not used in the recombination assay they were propagated in medium supplemented with 6-thioguanine to select against spontaneous reversion to the HPRT+ phenotype. Human SCLC OC-NYH cells have been described [72]. Hamster cells were propagated in DMEM medium and OC-NYH cells were propagated in RPMI-1640 medium. All cell culture media were supplemented with 10 % fetal calf serum and 100 U/ml penicillin-streptomycin. Cells were grown in a humidified atmosphere containing 5 % CO2 in the dark at 37°C.
Determination of topoisomerase II activity in crude cell extracts
Topoisomerase II activity in crude extract was determined by using a decatenation assay previously described [68]. Briefly, 200 ng 3H labeled kDNA isolated from C. fasciculata was incubated with increasing amounts of crude extracts in 20 μl reaction buffer containing 10 mM TRIS-HCl pH 7.7, 50 mM NaCl, 50 mM KCl, 5 mM MgCl2, 1 mM EDTA, 15 μg/ml BSA and 1 mM ATP for 20 min at 37°C. After addition of 5x stop buffer (5 % Sarkosyl, 0.0025 % bromophenol blue and 50 % glycerol), unprocessed kDNA network and decatenated DNA circles were separated by filtering, and the amount of unprocessed kDNA in each reaction was determined by scintillation counting. The amount of crude extract required to fully decatenate 200 ng of kDNA under these assay conditions (which is equivalent to 1 U of catalytic activity) was then determined, and the specific activity of the crude extract was calculated as U/μg protein.
Mammalian clonogenic assay
Four hours prior to continuous treatment with either ICRF-187 or m-AMSA, 250 cells of each of the hamster cell lines were plated onto 100 mm dishes. After 7 days colonies were fixed and strained in methylene blue in methanol (4 mg/ml), and colonies with more than 50 cells were counted. Finally, the relative survival compared to the no drug treatment was calculated, and plotted against drug concentrations to generate dose-response curves. 150 – 250 colonies were typically counted in the "no drug" dishes.
Mammalian homologous recombination assay
A mammalian recombination assay was performed as described [39]. Briefly, 1 × 106 SPD8 cells were inoculated into 75 cm2 flasks. When transferred, 6-thioguanine was omitted from the medium. Cells were trypsinised and resuspended in 10 ml medium at 100,000 cells/ml, and exposed to the indicated drug concentrations for 24 hours. To determine clonogenic survival, for each drug-treatment 500 cells were transferred to each of two 100 mm petri dishes containing 10 ml of non-selecting medium and the cells were cultured for 7 days. For selection of recombination events, 300,000 cells were transferred to each of three 100 mm petri dishes containing 10 ml medium supplemented with 50 μM hypoxanthine, 10 μM L-azaserine and 5 μM thymidine and selection was carried out for 10 days. Colonies were fixed by using methylene blue in methanol (4 mg/ml) and counted. Finally, the recombination frequency was determined as the plating efficiency in recombination selective medium divided by the plating efficiency in normal medium, for all concentrations of ICRF-187 or m-AMSA. To enable comparison of recombination frequency at equitoxic levels of m-AMSA and ICRF-187, the recombination frequency was plotted against the relative clonogenic survival of cells receiving only drug.
Histone purification
Human SCLC OC-NYH cells were grown to sub-confluence and histones were extracted as follows. After the relevant drug treatments, the cells were pelleted and washed in cold PBS, and lysed in lysis buffer (10 mM TRIS-HCl pH = 6.5, 50 mM Sodium Bisulphate, 1% Triton X-100, 10 mM MgCl, 8.6% sucrose) at 4°C by applying 20 strokes in a tight fitting Dounce homogenizer. Released nuclei were pelleted by centrifugation at 2500 g for 10 min at 4°C, and washed in lysis buffer followed by wash buffer (10 mM TRIS-HCl, 13 mM EDTA pH 7.4). The pellet was next resuspended in 100 μl ice-cold 0.4 M H2SO4, and incubated for 1 hour at 4°C prior to centrifugation. The supernatant was transferred to a clean tube and 1 ml ice-cold acetone was added followed by incubation overnight for histone precipitation. After centrifugation, the pellet was air-dried and resuspended in 40 μl H2O, and the protein concentration was determined by Bradford protein assay (Bio-Rad Laboratories)
γH2AX western blot
Western blotting was performed by loading 10 μg of total histones on a 4–12% gradient gel (NuPageTM Bis-Tris Gel, Invitrogen). Separated proteins were transferred to nitrocellulose membranes (Bio-Rad) which were blocked in 10% skimmed milk (Fluka) and incubated overnight with anti γH2AX primary antibody diluted 1:500 (Upstate Technology, cat no 16–193) followed by detection with goat-anti-mouse (Amersham) 1:2000 for one hour. Detection with ECL Plus™(Amersham) was performed by scanning on STORM™ 840 (Molecular Dynamics Inc), on which the image was optimized and bands quantified by Image Quant™ version 5.0 (Molecular Dynamics).
List of abbreviations used
BER, Base Excision Repair; CHO, chinese hamster ovary; HR, Homologous Recombination; ICRF-187, (+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane; m-AMSA, (N-[4-(9-acridinylamino)-3-methoxyphenyl]methanesulphonanilide); MMR, Mismatch Repair; NER, Nucleotide Excision Repair; NHEJ, Non-Homologous End Joining; PCR, Polymerase Chain Reaction; PRR, Post Replication Repair; SCLC, Small Cell Lung Cancer; SC-URA, Synthetic medium lacking uracil; SSA, Single Strand Anealing; YPD, Medium containing Yeast extract, Peptone and Dextrose.
Authors' contributions
Lars H. Jensen: Participated in planning the experiments, performed yeast transformations and clonogenic assays, and prepared the manuscript. Marielle Dejligbjerg: Performed γH2AX western blots, primer design, real-time PCR experiments, and data quantitation. Lasse T. Hansen: Performed mammalian clonogenic assays and recombination assays. Morten Grauslund: Performed RNA purification and microarray analysis. Peter B. Jensen: Participated in planning and monitoring the study. Maxwell Sehested: Participated in the initiation and conduction of the study. All authors read and approved the final manuscript.
Supplementary Material
Additional file 1
Clonogenic sensitivity of mutant single-gene deletion yeast strains towards ICRF-187 and m-AMSA. Clonogenic sensitivity of a panel of human topoisomerase II α-transformed haploid yeast deletion strains towards equitoxic (to wt cells) concentrations of ICRF-187 and m-AMSA. Error-bars represent SEM of 3 – 10 independent experiments.
Click here for file
Additional file 2
Level of killing of yeast cells used in transcriptional profiling experiments. Fresh colonies of pMJ1-transformed JN362At2–4 cells were inoculated into YPD medium and grown overnight at 34°C, 150 rpm. The cultures were then diluted into 50 ml YPD medium to obtain an OD600 of 0.2. After growing the cells for 2 hours to assure exponential growth, equitoxic concentrations of ICRF-187 and m-AMSA were applied and the cells were grown for an additional 2 hours before RNA was isolated. The figure depicts the clonogenecity of drug treated and untreated cells. Exposure of the cells to the two drugs resulted in a reduction of their clonogenecity of approx. 50 %. Error bars-represent SEM of three independent experiments.
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Additional file 3
Transcriptional response towards ICRF-187. A list of yeast genes whose average expression in two independent experiments is induced or repressed more than 1.5 fold by exposure to ICRF-187.
Click here for file
Additional file 4
Transcriptional response towards m-AMSA. A list of yeast genes whose average expression in two independent experiments is induced or repressed more than 1.5 fold by exposure to m-AMSA.
Click here for file
Additional file 5
Topoisomerase II activity levels in hamster cell lines. The levels of topoisomerase II catalytic activity in crude extracts from wt and recombination defective hamster cell lines. Error-bars represent SEM of two independent experiments. No difference in the level of topoisomerase II catalytic (DNA strand passage activity) is observed.
Click here for file
Acknowledgements
We thank dr. Thomas Helleday, Institute for Cancer Studies, University of Sheffield, Sheffield, UK, for providing hamster cell lines; John L. Nitiss, St. Jude Children's Research Hospital, Memphis TN, USA, for providing JN362At2–4 yeast cells and the pMJ1 construct; Sanne Christiansen (TopoTarget) for purifying kDNA and for determining the levels of topoisomerase II strand passage activity in crude cell extracts, and Christian B. Poulsen for help with the microarray data.
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| 15575955 | PMC545072 | CC BY | 2021-01-04 16:33:05 | no | BMC Pharmacol. 2004 Dec 2; 4:31 | utf-8 | BMC Pharmacol | 2,004 | 10.1186/1471-2210-4-31 | oa_comm |
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BMC Cell BiolBMC Cell Biology1471-2121BioMed Central London 1471-2121-5-481561324410.1186/1471-2121-5-48Research ArticleMAB21L2, a vertebrate member of the Male-abnormal 21 family, modulates BMP signaling and interacts with SMAD1 Baldessari Danila [email protected] Aurora [email protected] Renato [email protected] Vincenzo [email protected] G Giacomo [email protected] Dept. Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy2 Stem Cell Research Institute, San Raffaele Scientific Institute, 20132 Milan, Italy3 Dept. Molecular Biology and Functional Genomics, San Raffaele Scientific Institute, 20132 Milan, Italy4 National Research Center, Institute of Chemistry of Molecular Recognition, 20131 Milan, Italy2004 21 12 2004 5 48 48 28 8 2004 21 12 2004 Copyright © 2004 Baldessari et al; licensee BioMed Central Ltd.2004Baldessari 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
Through in vivo loss-of-function studies, vertebrate members of the Male abnormal 21 (mab-21) gene family have been implicated in gastrulation, neural tube formation and eye morphogenesis. Despite mounting evidence of their considerable importance in development, the biochemical properties and nature of MAB-21 proteins have remained strikingly elusive. In addition, genetic studies conducted in C. elegans have established that in double mutants mab-21 is epistatic to genes encoding various members of a Transforming Growth Factor beta (TGF-beta) signaling pathway involved in the formation of male-specific sensory organs.
Results
Through a gain-of-function approach, we analyze the interaction of Mab21l2 with a TGF-beta signaling pathway in early vertebrate development. We show that the vertebrate mab-21 homolog Mab21l2 antagonizes the effects of Bone Morphogenetic Protein 4 (BMP4) overexpression in vivo, rescuing the dorsal axis and restoring wild-type distribution of Chordin and Xvent2 transcripts in Xenopus gastrulae. We show that MAB21L2 immunoprecipitates in vivo with the BMP4 effector SMAD1, whilst in vitro it binds SMAD1 and the SMAD1-SMAD4 complex. Finally, when targeted to an heterologous promoter, MAB21L2 acts as a transcriptional repressor.
Conclusions
Our results provide the first biochemical and cellular foundation for future functional studies of mab-21 genes in normal neural development and its pathological disturbances.
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Background
The male-abnormal 21 (mab-21) gene was first characterized in Caenorhabditis elegans as part of the combinatorial genetic code affecting morphogenesis of the sensory rays, a male-specific sense organ located in the tail and involved in copulation. In that context, early studies [1] identified a hypomorphic mab-21 allele as a dominant enhancer of egl-5, a loss-of-function mutation affecting the nematode homolog of the Drosophila homeotic gene AbdominalB. Subsequent studies [2] demonstrated that mab-21(+) plays a key role in the formation of the nematode male tail. Homozygous mab-21 mutations act cell-autonomously on the identity of a sensory ray, leading to alterations in anteroposterior identities akin to a homeotic transformation. Furthermore, they act non-cell-autonomously on the binary switch between hypodermal and neuroblast specification. Finally, hypomorphic mab-21 mutants exhibit pleiotropic changes affecting movement, body shape and fecundity, suggesting that mab-21(+) also plays key developmental roles outside the male tail region.
The mab-21 gene encodes a 41 kD basic protein with no significant homologies to any other published proteins. Homologs of the mab-21 gene have been identified in many other species, from Drosophila to humans. The first vertebrate counterparts of mab-21 were isolated in amniote genomes: a human homolog, MAB21L1, was cloned during a systematic search for transcripts carrying trinucleotide repeats [3] and potentially involved in the pathogenesis of neuropsychiatric disorders. Interestingly, recent physical mapping data have located Mab21l1 within a chromosomal deletion discovered in a patient with autism and language deficit secondary to an auditory processing abnormality [4].
Our group, in parallel with others, identified two murine mab-21 genes (Mab21l1, Mab21l2) [5-8] and the human MAB21L2 gene, and showed that they encode nuclear proteins [6] that are 94% identical, 98% homologous to each other. In the mouse, Mab21l1 and Mab21l2 are expressed in largely overlapping territories [5-7], suggesting the possibility of a high degree of functional redundancy. More recently, only one vertebrate member of the mab-21 family was isolated in Xenopus laevis (Xmab21l2) [9], (and our unpublished data: GenBank entry AF040992) and Danio rerio [10,11] (Zmab21l2). Mab21l2 is an early marker of the tectum as well as primitive eye field, optic cup and retina in anamniotes, in agreement with observations previously made in mouse embryos.
To address the in vivo roles of mab-21 genes in vertebrates, loss-of-function studies have been carried out by different groups in Xenopus and mouse embryos. Lau et al. [9] interfered with the functions of Xmab21l2 in embryogenesis by applying antisense DNA and double-stranded RNAi techniques. As a result, they reported a high frequency of embryos arrested at late gastrula/early neurula, as well as a significant incidence of neural tube closure defects in tadpoles. Likewise, Wong and Chow [12] cultured mouse embryos in the presence of antisense oligos specific for Mab21l1 and Mab21l2, and reported that both treatments caused a sharp increase in the incidence of defective axial turning, incomplete notochord formation, and neural tube closure defects. However, Mab21l2-specific antisense oligos were more potent and more effective than Mab21l1-specific ones, suggesting that Mab21l2 may play a more irreplaceable role in early development than Mab21l1.
As said, mab-21 genes mark early stages of eye development in various species. Likely due to redundancy in the vertebrate mab-21 gene family a knock-out mouse carrying a null mutation of Mab21l1 featured an isolated, cell-autonomous defect in lens placode development, providing a novel and valuable model for the study of various eye defects in humans [13], but no overt retinal abnormalities. Conversely, Mab21l2 has been found to participate crucially in zebrafish retina formation acting as a downstream effector of Rx2 [14].
Some of these important advances point to a critical role for the mab-21 gene family as a whole in early embryogenesis on one hand, and in various aspects of neural development on the other. However, our knowledge on the molecular mechanisms through which mab-21 genes operate remains inadequate at best. Interestingly, by genetic analysis of loss-of-function mutations in C. elegans, Morita et al. have proposed mab-21 as a downstream target of a TGF-beta superfamily signaling pathway involved in sensory ray identity, the Small/Male-tail-abnormal pathway. This pathway is initiated by the secreted ligand CET-1 [15], also known as DBL-1 [16], and regulates body growth and male tail development. The CET-1/DBL-1 ligand transmits its signal through two receptor serine threonine kinases, DAF-4 and SMA-6, which in turn regulate the activity of the nuclear transducers SMA-2, SMA-3, and SMA-4. Hypomorphic mutations of mab-21 are epistatic to cet-1 pathway mutations affecting ray pattern formation (cet-1, sma-2, sma-3, sma-4), while they do not affect body size in double mutants [15]. Reportedly, this interaction did not reflect regulation of mab-21 gene transcription by the sma signaling pathway.
The above genetic studies prove the biological relevance of the mab-21 – TGF-beta interaction, but stop short of addressing several key questions. What is the molecular counterpart of the genetic interaction described in C. elegans between mab-21 and the sma pathway? Is this interaction conserved in vertebrate development, and what TGF-beta superfamily molecules does it involve? Does it play a relevant role in the establishment of vertebrate dorsoventral (DV) polarity? If so, at what level do MAB-21 and TGF-beta pathways converge into a single regulatory cascade? In the present study, through a gain-of-function strategy, we analyze the molecular interplay between MAB21L2, a vertebrate homolog of C. elegans MAB-21, and TGF-beta superfamily signaling molecules. In particular, we focus on the signaling pathway initiated by BMP4, a ligand structurally related to CET-1/DBL-1, that regulates DV axis formation and numerous key aspects of neural development in chordata.
Results
Xmab21l2 expression in development
It is well established that the BMP4 pathway is essential for proper formation of the dorsoventral axis; overexpression of BMP4 in Xenopus embryos can lead to the formation ventralized embryos, i.e. embryos devoid of axial structures, whereas BMP4 depletion can lead to dorsalized embryos, i.e. embryos exhibiting an expansion of dorsal mesoderm [17-20]. At tailbud stages, the expression of Xmab21l2 is restricted to the primitive eye field, optic recess, optic cup and retina (excluding the choroid fissure), to the tectum and dorsal neural tube, and to branchial arches (Figure 1A–E). The early-onset and sustained expression of the MAB21L2 protein in oocyte-, blastula, gastrula, tailbud and tadpole lysates suggests that Mab21l2 is expressed both maternally and zygotically, during and after gastrulation (Figure 1F).
Figure 1 Expression of Mab21l2 gene and protein in Xenopus laevis development. A) Double whole mount in situ hybridization for Xmab21l2 (blue) and Xkrox20 (brown). At the end of neurulation (dorsal view of a st. 19 embryo is shown) Xmab21l2 expression (arrows) is restricted to the eye primordium (bottom arrowhead) and midbrain (top arrowhead). Xkrox20 labels the third and fifth rhombomeres (arrows). B) Frontal view of a stage 24 embryo. Xmab21l2 expression (blue, arrows) is restricted to the eye (left, solid arrow) and midbrain (right, solid arrow), clearly posterior to the forebrain marker Emx2 expression domain (brown, empty arrow). C–E: Sections obtained from late tadpole embryos were wholemount-hybridized with Xmab21l2. Three transverse sections, from anterior to posterior, show Xmab21l2 expression in the retina and lens (Ey), in the branchial arches (BA), midbrain (arrow in C), hindbrain (arrow in D), and in the dorsal neural tube, (SC in E). F: Western blot analysis of MAB21l2 protein expression in Xenopus oocytes (stage 0) and Xenopus embryos (stages 3–23). ME: day 12 mouse embryo (positive control). Protein concentrations were quantitated through a Bradford assay and loaded on gel in equal amounts. The experiment shows maternal expression of MAB21l2 (41 kD, arrow), the levels of which decrease around blastula stage to resume zygotically thereafter. In particular, stage 11 gastrulae are positive for the MAB21l2 protein.
Mab21l2 coexpression rescues the dorsal axis in BMP4-injected Xenopus embryos
To determine whether Xmab21l2 and BMP4 have a synergistic or antagonistic interaction in vivo, we took a gain of function approach utilizing Xenopus laevis embryos. Preliminary RT-PCR experiments conducted on gastrula lysates injected at the one-cell stage with BMP4 mRNA had shown that, as previously reported by others in studies conducted in the nematode, Xenopus Mab21l2 expression is not controlled by BMP4 signaling (not shown).
Prior to addressing the nature of the interaction between Xmab21l2 and BMP signaling molecules, we analyzed the effects of isolated Xmab21l2 overexpression. Embryos were injected at the one-cell stage with 800 pg in vitro-transcribed Xmab21l2 alone. We analyzed the phenotype of injected tailbud embryos as well as the expression of dorsal markers at gastrulation. A significant percentage of injected embryos (48%, N = 200) featured various degrees of dorsalization/anteriorization (DAI 6–8), with a well formed, sometimes enlarged head, a broad cement gland, a short and/or bent axis (Figure 2B). We prepared embryos for histological examination by sectioning them along the longitudinal plane, both sagittally and horizontally. Histological analysis of horizontal sections revealed many tadpoles featuring an enlarged notochord (nc, fig. 2D), suggestive of a possible defect in convergent extension. Finally, wholemount in situ hybridization experiments conducted on gastrulae with markers of DV polarity revealed an increased percentage of embryos (50%, N = 22) with expanded and/or enhanced Chordin gene expression (blue signal, fig. 2F). Perturbation of Xvent2 gene expression was sporadic, with 5 out of 21 Xmab21l2-injected embryos featuring a reduced Xvent2 expression domain (not shown). Our results are in sufficient agreement with those previously obtained by others [9], confirming that Xmab21l2 overexpression produces neither signs of complete dorsalization, such as Janus twins or radial eyes [21], nor axis duplication.
Figure 2 Isolated overexpression of Xmab21l2 produces partially dorsalized embryos. A, C, E: control-injected embryos; B, D, F: Xmab21l2-injected embryos. A–D: tailbud-tadpole stages; E, F: gastrula stage. In B: external appearance of Xmab21l2-injected embryos. D: horizontal section of an Xmab21l2-injected section stained with orange-G/aniline blue. Note enlarged notocord (nc). F: expanded and enhanced Chordin expression in Xmab21l2-injected gastrulae (blue signal).
To determine whether Xmab21l2 can compensate the effects of BMP4 overexpression, Xenopus embryos were injected at the one-cell stage with in vitro-transcribed BMP4 mRNA alone (0.6 ng, n = 111), or coinjected with Xmab21l2 mRNA (0.8 ng, n = 83; 1.6 ng, n = 101). We scored the formation of dorsal structures in tadpole stage embryos by using the dorsoanterior index [21]. A complete loss of dorsal structures (DAI = 0) (top in Figure 3A) was observed in 11.4% of the embryos after BMP4 overexpression (0.6 ng) (Figure 3B). Coinjection with 0.8 or 1.6 ng Xmab21l2 drastically reduced this class (0% and 3.1%, respectively). Likewise, whilst complete dorsal structures (DAI = 5) (Figure 3A, bottom) were observed in only 34.1% of BMP4-injected embryos, coinjection with 0.8 or 1.6 ng Xmab21l2 increased this class significantly (65.9% and 59.4%, respectively), suggesting that Xmab21l2 antagonizes BMP4 in its ability to ventralize embryonic mesoderm in vivo (see Figure 3B for details).
Figure 3 Xmab21l2 rescues dorsal structures in BMP4 injected embryos. (A) As previously described, injection of 0.6 ng of BMP4 mRNA gave rise to ventralized embryos. The most severely ventralized phenotype of BMP4 injection, called bauchstück, was scored with a dorso-anterior index (DAI) of 0 (top), whereas normally dorsalized embryos were assigned a DAI of 5 (bottom). (B) Embryos were injected with 0.6 ng BMP4 (n = 111), with 0.6 ng BMP4 plus 0.8 ng Mab21l2 (n = 83), or with 0.6 ng BMP4 plus 1.6 ng Mab21l2 (n = 101). In each group, we scored the percentage of complete ventralization (DAI = 0, grey bars) and normal dorsal axis formation (DAI = 5, black bars) in embryos that completed development. Intermediate classes are omitted in the plot. Coinjection of embryos with Mab21l2 significantly increased the percentage of correctly dosalized embryos (see text for details). Statistical analysis was conducted using the Chi square algorithm (1 df). *: p = 0.0005; **: p = 0.0087.
Next, by wholemount in situ hybridization, we analyzed the expression of molecular markers of dorsal (chordin, Figure 4A–C) and ventral (Xvent2, Figure 4D–F) mesoderm in wildtype embryos, as well as embryos injected with BMP4 alone or coinjected with Xmab21l2. Whilst the expression domain of chd was sharply reduced in 88% of BMP4-injected embryos, 47% of the embryos co-injected with Xmab21l2 showed wild type chd expression (Figure 4B,C,G). Likewise, 64% of the BMP4-injected embryos showed a dorsal expansion of Xvent2 expression, that was rescued in 83% of the embryos co-injected with Xmab21l2 (Figure 4E,F,H).
Figure 4 Xmab21l2 restores the normal expression of Xvent2 and Chordin in BMP4-injected embryos. Chordin (A–C) and Xvent2 (D–E) expression were analyzed by whole mount in situ hybridization. Embryos are shown in a vegetal view, dorsal to the top, ventral to the bottom. (A, D) Wild type expression of Chordin (A) and Xvent2 (D) in stage 10.5 gastrulae; (B, E) embryos injected with 1.2 ng of BMP4 mRNA alone showed reduced Chordin expression (B) (22/25) and expanded Xvent2 expression (E) (16/25); (C, F) embryos co-injected with 1.2 ng of BMP4 and 1.6 ng Xmab21l2 mRNA showed a rescue of wild type Chordin (C) (17/36) and Xvent2 expression (F) (24/29). (G) Percentage of embryos exhibiting wild type (black bars) or reduced (grey bars) Chordin gene expression in embryos injected with 1.2 ng BMP4 alone, and in embryos coinjected with 1.6 ng Mab21l2. Coinjection of Mab21l2 significantly increased the number of embryos exhibiting a wild type Chordin expression pattern; *: p = 0.004. (H) Percentage of embryos exhibiting wild type (black bars) or expanded (grey bars) Xvent2 gene expression in embryos injected with 1.2 ng BMP4 and in embryos coinjected with 1.6 ng Mab21l2. Again, Mab21l2 significantly increased the number of embryos exhibiting a wild type Xvent2 expression pattern; **: p = 0.00044. Statistical analysis was conducted using the Chi square algorithm (1 df).
MAB21L2 interacts with SMAD1 in vitro and in vivo
The antagonistic interaction between Mab21l2 and BMP4 may be either interpreted as evidence of a molecular interaction between MAB21L2 and some BMP signaling transducer, or alternatively suggest that MAB21L2 and some component of the BMP signaling pathway compete for a common cofactor. It is well established that the DNA-binding protein SMAD1, once phosphorylated at the plasma membrane by the BMP receptor heterotetramer, assembles with the receptor-independent protein SMAD4, enters the nucleus, establishes interactions with various cofactors, and acts by regulating the transcription of a host of target genes [reviewed in [22]]. Because vertebrate MAB-21 proteins are mainly or exclusively distributed in the nucleus [6], we investigated the possibility of a molecular interaction between MAB21L2 and SMAD proteins. In order to obtain in vitro evidence of this interaction, P19 cells were either treated with BMP4 or left untreated, and subsequently lysed. P19 cell extracts were then incubated with a resin-bound His-ZZ-tagged form of MAB21L2, or with a control His-ZZ-coupled resin. Eluates were analyzed by immunoblotting with a polyclonal anti-SMAD1 antibody. The experiment revealed a clear interaction between synthetic His-ZZ-MAB21L2 and endogenous SMAD1 that was absent when P19 lysates were incubated with the control resin (Figure 5A). Although BMP4 treatment seemed to enhance it considerably, the interaction was not entirely BMP4-dependent, suggesting that conformational changes secondary to receptor-mediated phosphorylation may not be strictly required for SMAD1 to interact with MAB21L2.
Figure 5 Mab21l2 interacts with Smad1 in vitro and in vivo. (A) Lanes 1, 2: immunoblotting of total lysates from P19 cells, mock- and Smad1-transfected, respectively. P19 cells do not express detectable levels of endogenous SMAD1. Lanes 3–8: Affinity chromatography (pull-down) experiment. High-stringency eluates from untransfected P19 were separated by SDS-PAGE and transfered onto nitrocellulose filters. The blots were immunolabeled using an anti-SMAD1 antibody. Lanes 3–6: the in vitro interaction is not strictly dependent on activation of BMP signaling: the pull-down experiment was performed with 20 μl (3, 4) and 40 μl of P19 cell lysates (5, 6). Lysates came from P19 cells treated (4, 6) or untreated (3, 5) with BMP4. Lanes 7, 8: an His-ZZ-MAB21L2 protein (HisMab) synthesized in E. coli (lane 8) was coupled to a sepharose-Ig resin and incubated with cell lysates of P19 cells treated with BMP4. As a negative control, an in-vitro synthesized His-ZZ incubated with the same P19 lysates fails to pull down a 53 kD band. Arrows: SMAD1 (53 kD). (B) direct interaction between SMAD1 and MAB21L2; no direct interaction between SMAD4 and MAB21L2. An His-ZZ-MAB21L2 protein (+Mab) synthesized in E. coli (lane 2) was coupled to a sepharose-Ig resin and incubated with in vitro-translated SMAD1 (lanes 2), SMAD4 (lanes 4), and SMAD1 + SMAD4 (lanes 6). Negative controls were represented by His-ZZ-coupled resins (-Mab) incubated with the same in vitro-synthesized proteins (lanes 3, 5, 7). Lanes 1 and 8 contain in vitro-synthesized SMAD1 and SMAD4, respectively. (C) BMP4-dependent in vivo co-immunoprecipitation of flag-SMAD1 and myc-MAB21L2 in P19 cells. Cells were mock-transfected, transfected with flag-SMAD1 and/or with myc-MAB21L2, as indicated, and either treated with BMP4 or left untreated. In lanes 3–7, cell lysates were immunoprecipitated with a monoclonal anti-flag antibody. Cell lysates in lanes 1, 2 and immunoprecipitates in lanes 3–7 were gel fractionated and transfered to nitrocellulose filters. Blots were immunolabeled with an anti myc antibody. Arrow (42 kD) points to a band in lanes 2, 3) corresponding to myc-MAB21L2. (D) in vivo co-immunoprecipitation of flag-SMAD1 and myc-MAB21L2 in stage 11 Xenopus embryos, facilitated by BMP4 overexpression. Embryos were water-injected, injected with flag-Smad1 and/or with myc-Mab21l2 RNA, as indicated, and coinjected with BMP4 where indicated. In lanes 3–7, cell lysates were immunoprecipitated with a monoclonal anti-flag antibody. Embryo lysates in lanes 1, 2 and immunoprecipitates in lanes 3–6 were gel fractionated and transfered to nitrocellulase filters. Blots were immunolabeled with an anti myc antibody. Arrow (42 kD) points to a band in lanes 2–4) corresponding to myc-MAB21L2.
These results prompted more questions, such as whether the interaction between MAB21L2 and SMAD1 is direct and whether it is dependent on prior formation of a SMAD1-SMAD4 complex. Once again, we resorted to affinity chromatography, incubating in vitro-translated, 35S-Met-labeled SMAD1 and SMAD4 with a resin containing His-ZZ-MAB21L2. This experiment suggested that the interaction with SMAD1 is direct, and that MAB21L2 does not assemble with SMAD4 directly but only through SMAD1, indicating that the formation of a SMAD4-MAB21L2 complex is mediated by SMAD1 (Figure 5B). Indirectly, this experiment also showed that MAB21L2 does not obviously compete with SMAD4 to assemble with SMAD1.
Finally, we investigated whether the in vitro interaction observed between MAB21L2 and SMAD1 could be replicated in vivo. P19 cells co-transfected with myc-MAB21L2 and flag-SMAD1 were either treated with BMP4 or left untreated. In parallel, Xenopus laevis embryos were injected at the four-cell stage into the animal pole of each blastomere with myc-Mab21l2 and flag-Smad1 mRNAs, and coinjected with either BMP4 mRNA or H2O. The embryos were grown until mid-gastrula stage (st. 10.5/11). Both cell- and embryo lysates were subjected to immunoprecipitation, using an anti flag monoclonal antibody, whereas an anti-myc antibody was utilized for immunodetection in Western blotting. Experiments conducted in transfected P19 cells showed that SMAD1 coprecipitates with MAB21L2, and that the interaction is enhanced by activation of BMP signaling, which is required for nuclear localization of receptor-dependent SMADs (Figure 5C). Likewise, MAB21L2-SMAD1 co-precipitation took place especially, albeit not exclusively, in BMP4-coinjected embryos (Figure 5D). Interaction of the two proteins in the absence of exogenous BMP4 can likely be explained by the abundance of endogenous BMP2/4 ligands in stage 11 gastrulae.
When targeted to an heterologous promoter, MAB21L2 acts as a strong transcriptional repressor
Because in vivo MAB21L2 acts as a BMP4 antagonist, and because BMP4 signaling is transduced in the nucleus by SMAD transactivators, we asked whether MAB21L2 has any intrinsic repressor functions. To address this point, we generated fusion transcripts adding the GAL4 DNA binding domain (residues 1–147) [23], either to the N- or C-terminus of MAB21L2. For as yet unexplained reasons, the latter fusion protein proved extremely unstable and was discarded. Conversely, the N-terminal fusion protein is stable and was shown to bind DNA by gel-retardation assay (Figure 6A). Thus, we were able to assess its activity in cotransfection assays, using as a reporter system the GAL4 target promoter (5XUAS) fused to luciferase. Luciferase activity in Mab21l2-cotransfected COS7 cells was compared to the baseline reporter activity scored in cells transfected with the GAL4 DBD alone (G-D) or with a transcriptionally inactive GAL4 DBD fusion protein, (GAL4HOXB3NT, G-N) [24]. Reporter activity was clearly downregulated in Gal4-Mab21l2-transfected cells only, suggesting that MAB21L2 may function as a transcriptional repressor or co-repressor (Figure 6B). The same luciferase assay was performed in P19 cells, co-transfected with plasmid DNA, G-D, and G-M, revealing a 12-fold downregulation of 5XUAS-luc in G-M-transfected cells (not shown). This confirmed the evidence of a strong repressor activity of MAB21l2 when targeted to DNA.
Figure 6 When targeted to a heterologous promoter, Mab21l2 behaves as a strong transcriptional repressor. (A) An in vitro-translated chimeric protein containing the GAL4 DNA binding domain fused to the N-terminus of MAB21L2 was used in an electrophoretic mobility shift assay. An end-labeled ds-DNA containing the GAL4 nucleotide binding site (5XUAS) was incubated in native conditions with in vitro translated GAL4-MAB21L2 (G-M) and separated electrophoretically by nondenaturing PAGE. As negative controls, we used free oligonucleotides (F) and rabbit reticulocyte lysates (RRL). As a positive control we used a previously tested GAL4 fusion protein, GAL4-D9NT (G-D9) [40]. The arrow indicates a bandshift specific for GAL4-MAB21L2 and absent in negative controls. u: unbound. (B) 5XUAS-luc transcription is clearly downregulated in cells transfected with GAL4-MAB21L2 (G-M) vs. untransfected COS7 cells or same cells transfected with GAL4 DBD, G-D) alone (*: p = 0.0019). No downregulation of 5XUAS-luc activity was observed in COS7 cells transfected with a fusion of GAL4 and the N-terminal domain of the HOXB3 protein, which possesses no transcriptional activation or repression activity [24] (ns: not significant). Statistical analysis was conducted using the T test method (two tails).
Discussion
Mab-21 genes have been isolated in several species and have been shown, through genetic and epigenetic loss-of-function experiments, to play key roles in various developmental processes, ranging from gastrulation and neural tube closure to eye and lens formation [2,9,10,12,13]. However, no biochemical data have been available to identify the regulatory cascade(s) in which these elusive factors are involved. While epistatic analysis has proposed that C. elegans mab-21 undergoes inhibitory regulation by TGF-beta superfamily signals [15], the molecular nature of this regulation remained obscure. Likewise, no information has been published as to the conservation and relevance of the sma/mab-21 interaction in vertebrate development.
Our results, obtained in an anamniotic model system, the Xenopus embryo, and in murine embryonic carcinoma cells, indicate that MAB21L2 interacts functionally with SMAD1, a nuclear transducer of BMP2/4/7 signaling. Overexpression of Mab21l2 complements the effects of BMP4 overexpression, in keeping with the epistatic interactions observed in nematodes [15]. Thus, genetic interactions first described in pseudocoelomates appear to be strongly conserved in evolution.
As the nuclear localization of MAB21L2 suggested, our pull-down and immunoprecipitation results indicate that MAB21L2 is a new interactor of the receptor-activated transducer SMAD1. Whilst the interaction between MAB21L2 and SMAD1 appears to be direct, our results do not seem to favor a direct contact between MAB21L2 and SMAD4.
The interaction of MAB21L2, a transcriptional repressor, with nuclear transducers of BMP signaling offers a possible explanation for the results of loss-of-function experiments conducted by other investigators in Xenopus laevis and mouse embryos. The increasing levels of MAB21L2 protein observed starting at midblastula transition may be required to compensate mesoderm-ventralizing signals triggered by BMP2/4. In the absence of MAB21L2 genes, completion of gastrulation may be hampered, resulting in various abnormalities of dorsoventral polarity [9,12]. Results obtained by others have demonstrated that SMAD complexes can be turned into negative regulators of transcription through a physical interaction with transcriptional repressors [25]. Our results indicate that MAB21L2 possesses a considerable transcriptional repressor activity. However, the antagonistic interactions between MAB21L2 and BMP signaling may not necessarily depend on the formation of transcriptional regulation complexes. Further experiments will be required to characterize the mechanism(s) of transcriptional regulation mediated by MAB21L2. Importantly, the identification of Mab21l2 downstream genes will make it possible to investigate mab-21 – BMP4 interactions from the opposite standpoint, i.e. by looking at the modulatory effect of BMP signaling on the expression of Mab21l2 targets.
Although our results are more directly relevant to understanding the functions of Mab21l2 in early embryogenesis, the gene is clearly expressed at high levels and in a tightly localized fashion in neurulation and morphogenesis as well, likely contributing to neural tube and eye formation. Indeed, because of redundancy in the mab-21 gene family, many of the early effects of mab-21 gene mutation may be masked, uncovering these genes' roles in later stages of development and postnatal life.
In late developmental processes, well beyond the end of gastrulation, MAB-21 proteins may prolong their dynamic interaction with BMP signaling transducers. As mentioned, a cell-autonomous requirement for Mab21l1 in lens formation has been recently documented in knock-out mice [13] that develop otherwise normally in the nervous system. In the same mice, genetic redundancy has hampered the analysis of mab-21 gene function in retinal development. However, antisense studies have shown that inhibition of mab-21 leads to disruption of the retinal anlage, where the gene is strongly expressed in a territory overlapping with the Bmp4 and Xvent2 expression domain. Recent studies have shown that BMP signaling plays a relevant role in guiding morphogenesis along the dorsoventral axis of the chick [26] and Xenopus [27] retina. In this context, it would be interesting to determine if mab-21 genes act by balancing BMP signals in the establishment of retinal polarity.
Mab21l1 and Mab21l2 are prominently expressed in the embryonic midbrain, both dorsally and ventrally, and in prosomere 1 [5,6]. Roles exerted by BMP signaling molecules in these territories are starting to emerge, and appear relevant to the induction and patterning of dorsal mesencephalic structures (roof plate) [28], and, more ventrally, to the differentiation of dopaminergic precursors [29].
Throughout the hindbrain and spinal cord, Mab21l2 is expressed mostly in dorsal territories. In cerebellar development, it is expressed in the cerebellar plate and, later on, in granule cell neurons [5]. BMP signaling molecules are coexpressed with Mab21l2 at several of those sites, and their developmental roles have been at least partially addressed: as an example, granule cell specification can be induced by BMP7 overexpression in the dorsal and ventral rhombencephalon [30]. BMP7 is homologous to BMP2 and BMP4, and its signal can be transduced by SMAD1 [31]. Likewise, in the spinal cord Mab21l2 is expressed in a dorsal territory, where BMP2/4 signaling plays a key role in the specification of neuronal identities alongside the dorsoventral axis of the neural tube [32-34].
Finally, in postmigratory neural crest, the interplay between BMP2/4 and Mash1 maintains competence for neuronal differentiation [35]. In neural crest derivatives, including the branchial arches, Mab21l2 may thus participate in specifying neuronal and non-neuronal cell fates by interacting with BMP signals.
Conclusions
Our results provide a biochemical and molecular foundation for future studies of mab-21 gene function in vertebrate systems, demonstrating that MAB21L2 interacts functionally with the BMP4 signalling pathway and physically with its best characterized nuclear transducer, SMAD1. Furthermore, we show that MAB21l2 can act as a powerful transcriptional repressor when targeted to an heterologous promoter. Further work is clearly required to determine if MAB21L2 binds DNA, what its binding specificity is, or if it only exerts its effect in a DNA-binding-independent fashion. More broadly, additional studies are required to clarify the role of mab-21 genes in the context of BMP signaling, and their likely function as a molecular switch linking different regulatory pathways in development and disease.
Methods
Image acquisition and processing
Images were obtained either by traditional photography (slides 1–3) or by electronic scanning of autoradiography films. All images were processed through the Adobe Photoshop 7.0 software or through the Adobe Illustrator 10 software.
Isolation of Xmab21 cDNA and constructs
The Xenopus laevis homolog of mouse Mab21L2 was isolated by screening a Xenopus stage 28–30 lambdaZapII library (Courtesy of Richard Harland) using the murine homolog as a probe. The 2.8 Kb clone isolated contained the full length Xmab21l2 cDNA (GenBank AF040992) as well as 5' and 3' regions. The coding region of Xmab21 was excised using NotI and BalI (1.3 Kb) and subcloned into pBluescript for whole mount in situ hybridization, or BamHI-StuI (1.1 Kb) and subcloned into pT7TS for overexpression in embryos and into pCDNA3 for cell transfection.
Embryos
All animal experimentation was conducted according to the stipulations of the Institutional Animal Care and Use Committee, San Raffaele Scientific Institute. In order to obtain embryos, Xenopus females were primed 1000 U of human chorionic gonadotropin (Profasi HP 5000, Serono) the night before collection. Ovulated eggs were fertilized with testis homogenates and allowed to develop in 0.1X MMR (1X MMR is 0.1 M NaCl, 2.0 mM KCl, 1.0 mM MgCl2, 2.0 mM CaCl2, 5 mM HEPES, pH7.4). Jelly coats were removed in 3.2 mM DTT, 0.2 M Tris pH 8.8. Embryos were staged according to Nieuwkoop and Faber [36] and fixed in MEMFA [37] for in situ hybridization.
Histology
For histological examination, wt or injected and stained embryos were fixed in MEMFA, embedded in wax, cut into 10 μm sections, dried onto slides, dewaxed with xylene and dehydrated with alcohol. Sections were rehydrated and stained with an orange G solution (2 g orange G, 8 ml glacial acetic acid, 100 ml water) and subsequently with an orange G-aniline blue solution (2 g orange G, 0.5 g aniline blue, 8 ml glacial acetic acid, 100 ml water), and dehydrated [36,38].
Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed on staged embryos as described by [37]. The antisense or control sense-strand were generated from the following linearized plasmids: pBS-Xmab21 (antisense linearized NotI, trancribed from T7; sense linearized HindIII, transcribed from T3), Engrailed 2, Krox20, Emx2 (Maria Pannese), CS2+-Chordin (a gift of Stefano Piccolo), Xvent1, Xvent2 (a gift of Christof Niehrs). In single whole-mount hibridizations the probe was labeled with digoxigenin. Digoxigenin-labeled probes were immunolabeled with alkaline phosphatase-conjugated anti-digoxigenin antibody and with the BM Purple substrate (Boehringer). In the double whole-mount one probe was digoxigenin-labeled the second was labeled with fluorescein-UTP. Each probe was used at a concentration of 1 g/ml. Sequential detection with alkaline phosphatase-conjugated anti-fluorescein antibodies (1:8000) and anti-digoxigenin (1:2000) was done. The first color reaction was revealed with NBT/BCIP, while for the second color reaction the Vector Black kit II (Vector Laboratories) was used.
Selection of peptide and production of Mab-21 antisera in rabbits
A 14aa C-terminal stretch from the mMab21l2 protein was chosen as immunogenic peptide to obtain antisera in rabbits. Peptide was synthesized, lyophilized and coupled to Keyhole limped hemocyanin (KLH). Coupled peptide was used for immunization of two rabbits. Immune and preimmune serum were controlled by ELISA using the same peptide coated at different serial dilutions. Immune serum was then purified by affinity chromatography: briefly, 20 mg peptide was coupled on CNBr-Sepharose-4B resin (Pharmacia), according to the standard procedure suggested. 50 ml antiserum diluted in 1:1 in 1X PBS was applied on the peptide/CNBr-Sepharose-4B over night at 4°C. After washing the resin with 50 ml PBS and 50 ml 3 M NaCl, bound antibodies were eluted using 100 mM Glycine pH 1.8. Amount, specificity and purity of the antiserum was tested by the Bradford assay, ELISA and SDS-PAGE/Coomassie staining. Purified antibody was tested for specificity in western blot on total protein extracts from cos-7 cells transfected with mMab21l2-myc fusion protein or mock vector, and on total brain extract from mouse embryos. One single band of 41 KDa was detected by anti Mab21l2 antibody. The same 41 kD band was detected by WB in lysates from transfected COS-7 cells, and was missing in untransfected cells. Cross-reactivity of anti mouse Mab21l2 antibody with the Xenopus Mab21l2 protein wastested by Western blot on total extracts from X. laevis embryos. The antibody did not work successfully in IHC or IP experiments, suggesting that it fails to recognize the native epitope.
Western blotting
Protein concentrations in extracts were quantitated through Bradford assays. Extracts were gel-fractionated by denaturing polyacrylamide gel electropohoresis SDS-PAGE. Gels were transfered onto nitrocellulose filters. Even loading was confirmed by Ponceau staining of nitrocellulose filters. Imunostaining was conducted as described [39].
Embryo injections
The vector Xmab21-T7TS was linearized with EcoRI and transcribed with the T7 RNA polimerase, N-MycMab21L2-CS2+ was linearized with Asp718 and transcribed with the SP6 RNA polymerase. The Flag-Smad1 was excised from the human construct Flag-Smad1-CMV5 (Courtesy of J. Massagué) and subcloned into the vector pCS2+. The Flag-Smad1-pCS2+ vector was linearized with Asp718 and transcribed with SP6. BMP4 expression constructs were a kind gift of N. Ueno. Capped mRNA was synthesized using the Ambion Message Machine kit according to manufacturer's instructions. Injections were performed in 4% Ficoll in 1X MMR. Embryos were injected animally at the one-cell or two-cell stage into one or both blastomeres. The amount of mRNA injected is given in the text.
Luciferase activity assay
COS7 cells were maintained in DMEM Medium (Gibco Brl) supplemented with 10% FBS (Euro Clone), 2 mM L-glutamine (Euro Clone), 100 IU/ml penicillin, and 100 É g/ml streptomycin. Calcium phosphate transfection was performed in a 60 mm Petri dish with different amounts of the expression constructs, 5 É g of reporter plasmids, and 100 ng of pRL-TK (Promega) as an internal control. 18 h after transfection the cells were washed twice with PBS1X and the medium was replaced with DMEM Medium supplemented with 10% FBS, 2 mM L-glutamine (Euro Clone), 100 IU/ml penicillin, and 100 É g/ml streptomycin. Cells were harvested 36 h after the transfection, lysed, and assayed for luciferase activity (Dual Luciferase Reporter Assay System Kit, Promega). All luciferase assays were performed in triplicate.
Immunoprecipitations
P19 cells were maintained in MEM alpha medium (Gibco Brl) supplemented with 10% FBS (Euro Clone), 2 mM L-glutamine (Euro Clone), 100 IU/ml penicillin, and 100 μg/ml streptomycin. Calcium phosphate transfection was performed in a 100 mm Petri dish with 10 μg of mycMab21l2 expression construct and 10 μg of flagSmad1 expression construct. 24 h after transfection the medium was replaced with MEM Alpha Medum supplemented with 1% FBS 2 mM L-glutamine (Euro Clone), 100 IU/ and 40 ng/ml BMP4 (R&D Systems). Cells were harvested 36 h after the transfection with TEN Solution (40 mM Tris pH7.5; 1 mM EDTA; 150 mM NaCl) and centrifugated for 15 minutes at 5000 RPM. Cell pellets were frozen at -80°C o/n, thawed at RT and resuspended in 5 volumes of Extraction Buffer (10 mM Hepes pH 7.9; 400 mM NaCl; 0,1 mM EGTA, 5% glicerol). The samples were centrifugated at 34000 RPM for 30 minutes at 4°C and the supernatants were harvested. The concentration of the proteins was determined by BCA assay (Pierce). Xenopus laevis lysates were obtained in the same way starting from injected embryos harvested by centrifugation.
After preclearing with 50 μl of protein G-sepharose o/n at 4°C, lysates were incubated with 25 μg of M2 anti-Flag monoclonal antibody (Sigma) and 50 μl of protein G-Sepharose o/n at 4°C. The resin was washed twice with Dilution Buffer (10 mM Tris-Cl pH8; 140 mM NaCl; 0.025% NaN3; 0.1% triton), once with TSA solution (10 mM Tris-Cl pH8; 140 mM NaCl; 0.025% NaN3) and once with 50 mM Tris-Cl pH 6.8. The protein complexes were eluted by addition of Sample buffer (Tris-Cl 125 mM pH 6.8; 0.1 M 2-mercaptoethanol; 2% SDS; 20% glycerol; 25 mg/ml Bromphenol Blue) followed by boiling for 5 minutes and separated on an SDS-polyacrylamide gel followed by anti-myc (1 μg/ml of clone n°9E10) immunoblotting. The secondary antibody was a goat anti-mouse antibody HRP-conjugated (1:30000 Bio-Rad). The blots were developed with the Supersignal West Pico reagent (Pierce).
Purification of His-ZZ fusion proteins
The protocol was based on manufacturer's recommendations for Ni-NTA Agarose (Qiagen). His-ZZ fusion protein expression in bacterial cultures (37°C, BL21 E. coli) was induced by β-D-thiogalactopyranoside (IPTG) (0.4 mM for His-ZZ-Mab21; 1 mM for His-ZZ and for His-ZZ-Smad1) when the optical density (OD600) reached 0.7–0.9. Cells were harvested after 3 hours, resuspended in lysis buffer (50 mM NaH2PO4; 300 mM NaCl; lysozyme 2 mg/ml), and sonicated. The purification protocol was performed with Ni-NTA Agarose (Quiagen) beads and the elution with 250 mM Imidazole (Sigma).
His-ZZ-Mab21l2 fusion protein pull-down assays
120 μg of His-ZZ-Mab21l2 fusion protein was incubated 1 h at 4°C with 25 μl of IgG-Sepharose beads (Amersham). The beads were recovered by centrifugation and washed ten times with 0.4 M KCl. The beads were incubated 1 h with 250 μg of precleared lysates of P19 treated and untreated with 40 ng/ml of BMP4. The beads were washed 5 times with 0.2 M KCl; the protein complexes were eluted by addition of Sample buffer followed by boiling for 5 minutes, and separated on an SDS-polyacrylamide gel for anti-Smad1 (1:2000 Santa Cruz) immunoblotting. The secondary antibody was a goat anti-rabbit HRP-conjugated antibody (Bio-Rad). The blots were developed with the Supersignal West Pico reagent (Pierce).
His-ZZ-Smad1 fusion protein pull-down assays
120 μg of His-ZZ-Smad1 fusion protein was incubated 1 h at 4°C with 25 μl of IgG-Sepharose resin (Amersham). The beads were recovered by centrifugation and washed ten times with 0.4 M KCl.
The beads were incubated 1 h with 250 μg of precleared lysates of COS7 cells transfected with mycMab21l2. The beads were washed 5 times with 0.2 M KCl; the protein complexes were eluted by addition of Sample buffer,boiled for 5 minutes and separeted on a SDS-polyacrylamide gel for anti-myc immunoblotting.
His-ZZ-Mab21l21 fusion protein pull-down assays with in-vitro translated proteins
120 μg of His-ZZ-Mab21l2 fusion protein was incubated 1 h at 4°C with 25 μl of IgG-Sepharose beads (Amersham) in Binding buffer (50 mM Tris pH8; 50 mM KCl; 5 mM MgCl2; 1 mM DTT; 0.2% NP-40, 10% glycerol). The beads were recovered by centrifugation and washed with Binding buffer. In-vitro translated, 35S-methionine-labeled Smad1 and Smad4 were prepared with the TNT coupled transcription/translation system (Promega). 45 μl of the TNT reaction were mixed with the His-ZZ-Mab21l2 bound to the IgG-Sepharose beads. The mixture was incubated for 2 hours at 4°C in Binding buffer. The Sepharose-protein complex was washed four times with Wash buffer (50 mM Tris pH7.5; 150 mM NaCl; 1 mM EDTA; 0.2% NP-40), eluted by addition of Sample buffer followed by boiling for 5 minutes and separated on SDS-polyacrylamide gels. Gels were fixed and stained with 50% methanol, 10% acetic acid and 0.1% Comassie Blue for 20 minutes. After the incubation with Sodium salicylate 1 M (Fluka) for 20 minutes to improve the radioactive signal the gels were dried and exposed to Kodak X-Omat film o/n at -80°C.
Authors' contributions
DB did in situ hybridizations and in vivo overexpression experiments.
AB did pull-downs, immunoprecipitations and luciferase assays.
RL designed peptides and produced an anti MAB-21 polyclonal antibody.
VZ produced constructs for, and carried out DNA-binding assays and luciferase assays. In addition he helped design and evaluate all cellular assays.
GGC designed and coordinated the experimental work.
All authors read and approved the final manuscript
Acknowledgements
We thank Elena Moneta, Margherita Mariani and Laura Croci for their collaboration to this project; Ombretta Pozzoli and Franco Cotelli for their help with unpublished experiments; Maria Pannese for her assistance during our initial overexpression studies. The support of the Italian Telethon Foundation (Grant 740) is gratefully acknowledged. Additional support came from the Italian Ministry of University (FIRB Neuroscienze RBNE01WY7P).
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| 15613244 | PMC545073 | CC BY | 2021-01-04 16:39:07 | no | BMC Cell Biol. 2004 Dec 21; 5:48 | utf-8 | BMC Cell Biol | 2,004 | 10.1186/1471-2121-5-48 | oa_comm |
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BMC Cell BiolBMC Cell Biology1471-2121BioMed Central London 1471-2121-5-501562500710.1186/1471-2121-5-50Research ArticleThe two neutrophil members of the formylpeptide receptor family activate the NADPH-oxidase through signals that differ in sensitivity to a gelsolin derived phosphoinositide-binding peptide Fu Huamei [email protected]örkman Lena [email protected] Paul [email protected] Anna [email protected] Jennie [email protected] Charlotta [email protected] Claes [email protected] Department of Rheumatology and Inflammation Research, University of Göteborg, Sweden2 Department of Physiology, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA 19063 USA3 Department of Virology, University of Göteborg, Sweden2004 29 12 2004 5 50 50 5 10 2004 29 12 2004 Copyright © 2004 Fu et al; licensee BioMed Central Ltd.2004Fu 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 formylpeptide receptor family members FPR and FPRL1, expressed in myeloid phagocytes, belong to the G-protein coupled seven transmembrane receptor family (GPCRs). They share a high degree of sequence similarity, particularly in the cytoplasmic domains involved in intracellular signaling. The established model of cell activation through GPCRs states that the receptors isomerize from an inactive to an active state upon ligand binding, and this receptor transformation subsequently activates the signal transducing G-protein. Accordingly, the activation of human neutrophil FPR and FPRL1 induces identical, pertussis toxin-sensitive functional responses and a transient increase in intracellular calcium is followed by a secretory response leading to mobilization of receptors from intracellular stores, as well as a release of reactive oxygen metabolites.
Results
We report that a cell permeable ten amino acid peptide (PBP10) derived from the phosphatidylinositol 4,5-bisphosphate (PIP2) binding region of gelsolin (an uncapper of actin filaments) blocks granule mobilization as well as secretion of oxygen radicals. The inhibitory effect of PBP10 is, however, receptor specific and affects the FPRL1-, but not the FPR-, induced cellular response. The transient rise in intracellular calcium induced by the active receptors is not affected by PBP10, suggesting that the blockage occurs in a parallel, novel signaling pathway used by FPRL1 to induce oxygen radical production and secretion. Also the FPR can activate neutrophils through a PBP10-sensitive signaling pathway, but this signal is normally blocked by the cytoskeleton.
Conclusions
This study demonstrates that the two very closely related chemoattractant receptors, FPR and FPRL1, use distinct signaling pathways in activation of human neutrophils. The PIP2-binding peptide PBP10 selectively inhibits FPRL1-mediated superoxide production and granule mobilization. Furthermore, the activity of this novel PBP10 sensitive pathway in neutrophils is modulated by the actin cytoskeleton network.
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Background
The molecular basis for cellular recognition of signal molecules is their binding to specific cell surface receptors [1]. Despite large structural differences between the huge numbers of extracellular ligands, many bind to (and activate) specific receptors belonging to a large family of pertussis toxin-sensitive G-protein linked receptors (GPCRs) [2]. These receptors possess a high degree of similarity and although activated by different agonists, they transduce downstream signals that have many common features [3]. Nevertheless, it is clear that there are also important differences between receptor-ligand pairs regarding their functional repertoire [4]. The pattern recognition, formyl peptide receptor (FPR) family, belongs to the larger GPCR group of chemoattractant receptors [5]. The FPR gene family has a complex evolutionary history and the number, function, and specific cell expression of genes comprising the family vary considerably between different mammalian species [5,6]. Human neutrophil granulocytes express two FPR members [7], the FPR and the FPRL1 (the formyl peptide receptor like 1). FPRL1 was originally defined as an orphan receptor, cloned from an HL-60 cell cDNA library by low-stringency hybridization with the FPR sequence [7]. In the past few years several neutrophil activating ligands specific for FPRL1 have been identified [8], but knowledge on the precise functional activities and the signal transduction pathways utilized by FPRL1 are still somewhat limited. In contrast to FPRL1, a large number of studies on FPR induced cell function and signaling have been performed in neutrophils and in receptor-expressing transfected cell lines. These studies reveal that FPR signaling shows all the basic characteristic of a GPCR. Binding of the prototype FPR agonist fMLF to its receptor initiates a chain of events starting with dissociation of the G-protein α subunit from its βγ subunit. These subunits directly or indirectly activate downstream signaling molecules such as protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase (PI3K) that uses the membrane phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate [9]. The dissociated subunits of the G-protein also activate the phosphoinositide-specific phospholipase C (PLC) that upon cleavage of PIP2 produces the second messengers responsible for an elevation of intracellular free calcium [8]. It has been assumed that FPRL1 shares signal transduction features with FPR, since both receptors are sensitive to pertussis toxin and possess a high degree of amino acid identity in the signaling cytoplasmic domains [7]. Further, the functional responses induced by the FPRL1 specific hexapeptide agonist WKYMVM is in most respects similar to (or even indistinguishable from) those induced by the prototype FPR agonist fMLF [10-15]. However, in this study we have used a membrane permeant polyphosphoinositide-binding peptide (PBP10 [16]) derived from the cytoskeletal protein gelsolin, and we show that with respect to messengers generated by the these receptors leading to mobilization of secretory granules and NADPH-oxidase activation, two totally different signaling routes are used by FPR and FPRL1, one being sensitive (the FPRL1 route) the other insensitive (the FRP route) to the PIP2-binding peptide.
Results
NADPH-oxidase activity induced by fMLF and WKYMVM and effects of PBP10
The peptides fMLF and WKYMVM, agonists for FPR and FPRL1, respectively, both induced a robust oxidative burst measured as a release of superoxide anions (Fig 1). In accordance with the known receptor specificity [10,17], the WKYMVM response was totally inhibited by the FPRL1 specific antagonist WRWWWW whereas the FPR specific antagonist (cyclosporine H)was without effect. The effects were reversed for fMLF-triggered activity, that is, this response was totally inhibited by cyclosporine H but not affected by WKWWWW (data not shown). The time-courses of the responses were very similar, as were the EC50 values (Fig. 1A,B). Both the FPR and FPRL1 mediated response was inhibited by pertussis toxin (whereas no reduction in oxidase activity was seen with PMA, a PKC activator that bypasses the G-protein), showing that a heterotrimeric G-protein is involved in the signal transduction of both receptors (Fig. 1C). These indistinguishable responses were expected, based on the fact that the two receptors are very similar in the regions suggested to be of importance for intracellular signaling.
Figure 1 Oxidase activity induced by peptide agonists and the effect of PBP 10. Neutrophils were activated by fMLF or WKYMVM and the extracellular release of superoxide anions was recorded by chemiluminescence (expressed in Mcpm). (A) The figure shows the kinetics of the neutrophil response to two concentrations (100 nM and 20 nM) of fMLF or WKYMVM. (B) Dose dependent oxidase activation induced by the two agonists. The peak values were measured and the responses are given as percent of the maximal response. (C) Neutrophils were incubated for 120 minutes in the absence (control) or presence of pertussis toxin (PTX; 500 ng/ml) and the cells were then activated with fMLF (100 nM), WKYMVM (100 nM) or the receptor independent PKC activator PMA (100 nM) (inset). For comparision, the response induced by the two peptides after 90 minutes long incubation time with pertussis toxin is also included. (D) Effect of different concentrations of PBP10 on the neutrophil NADPH-oxidase response induced by fMLF or WYMVM, respectively. Data are expressed as percent of control (without PBP10; mean ± SD of three independent experiments).
Despite the indistinguishable activation by FPR and FPRL1 shown in Fig. 1A–C, functional differences between these two highly homologous receptors emerge when they are treated with the membrane permeant polyphosphoinositide-binding peptide rhodamine-B-QRLFQVKGRR (PBP10; Fig. 1D) prior to activation. The neutrophil NADPH-oxidase activity was totally inhibited by PBP10 when FPRL1 was stimulated by WKYMVM, whereas there was no effect of the peptide on the fMLF-induced, FPR-mediated neutrophil response. The IC50 value for the WKYMVM induced response was around 0.05 μM whereas no effect was seen on the fMLF induced activity even at concentrations up to 10 μM. There was no effect on WKYMVM or fMLF induced superoxide production of rhodamine alone (data not shown).
The neutrophil response is inhibited by Wortmannin
The amino acid sequence of PBP 10 peptide corresponds to the phosphatidylinositol 4,5-bisphosphate (PIP2) binding region segment 2 of the cytoskeletal protein gelsolin [16]. Following G-protein coupled receptor (GPCR) activation, the dissociated G-protein subunits activate the downstream phosphoinositide remodeling enzyme phosphatidylinositol 3-kinase (PI3K) [18], an enzyme that uses the membrane phosphoinositide PIP2 as substrate to generate the signaling molecule phosphatidylinositol 3,4,5-trisphosphate (PIP3) [9]. The oxidase activity induced by fMLF as well as by WKYMVM was largely inhibited by the specific PI3K inhibitor Wortmannin (Fig. 2A) suggesting that this signaling pathway is of importance in the cellular response. In contrast to the effect of PBP10, the effects of the PI3K inhibitor showed no specificity in the inhibition of the WKYMVM/FPRL1 triggered response.
Figure 2 Effect of different inhibitors on the neutrophil NADPH-oxidase activity. (A) Neutrophils were incubated with different concentrations of the PI3K specific inhibitor Wortmannin at 37°C for 30 minutes followed by stimulation with fMLF (100 nM) or WKYMVM (100 nM). A representative experiment is shown. (B) Neutrophils were incubated with the cell impermeable PIP2 binding peptide QRLFQVKGRR (1 μM final concentration) at 37°C for 5 minutes followed by stimulation with WKYMVM (100 nM). The effect of PBP10 (1 μM) is included for comparison.
PBP 10 inhibits the response to FPRL1 but not to FPR
The inhibition induced by PBP10 on neutrophil oxidase activity was linked to the receptor FPRL1 rather than to an FPRL1-specific agonist, since the same inhibition pattern was obtained when WKYMVM was replaced by serum amyloid A (SAA) (Table 1), an agonist that activates cells through binding to FPRL1 [19]. Moreover, no effect was induced by PBP10 when fMLF was replaced by annexinI9–25 (Table 1), a 17 amino acid peptide derived from the N-terminus of annexin I that has been shown to be a FPR agonist ([20] and our own observation). Moreover, the cellular responses mediated through other receptors, such as the chemokine receptor CXC or the C5a-receptor were not affected by PBP10 (Table 1).
Table 1 Effect of PBP10 on neutrophil NADPH-oxidase activity induced by receptor agonists#.
Neutrophil NADPH-oxidase activator (Concentration) Neutrophil receptor(s) Inhibition of NADPH-oxidase by PBP10 (1 μM)
WKYMVM (100 nM) FPRL1 +
SAA (5 μM) FPRL1 +
fMLF (100 nM) FPR -
Annexin9–25 (50 μM) FPR -
C5a (100 ng/ml) C5aR -
IL-8 (100 ng/ml) CXCR1, CXCR2 -
Abbreviations: C5a, complement 5a; FPR, formyl peptide receptor; IL-8, interleukin 8; SAA, serum amyloid A.
The membrane permeability of PBP10 is necessary for FPRL1 inhibition
Interaction of PBP10 with PIP2 depends on the peptide sequence, as truncation of the peptide either at the N terminus or C terminus reduces the PIP2 binding affinity [21]. Accordingly, Rh-VKGRRG affected the oxidase activity induced by WKYMVM only when higher concentrations of peptide were used (IC50 = 1 μM compared to 0.05 μM for PBP10) suggesting that PIP2 binding is of importance for inhibition of the neutrophil oxidase response induced by FPRL1.
The PBP10 peptide possesses not only PIP2 binding capacity but is also membrane permeable. This feature has been shown to be coupled to the rhodamine B part of the molecule, i.e., the unconjugated peptide QRLFQVKGRR exhibits the same PIP2-binding characteristics as PBP10 but lacks the ability to cross the plasma membrane [16]. The unconjugated peptide had no effect on the WKYMVM induced neutrophil oxidase activity (Fig. 2B) suggesting that membrane permeation is required for peptide-induced inhibition.
No effect of PBP10 on the transient calcium response
PIP2 is utilized for generation of IP3 that induces a transient increase in intracellular calcium [22], we thus investigated the effect of PBP10 on the neutrophil calcium response. Stimulating the cells with fMLF or WKYMVM induced a rapid and transient increase in cytosolic calcium. These responses were not significantly reduced by the removal of extracellular Ca2+ with EGTA (data not shown), suggesting that the rise in intracellular free Ca2+ relates to a mobilization from calcium storing organelles rather then from an influx through ion channels in the plasma membrane. Treatment of the cells with PBP10 prior to stimulation did not alter the calcium responses, neither induced by fMLF nor by WKYMVM (Fig. 3). Hence, the PBP10-induced effect on FPRL1 signaling is not due to a specific inhibition of the calcium transients induced by the activated GPCRs.
Figure 3 Effect of PBP10 on cytosolic calcium mobilization induced by peptide agonists. Neutrophil intracellular calcium mobilization was analyzed by monitoring Fura-2 fluorescence upon stimulation with the FPR agonist fMLF (100 nM; left) or the FPRL1 agonist WKYMVM (100 nM; right) in the absence (upper curves) or presence (lower curves) of PBP10 (1 μM). The curves are derived from a representative experiment. The calcium concentrations given in the right hand part of the figure are valid for the fMLF as well as the WKYMVM induced responses.
PBP10 inhibits mobilization of CR3
The Ca2+ elevation has been claimed to be required but not sufficient for the generation of an NADPH-oxidase activating signal from FPR [23,24]. Likewise, the mobilization through regulated exocytosis of neutrophil secretory storage organelles containing reserve pools of cell-surface receptors [25] has been suggested to be directly regulated by the cytosolic concentration of free Ca2+ [26,27]. We found that despite the fact that PBP10 was without effect on the transient rise in intracellular Ca2+ induced by WKYMVM, it blocked the secretory response (Fig. 4). In accordance with the inhibition of the NADPH-oxidase activity, PBP10 was without effect on the FPR induced granule mobilization (Fig. 4). This suggests that degranulation and NADPH-oxidase activity induced by FPRL1 both are on the same, PBP10-sensitive, signal transduction pathway in contrast to FPR, which induces both cellular functions by a PBP10-insensitive pathway.
Figure 4 Effect of PBP10 on fMLF and WKYMVM induced mobilization of CR3 to the neutrophil cell surface. Neutrophils were incubated with or without PBP10 (1 μM) at 37°C for 5 minutes after which the cells were challenged with fMLF (100 nM) or WKYMVM (100 nM) for another 10 minutes. After fixation of the cells with paraformaldehyde and labeling with anti-CR3 antibody, the amount of CR3 on the cell surface was determined by FACS analysis. A representative histogram of PBP10 effect on WKYMVM mediated CR3 exposure is shown (inset), and the exposure of CR3 after cell stimulation with fMLF and WKYMVM is expressed as percent of control (without PBP10; mean ± SD of four independent experiments). *p < 0.05 compared to WKYMVM without PBP10.
Regulation by the cytoskeleton
Regulation of FPR signaling has been suggested to involve mechanisms that depend on direct receptor interaction with the membrane cytoskeleton [28]. It is well known that the neutrophil response to FPR and FPRL1 agonists is both augmented and prolonged in the presence of cytochalasin B [15], a fungal metabolite that inhibits re-organization of actin polymers and uncouples receptors from the cytoskeleton [13].
To investigate the involvement of cytoskeleton in the PBP10-sensitive signal transduction pathway, neutrophils were treated with cytochalasin B prior to activation by a receptor agonist (in the presence or absence of PBP10). Interestingly, the receptor selectivity of PBP10's inhibitory effect was lost when the receptors were first uncoupled from the cytoskeleton by cytochalasin B (Fig. 5). In accordance with the findings reported above, the NADPH-oxidase activity induced by WKYMVM was largely inhibited by PBP10 also in the presence of cytochalasin B. However, the drug introduced a PBP10 sensitivity also in the FPR-induced response. The inhibition of the FPR-mediated response required higher concentrations of PBP10, and the response was only partly inhibited suggesting that the PBP10-insensitive as well as the PBP10-sensitive signaling pathways were triggered simultaneously during activation of FPR uncoupled from the cytoskeleton. The transient rise in intracellular calcium induced by the active receptors was not affected by cytochalasin B (data not shown). In conclusion, both FPRL1 and FPR appear to possess the ability to activate neutrophils via a signaling route that is sensitive to PBP10, but that this signaling pathway normally is blocked for FPR, through association of the receptor with the cytoskeleton.
Figure 5 Effect of PBP10 on oxidase activity induced by peptide agonists in the presence of cytochalasin B. Neutrophils were activated by fMLF (100 nM) or WKYMVM (100 nM) in the presence of cytochalasin B (2.5 μg/ml, final concentration) and the extracellular release of superoxide anion was recorded (expressed in Mcpm). (A) The figure shows the kinetics of the neutrophil response to fMLF in the absence (solid line) and presence (broken line) of PBP10 (5 μM). The results obtained are summarized in the inset, expressed as the integral values of oxidase activities from the controls (presence of cytochalasin B but not PBP10) and the activities in the presence of both cytocalasin B and PBP10. (B) The figure shows the kinetics of the neutrophil response to WKYMVM in the absence (solid line) and presence of PBP10 (broken line). The results obtained are summarized in the inset and expressed as the integral values of oxidase activities from the controls (presence of cytochalasin B but not PBP10) and the activities in the presence of both cytochalasin B and PBP10.
Discussion
The two formyl peptide receptor family members FPR and FPRL1 possess a high degree of amino acid identity in the signaling cytoplasmic domains [7], and the cell functions induced by the FPR and FPRL1 agonists are in most respects identical [10-15]. The indistinguishable responses of activated FPR and FPRL1 respectively, are expected, based on the fact that the two receptors are very similar in the regions suggested to be of importance for their interaction with the signaling, pertussis toxin-sensitive heterotrimeric G-protein. Dysfunctional variant FPR alleles (F110 replaced by an S and C126 replaced by a W) have been described that are associated with juvenile periodontitis and a deficiency in G-protein coupling [29,30], but FPRL1 contains the functional amino acids of FPR both in position 110 and in 126. Other G-protein coupling structures, identified through expression of different FPR mutants, suggest that the N-terminal part of the second transmembrane domain (S63 and D71) and the C-terminal interface of the third transmembrane domain (R123, C124 and C126) may be sites of interaction between the receptor and the G-protein [31]. In FPRL1 all but one of the amino acids in these suggested interaction sites are identical with those in FPR, and the signal-regulating NPXXY motif in the seven transmembrane domain (highly conserved among all GPCRs) is identical (sequence NPMLY [32]) in the two receptors. The exception is the serine in position 63 that in FPRL1 is replaced by a cysteine, but this difference seems to be of minor importance with respect to the effect of pertussis toxin, since both receptors are sensitive to the toxin.
Despite the indistinguishable activation by FPR and FPRL1, functional differences between these two highly homologous receptors emerge when they are challenged by the membrane permeant polyphosphoinositide-binding peptide PBP10 (rhodamine B-QRLFQVKGRR) prior to activation. The FPRL1-mediated neutrophil activity was totally inhibited by the peptide PBP10 whereas there was no effect on FPR-mediated responses. As mentioned, the amino acids in the PBP 10 peptide correspond to the PIP2 binding region segment 2 of the cytoskeletal protein gelsolin [16]. One of the enzymes competing with PBP10 for PIP2 would be the phosphoinositide remodeling enzyme PI3K that converts PIP2 into phosphatidylinositol 3,4,5-trisphosphate (PIP3) which is of importance for cell locomotion and the associated dynamic reorganization of cytoskeletal components. The precise target for PIP3 has however not yet been defined [33]. The inhibition of neutrophil function by Wortmannin, an inhibitor of PI3K, suggests that this signaling pathway is of importance for the NADPH-oxidase activity but the PI3K inhibitor lacked receptor specificity (i.e., both FPR and FPRL1 induced responses were inhibited) and we can thus rule out that the mechanism behind the PBP10 effect is a direct interference with the PI3K pathway.
The basic properties of PBP 10 have been described earlier [16,21,34], and it has been shown that when coupled to rhodamine B the peptide possesses not only PIP2 binding activity but also crosses the cell membrane of neutrophils and other cells. Interaction of rhodamine labeled peptides with PIP2 depends on the peptide sequence, and truncation of the peptide reduces the PIP2 binding affinity [21]. The truncated peptides were still membrane permeable and affected the oxidase activity only at higher concentrations. The unconjugated peptide QRLFQVKGRR exhibiting the same PIP2-binding characteristics as PBP10 but lacking the ability to cross the plasma membrane [16] had no effect on neutrophil oxidase activity. Taken together these data suggest that membrane permeation is required and PIP2 binding is of importance for peptide-induced inhibition.
Plasma membrane localized PIP2 is utilized for generation of IP3 which in turn is responsible for inducing transients in intracellular calcium [22]. Triggering of cells with fMLF or WKYMVM induced a rapid and transient increase in cytosolic calcium, but PBP10 did not have any inhibitory effect on the calcium transient. The Ca2+ elevation has been claimed to be required but not sufficient for the generation of an NADPH-oxidase activating signal from FPR [23,24]. Likewise the mobilization of granule localized reserve pools of cell-surface receptors [25], has been suggested to be directly regulated by the cytosolic concentration of free Ca2+ [26,27]. These experimental evidences rely, however, on methods that cannot distinguish a dependency on basal Ca2+ levels from a requirement for a Ca2+ transient, and we have earlier shown that receptor mobilization can occur and the oxidase can be activated without any transient rise in cytosolic Ca2+ [13,35,36]. Despite the fact that PBP10 was without effect on the transient rise in intracellular Ca2+ induced by WKYMVM, the secretory response was blocked and PBP10 selectively inhibited the FPRL1 induced granule mobilization.
The GPCR family is very diverse and the transmission of signals by such receptors is a critical function in many cell/organ systems. Signaling through GPCRs is highly complex, evidently not only with respect to the wide variety of mechanisms that regulate different functional responses [2], but also with respect to the pathways used to regulate a defined cellular response through closely related receptors. The two FPR and FPRL1 genes, although originating from a common ancestral gene, appear to have undergone markedly different evolutionary events [37]. In contrast to FPR, which is characterized by a relatively high degree of single nucleotide polymorphism (five non-synonymous and two synonymous identified [37]), no FPRL1 polymorphism has been found. Only one of the FPR polymorphisms is located in the cytoplasmic regions; and the variant (containing the A346 → E exchange) has the same amino acid in that position in the cytoplasmic tail as FPRL1. A direct comparison of the amino acid sequences of FPR and FPRL1 reveal very small differences between the receptors in all intracellular domains except for the C-terminal tail. In the first intracellular loop the H57 in FPR is replaced by an R (H57 → R) in FPRL1, in the second loop differences are found in V125 → I and T133 → A, and in the third loop the Q231 → K and L233 → M exchanges are found. The only major differences between the two receptors are found in the cytoplasmic C-terminal tail in which 13 out of 45 amino acids differ, and it is worth noting that the amino acid exchange in nine of the positions that differ between FPR and FPRL1 are potential targets for phosphorylation. Five potential phosphorylation sites, suggested to be of importance for arrestin-binding and receptor desensitization in FPR [38], are missing in FPRL1 while two new sites have been added in this receptor. It seems reasonable to assume that the signaling route that is sensitive to PBP10 originates from this region of the FPRL1 receptor. Despite the fact that the differences between FPR and FPRL1 are limited, the identification of putative sites in FPRL1 of importance for the PBP10 sensitivity cannot be achieved through experiments performed with receptor chimeras, site-directed receptor mutants or deletions, since no cells equipped with the required effector functions are available for expression of the receptors.
Regulation of FPR signaling has been suggested to involve mechanisms that depend on direct receptor interaction with the membrane cytoskeleton [28]. It is well known that the neutrophil response to FPR and FPRL1 agonists is both augmented and prolonged in the presence of cytochalasin B [15], a drug that inhibits re-organization of actin polymers and uncouples the receptors from the cytoskeleton [13,36]. The cytoskeleton is part of the signaling modulating machinery and we show that the receptor selectivity of the PBP10 inhibitory effect was lost when the receptors were uncoupled from the cytoskeleton. In accordance with the earlier described findings, the NADPH-oxidase activity induced by WKYMVM was largely inhibited by PBP10 also in the presence of cytochalasin B, but the drug introduced this sensitivity when fMLF was used as the triggering agent. The inhibition of the FPR-mediated response required higher concentrations of PBP10, and the response was only partly inhibited suggesting that the PBP10-insensitive as well as PBP10-sensitive signaling pathways were activated simultaneously by FPR's uncoupled from the cytoskeleton. It is interesting to note that when FPR was uncoupled from the cytoskeleton by cytochalasin B, PBP10 affected the sustained generation of superoxide but not the initial rate of production. This suggests that different signals are responsible for the triggering of the oxidase in the early and late phases of the response, respectively. It is reasonable to assume that the PBP10 sensitive signal generated by the uncoupled FPR is identical to that generated by FPRL1, however, we cannot at present exclude the possibility that also a novel FPR-triggered pathway is blocked by PBP10. The transient rise in intracellular calcium induced by the active receptors was not affected by cytochalasin B (data not shown), suggesting that this signaling route does not depend on interaction with the cytoskeleton. A possible explanation for the effects when PBP10 and cytochalasin B are combined is that signaling G-proteins compete with cytoskeletal proteins for the same site on FPR, and that this interaction involves a region of the receptor that differs between FPR and FPRL1. A 15 amino acid long sequence in FPR (322FPR336) has a fairly high (45–50%) identity with the actin-binding cytoskeletal proteins vinculin and coronin, and this region also participates in FPR interaction with the G-protein [28]. It is of interest to notice that the amino acid sequence in this region of FPRL1 differs from that of FPR in five positions and four of these are (in FPR) potential phosphorylation sites. Phagocytes express predominantly the Giα2 complex of the pertussis toxin sensitive G-proteins and, to a lesser extent Giα3 [39]. The molecular mechanism behind the difference in sensitivity to PBP10 between FPR and FPRL1 could possibly be that the receptors couple to different G-protein subtypes. FPR has, however, been shown to couple to both G-protein subtypes with similar efficiency [40]. It is however important to note that these experiments were performed in receptor-expressing cells in which nothing is known about the linkage between the receptors and the cytoskeleton and in which the proper cell function repertoire is missing.
The relation of the biochemical/biophysical activities of the PBP10 peptide to its effect on the FPRL1-triggered cell function is not obvious and is likely to be complex. The sensitivity to PBP10 seems to be unique to FPRL1, but this receptor is expressed also in other cells such as astrocytes, neuroblastoma, and microglia cells [8]. FPR is also expressed in other cell types and whether the receptor selectivity/specificity of PBP10 is maintained in other cells remains to be determined. The biochemical/biophysical characterization of the ten strategically organized basic and hydrophobic amino acids of the gelsolin molecule included in PBP10 reveal that it may interact with a broad range of negatively charged phosphomonoesters and hydrophobic acyl chains of anionic phospholipids [41,42]. This suggests that in addition to blocking/competing activities which involve proteins that are regulated by cellular phosphoinositides, the peptide may function as a buffer of bioactive and signaling lipids. Although elucidation of the step in signal transduction that is disrupted by PBP10 requires much additional work, the receptor-specific and signal-selective effects of this peptide on neutrophil functions suggest that it has a potential as a tool to manipulate and help define how GPCRs produce and integrate the signals generated from activated receptors and to probe new signaling functions of polyphosphoinositides as well as defining the role as promotor/blocker of G-protein signaling of different cytoskeletal proteins.
Conclusions
The neutrophil formyl peptide receptor family members FPR and FPRL1 share 69% of amino acid identity and mediate almost indistinguishable cellular responses. Thus, the assumption that FPR and FPRL1 use the same signaling pathways has been generally accepted. However, in this study we clearly demonstrate a fundamental difference in intracellular signaling between these two very closely related neutrophil formyl peptide receptor members, one being PBP10 sensitive and the other not. This novel PBP10 sensitive signaling pathway utilized by FPRL1 is also used by FPR but only when the cytoskeleton network is disrupted.
Methods
Isolation of human neutrophils
Neutrophil granulocytes were isolated from buffy coats obtained from healthy adults. After dextran sedimentation at 1 × g, hypotonic lysis of the remaining erythrocytes, and centrifugation in a Ficoll-Paque gradient [43], the neutrophils were washed twice and resuspended (1 × 107 /ml) in Krebs-Ringer phosphate buffer containing glucose (10 mM), Ca2+ (1 mM), and Mg2+ (1.5 mM) (KRG; pH 7.3). The cells were stored on melting ice and used within 120 min of preparation.
Chemoattractants and stimuli
The hexapeptide Trp-Lys-Tyr-Met-Val-Met-NH2 (WKYMVM) and the annexin I peptide (annexin9–25 Ac-QAWFIENEEQEYVQTVK) were synthesized and HPLC-purified by Alta Bioscience (University of Birmingham, Birmingham, United Kingdom), and Ross-Petersen ApS (Holte, Denmark). The formylated peptide N-formyl-Met-Leu-Phe (fMLF), C5a, and, phorbol myristate acetate (PMA) were from Sigma Chemical Co. (St. Louis, Missouri). IL-8 was from R&D systems (Minneapolis, MN) and dissolved in KRG containing 0.5% (w/v) bovine serum albumin. Serum amyloid A (SAA) was from Pepro Tech Inc. (UK). The C5a and SAA were dissolved in water while the other peptide agonists were dissolved in dimethyl sulfoxide to 10-2 M and stored at -70°C until use. Further dilutions were made in KRG.
PBP10 synthesis
The peptide QRLFQVKGRR (gelsolin residues 160–169) and related peptides were prepared by solid phase peptide synthesis and coupled to rhodamine as described earlier [16].
Neutrophil NADPH-oxidase activity
Neutrophil production and release of superoxide anions was measured by means of an isoluminol-enhanced chemiluminescence (CL) assay [44]. The CL activity was measured in a six-channel Biolumat LB 9505 apparatus (Berthold Co., Wildbad, Germany), using disposable 4-ml polypropylene tubes with a 900-μl reaction mixture containing 105–106 neutrophils, horseradish peroxidase (4 U) and isoluminol (20 μM) with and without cytochalasin B or PBP10. The measuring tubes were equilibrated for 5 to 10 minutes at 37°C and the cells were activated by addition of a receptor specific peptide agonist, fMLF for FPR and WKYMVM for FPRL1. The light emission was recorded continuously. Details about the CL technique are given in [45].
Determination of changes in cytosolic calcium
Neutrophils at a density of 2 × 107/ml in Ca2+-free KRG supplemented with bovine serum albumin (BSA, 0.1%) were incubated with the acetoxymethylated derivative fura-2/AM (2 μM) at room temperature for 30 minutes. The cells were washed twice and resuspended in KRG, adjusted to 2 × 107/ml and kept protected from light on ice until use. Cells with or without PBP10 were equilibrated for 5 minutes at 37°C, after which the peptide agonist was added. The fura-2 fluorescence was followed with a luminescence spectrometer (LS50B; Perkin Elmer Corp.) using excitation wavelengths of 340 nm and 380 nm, and an emission wavelength of 510 nm and the [Ca2+]i was calculated as described earlier [46].
Determination of receptor exposure by FACS analysis
The exposure of CR3 (CD11b/CD18) on the neutrophil cell surface was assessed by immunostaining and FACS-analysis. Cells challenged with peptide agonists with or without PBP10 were fixed with ice-cold paraformaldehyde and washed with FACSwash (PBS, 0.02% NaN3), after which the cells were incubated at 4°C with phycoerythrin-conjugated anti-CR3 antibodies (CD11b; Becton Dickinson 10 μl/106 cells) before analysis using a FACScan (Becton Dickinson, Mountain View, CA).
Reagents
Horseradish peroxidase (HRP) was from Boehringer-Mannheim (Mannheim, Germany). Isoluminol, cytochalasin B, and pertussis toxin were purchased from Sigma. Dextran and Ficoll-Paque were from Pharmacia (Uppsala, Sweden), and Wortmannin was from Calbiochem (La Jolla, CA). Fura-2AM was from Molecular Probes Inc. (Eugene, OR).
Statistic analysis
Two-tailed, paired Students's t-tests were performed to determine statistical significance, and a P-value of < 0.05 was regarded as significant.
Abbreviations
GPCR – G-protein coupled receptor
PBP – Phosphoinositide binding peptide
FPR – formyl peptide receptor
FPRL1 – formyl peptide receptor-like 1
PIP2 – phosphatidylinositol 4,5-bisphosphate
PI3K – phosphatidylinositol 3-kinase
fMLF – formyl-methionly-leucyl-phenylalanin
Authors' contributions
The scientific question raised in the paper was formulated during discussions between HF and CD, about the mechanisms behind receptor activation/deactivation/reactivation (see ref [38]). HF was responsible for most of the experiments but LB, JK and CM performed some of the experiments using techniques developed by CD and AK. PJ provided the PIP-binding peptides as well as suggestions for experiments. HF and CD wrote the first version of the paper, but contributions from all authors were important for the final outcome of the paper.
Acknowledgements
This work was supported by the Swedish Medical Research Council, King Gustaf the V's 80-year foundation and the Swedish Society for Medical Research.
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| 15625007 | PMC545074 | CC BY | 2021-01-04 16:39:07 | no | BMC Cell Biol. 2004 Dec 29; 5:50 | utf-8 | BMC Cell Biol | 2,004 | 10.1186/1471-2121-5-50 | oa_comm |
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BMC Dev BiolBMC Developmental Biology1471-213XBioMed Central London 1471-213X-4-161561559510.1186/1471-213X-4-16Methodology ArticleIdentification of cardiac malformations in mice lacking Ptdsr using a novel high-throughput magnetic resonance imaging technique Schneider Jürgen E [email protected]öse Jens [email protected] Simon D [email protected] Achim D [email protected] Carol [email protected] Kieran [email protected] Stefan [email protected] Andreas [email protected] Shoumo [email protected] Department of Cardiovascular Medicine, University of Oxford, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford, OX3 7BN UK2 German Research Centre for Biotechnology, Division of Microbiology, Junior Research Group Infection Genetics, Mascheroder Weg 1, 38124 Braunschweig, Germany3 Department of Pathology, School of Veterinary Medicine Hannover, Bünteweg 17, 30559 Hannover, Germany4 Department of Physiology, University of Oxford, Parks Road, Oxford OX1 3PT UK2004 22 12 2004 4 16 16 14 10 2004 22 12 2004 Copyright © 2004 Schneider et al; licensee BioMed Central Ltd.2004Schneider 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
Congenital heart defects are the leading non-infectious cause of death in children. Genetic studies in the mouse have been crucial to uncover new genes and signaling pathways associated with heart development and congenital heart disease. The identification of murine models of congenital cardiac malformations in high-throughput mutagenesis screens and in gene-targeted models is hindered by the opacity of the mouse embryo.
Results
We developed and optimized a novel method for high-throughput multi-embryo magnetic resonance imaging (MRI). Using this approach we identified cardiac malformations in phosphatidylserine receptor (Ptdsr) deficient embryos. These included ventricular septal defects, double-outlet right ventricle, and hypoplasia of the pulmonary artery and thymus. These results indicate that Ptdsr plays a key role in cardiac development.
Conclusions
Our novel multi-embryo MRI technique enables high-throughput identification of murine models for human congenital cardiopulmonary malformations at high spatial resolution. The technique can be easily adapted for mouse mutagenesis screens and, thus provides an important new tool for identifying new mouse models for human congenital heart diseases.
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Background
Congenital malformations are a major cause of death in childhood, and are typically characterized by lesions that do not compromise fetal survival. For instance, congenital heart disease (CHD) typically consists of lesions such as ventricular and atrial septal defects, which are compatible with fetal hemodynamics [1]. Although human genetic studies have identified some genes that cause congenital cardiac malformations, the molecular and developmental mechanisms underlying most of these defects remain largely unknown. Despite the high incidence of CHD (~1% of live births), only a handful of genes have been identified that when mutated, result in congenital heart disease [2-4].
The mouse is a particularly good model for studying mechanisms of cardiac diseases as its anatomy and development resembles that of the human more closely than any other genetically tractable organism. Importantly, the mouse is amenable to genotype-driven approaches such as transgenic knockouts of defined candidate genes [5], genome wide mutagenesis approaches using gene-trap [6] or transposon insertion screens [7], and high-throughput phenotype-driven screens that rely, for instance, on N-ethyl-N-nitrosourea (ENU) mutagenesis [8,9]. However, high-throughput cardiovascular genomic approaches in the mouse have been hampered by the paucity of phenotyping tools that allow efficient identification of complex cardiac malformations. As mouse embryos are opaque, late developmental defects are particularly difficult to identify. For instance, cardiac septal defects or outflow tract abnormalities can only be confidently identified after 14.5 days post coitum (dpc) when cardiac and outflow tract septation are completed in normal embryos. The identification of malformations in late gestation embryos typically relies on serial histological sectioning, which is extremely labor intensive. Furthermore, this often results in the irretrievable loss of 3D information, which is essential for the interpretation of complex cardiac malformations. In addition, standard pathological analysis is not amenable to high-throughput phenotype screening protocols that are required for any mutagenesis screen aiming at the functional dissection of the developmental biology of cardiac diseases. Therefore, new technological approaches must be harnessed that allow an efficient phenotyping of heart defects and also of subtle cardiac abnormalities that are at danger of being overseen in traditional histopathology screens. This is even more important in the light of upcoming new endeavors in functional mouse genome annotation [10]. Currently, new large-scale mouse mutagenesis screens are being set up in the US and in Europe that aim to produce heritable mutations in every gene in the mouse genome [11-13]. To make full use of these new mouse mutant resources more precise and efficient phenotyping methods are urgently needed [12-14]. The success of genome wide saturation mutagenesis screens depends therefore on improved phenotyping, and new high-resolution imaging approaches for mouse mutants are one of the most important which need to be established.
We previously reported the development of fast gradient-echo MRI of single mouse embryos [15-17]. This resulted in the acquisition of a 3D dataset in under 9 hours, with an experimental image resolution of 25 × 25 × 26 μm/voxel. We showed that MRI is capable of accurately identifying normal embryonal structures, and cardiac and adrenal malformations in knockout mouse embryos, and we have validated this technique by performing in depth histological examinations of imaged embryos [15-17]. These experiments showed that single-embryo MRI could correctly identify all cardiac lesions (atrial septal defects, ventricular septal defects, outflow tract defects such as double-outlet right ventricle, and aortic arch defects) except those under 20 μm – which is below the resolution of the MRI technique. As this method images single embryos in overnight runs, it still lacks the throughput required for phenotype-driven mutagenesis screens. For instance, in a typical recessive ENU mutagenesis screen, to screen 50 ENU mutant lines using a 3-generation breeding scheme would require the analysis of ~1200 embryos [18]. We now report the development of a method of imaging up to 32 embryos simultaneously in a single unattended overnight run, at high spatial resolution. Allowing ~30 minutes per embryo, the analysis of 1200 embryos would take 75 working days for a single trained individual. We show that this high-throughput multi-embryo MRI technique can be used to rapidly identify unsuspected embryonal cardiac and visceral malformations. Using this technique we could identify a novel, and hitherto unsuspected role for the phosphatidylserine receptor (Ptdsr) in controlling ventricular septal, outflow tract and pulmonary artery development. In addition, we found thymus hypoplasia in Ptdsr-deficient embryos. These findings suggest that a novel Ptdsr-mediated pathway is required for cardiac and thymus development.
Results
Multi-embryo imaging
We modified our previously described fast gradient echo magnetic resonance imaging technique [15-17] to image embryos embedded in four to eight layers (16 – 32 embryos total) in 28 mm nuclear magnetic resonance tubes using a single quadrature driven birdcage coil (Figure 1a). Preparation and embedding of embryos typically took less than an hour. In initial experiments we imaged up to 16 embryos simultaneously in overnight runs of <9 hours, with an experimental resolution of 51 × 51 × 39 μm. Subsequently, we used a custom made optimized probe with an increased sensitivity range to enhance imaging throughput. This allowed us to image 32 embryos simultaneously (Figure 1a,b), but with a larger matrix size and an increased field-of-view in the long axis of the tube. For these experiments we imaged embryos for ~12 hours, and achieved an improved experimental resolution of 43 × 43 × 36 μm. The optimized coil used for 32 embryos MRI (Figure 1) has a sensitivity range in z-direction of close to 50 mm. The artefacts seen at both ends in the longitudinal image (Figure 1b) are caused by B1-inhomogeneities at the end-rings of the coil. However, accurate image analysis for the entire data set remains possible if the height of the embryo stack does not exceed approximately 47 mm as demonstrated in the corresponding axial views of the top and the bottom layer (Figure 1c,d). The resolution achieved with the multi-embryo MRI technique allowed us to visualize the heart, cardiac septa, central nervous system, and visceral organs in fine detail (Figure 1d–f), in embryos taken from each layer. The data are permanently archived on DVDs, for subsequent analysis. Construction of 3D reconstructions of the heart typically takes ~4 hours, but was not necessary for the identification of cardiovascular defects.
Figure 1 High-throughput high-resolution magnetic resonance microscopy. (a) Stack of 32 embryos embedded in a NMR tube. (b) Section through the long axis of the NMR tube showing embryos in eight layers. (c) Sagittal section through layer 8 showing the four embryos in this layer. (d–f) Transverse, sagittal, and coronal sections through individual embryos in layers 5, 1 and 4 respectively. The voxel size is 25.4 × 25.4 × 24.4 μm. Structures indicated are the spinal cord (sc), the right and left lungs, atria and ventricles (rl, ll, ra, la, rv, lv), primary atrial and interventricular septa (pas, ivs), mitral valve (mv), midbrain roof (mbr), midbrain (mb), mesencephalic vesicle (mes), thalamus (tha), hypothalamus (hy), pons (po), cerebellum (c), medulla oblongata (mo), pituitary (pit), tongue (t), thymus (th), left superior vena cava and main bronchus (lsvc, lmb), aorta (ao), liver (li), stomach (s), left adrenal and kidney (lad, lk), pancreas (pa), intestines (i), umbilical hernia (uh), aqueduct of Sylvius (aq), fourth ventricle (fv), inner ear (ie), larynx (lar), right ventricular outflow tract (rvot), spleen (sp), and testes (te). Scale bars = 500 μm; axes: d – dorsal; v – ventral; r – right; l – left; a – anterior, p – posterior.
Sensitivity and specificity of multi-embryo imaging
To assess the sensitivity and specificity of multi-embryo imaging in comparison to single-embryo imaging, we used a model of Cited2 deficiency [19]. Embryos lacking Cited2 (Cited2-/-) have diverse cardiac malformations, including atrial and ventricular septal defects, outflow tract and aortic arch malformations, and adrenal agenesis [15,17,19]. As the Trp53-repressor gene Cdkn2aP19ARF is a target of Cited2 [20], we also examined embryos lacking both Cited2 and Trp53 to determine if this would rescue the heart and adrenal defects in Cited2-/- mice. We imaged 50 embryos using the multi-embryo technique in the 16-embryo mode. Embryonal genotypes included 12 wild-type, 13 Cited2+/-, 14 Cited2-/-, three Cited2-/-: Trp53-/-, four Cited2-/-: Trp53+/-, two Trp53+/-, and two Trp53-/-. We analyzed the data from each embryo for cardiac malformations without knowledge of the genotype. This typically took a maximum of 30 minutes per embryo. We scored each embryo for atrial and ventricular septal defects (ASD, VSD), outflow tract (e.g. double-outlet right ventricle, common arterial trunk), and aortic arch malformations (e.g. right-sided or bilateral aortic arch, retroesophageal subclavian artery), and for adrenal agenesis. Each embryo was then re-imaged singly at high resolution, and the data re-analyzed as before. In this group we identified 20 embryos with ASD, 19 with VSD, 18 with outflow tract defects, 11 with aortic arch defects, and 21 with bilateral adrenal agenesis, using high-resolution single embryo imaging. In comparison to single embryo imaging, the overall sensitivity and specificity of multi-embryo imaging for cardiac malformations was 88% and 92% respectively. For ASD (18 identified by single embryo imaging) the sensitivity and specificity was 85% and 95%; for VSD 94% and 94%; for outflow tract malformations 94% and 100%; and for aortic arch malformations 91% and 100% respectively (Figure 2). For bilateral adrenal agenesis, the sensitivity was 100% and specificity was 95% (Figure 3). Embryos lacking both Cited2 and Trp53 had cardiovascular defects and adrenal agenesis, indicating that Trp53 does not play a major role in the genesis of these defects in mice lacking Cited2. These results indicate that multi-embryo MRI is a potentially powerful high-throughput tool for efficiently characterizing cardiovascular malformations and identifying other defects in organogenesis.
Figure 2 Identification of septal, outflow tract, and aortic arch malformations using multi-embryo MRI (a – e') Images of transverse sections from 5 Cited2-/- embryos obtained using the multi-embryo technique (a–e) compared with images from the same embryos obtained subsequently using the single embryo technique (a'–e'). (a, a') Section showing left and right atria and ventricles (la, ram, live, rave). The atria are separated by the primary atria septum (pas), which is deficient at its ventral margin creating an osmium premium type of atria septal defect (ASD-P). (b, b') Section showing a ventricular septal defect (VSD) in the interventricular septum (ivs). (c, c') Section showing double outlet right ventricle, wherein the ascending aorta (a-ao) and the pulmonary artery (pa) both arise from the right ventricle (rv). The aortic valve (ao-v) is indicated. (d, d') Section showing a right-sided aortic arch (ao-a) passing to the right of the trachea (tr) and the esophagus (es). (e, e') Section showing bilateral aortic arches (ao-a) forming a vascular ring around the trachea (tr) and the esophagus (es). Also indicated are the thymus (th) and the right superior vena cava (r-svc). (f – j) Serial transverse sections through a wild-type heart obtained using single embryo MRI, demonstrating corresponding normal structures, including the systemic venous sinus (svs), left superior vena cava (l-svc), pulmonary vein (pvn), descending aorta (d-ao), mitral and tricuspid valves (mv, tv), the secondary atrial septum (sas), left and right ventricular outflow tracts (lvot, rvot), pulmonary valve (pv), and arterial duct (ad) of the pulmonary artery. Scale bars = 635 μm for multi-embryo, and 317 μm for single embryo images; axes: d – dorsal; v – ventral; r – right; l – left.
Figure 3 Identification of adrenal agenesis using multi-embryo MRI Images of coronal sections from 2 embryos obtained using the multi-embryo technique (a, b) compared with images from the same embryos obtained subsequently using the single embryo technique (a', b'). (a, a') Normal right adrenal gland (rad) anterior to the right kidney (rk) in a wild-type embryo. The right lung (rl) is indicated. (b, b') Agenesis of right adrenal gland in a Cited2-/- embryo. Scale bars = 635 μm for multi-embryo, and 317 μm for single embryo images; axes: d – dorsal; v – ventral; a – anterior, p – posterior.
Cardiac malformations in mice lacking Ptdsr
We next evaluated the role of multi-embryo MRI in analyzing unexplained lethality in embryos generated in collaborating laboratories. Recently, we have generated mice lacking the phosphatidylserine receptor (Ptdsr-/-) on a C57BL/6J background, by gene targeting in embryonic stem cells [21]. Ptdsr is a nuclear protein of unknown function, which is essential for the development and differentiation of multiple organs during embryogenesis [21-24]. Ablation of Ptdsr function in knockout mice causes perinatal lethality, growth retardation [21,22,24] and a delay in terminal differentiation of the kidney, intestine, liver and lungs during embryogenesis [21]. In addition, Ptdsr-/- embryos develop complex ocular lesions [21] as well as haematopoietic defects [24]. However, as many malformations have been described in Ptdsr mutants, none of those detected could explain the observed perinatal lethality of Ptdsr-/- mice. In the process of phenotypical characterization of our Ptdsr-deficient mouse line, we frequently observed subcutaneous edema of varying sizes in Ptdsr-/- embryos by gross inspection ([21] and Figure 4). As the development of edema in various mouse mutants is frequently associated with cardiovascular defects [25] we started to investigate if this also holds true for Ptdsr-deficient mice. We examined 8 embryos lacking Ptdsr, and 8 littermate wild-type or heterozygous controls using multi-embryo MRI. We found that 5 of 8 Ptdsr-/- embryos had cardiac malformations, which included ventricular septal defects, double outlet right ventricle, and pulmonary artery hypoplasia (Figure 5). None of the wild-type or Ptdsr+/- embryos had cardiac malformations. These findings were confirmed on single embryo imaging (Figure 6). Furthermore, to verify the identified cardiac defects in the Ptdsr-/- mice we performed serial transverse sectioning of all analyzed embryos. In all cases, we could recognize again the same heart defects that were identified before using the multi-embryo MRI technique (Figure 7). In addition, we analyzed by multi-embryo MRI a second Ptdsr-knockout mouse line (Ptdsrtm1.1 Gbf), which is identical to the initially analyzed Ptdsr mutant except that the loxP-flanked neomycin selection cassette was removed by breeding the original Ptdsrtm1 Gbf knockout line [21] to a CMV-Cre deleter mouse line [26]. From this Ptdsrtm1.1 Gbf knockout mouse line we analyzed 8 Ptdsr-/- embryos, and as littermate controls, 3 Ptdsr+/+ and 1 Ptdsr+/- embryos. We found ventricular septal defects in five out of the eight Ptdsr-/- embryos (data not shown). Again we found no evidence for cardiac malformations in wild-type or heterozygous littermate control embryos.
Figure 4 Edema in Ptdsr-/- mice (a) The Ptdsr-/- mutant (15.5 dpc) is growth retarded and the severe edema along the back of the embryo is visible. (b, c) Sagital sections of embryos at 16.5 dpc. The mutant embryo (c) exhibits massive subcutaneous edema compared to a wild-type (b) littermate. Scale bar = 100 μm in (b) and (c).
Figure 5 Identification of cardiac malformations in Ptdsr-/- embryos using multi-embryo MRI (a–e) Transverse thoracic sections showing the heart of heterozygous or wild-type control embryos from each litter. The left and right ventricles (lv, rv) are separated by the interventricular septum (ivs). The left and right atria (la, ra) are also indicated, separated by the primary atrial septum (pas). (f–i) Corresponding sections through littermate Ptdsr-/- embryos, showing ventricular septal defects (VSD). Scale bar = 635 μm; axes: d – dorsal; v – ventral; r – right; l – left; a – anterior, p – posterior. Individual embryos are indicated by number.
Figure 6 Cardiac malformations and thymus hypoplasia in Ptdsr-/- embryos. (a–c) Transverse and oblique (through the plane of the ascending aorta) sections, and 3D reconstruction (left-ventral oblique view) of a heart of a wild-type embryo at 15.5 dpc. The left and right ventricles (lv, rv) are separated by the interventricular septum (ivs). The left and right atria (la, ra), and the trachea (tr) are also indicated. The ascending aorta (a-ao) arises from the left ventricular outflow tract (lvot), via the aortic valve (ao-v), and continues on as the aortic arch (ao-a), which joins the descending aorta (d-ao). The pulmonary artery (pa) arises from the right ventricular outflow tract (rvot), and continues as the arterial duct (ad), which joins the descending aorta. (d–f) Corresponding images of a Ptdsr-/- embryo, showing a smaller heart with a ventricular septal defect (VSD). The aorta arises from the right ventricle. The pulmonary artery is small and its connection to the descending aorta (arterial duct) could not be identified. (g–i) Corresponding images of another Ptdsr-/- embryo, showing a ventricular septal defect (VSD). The aorta overrides the VSD resulting in a double-outlet right ventricle. (j, k) Coronal sections of Ptdsr+/+ and Ptdsr-/- embryos, showing the two lobes of the thymus (th). The arterial duct of the pulmonary artery in the Ptdsr-/- embryo is narrowed. (l) Correlation between embryo weight and volume. Scattergram of embryo weight versus embryo volume measured from multi-embryo MRI datasets for 16 embryos using Amira. The co-efficient of regression (r) is indicated. (m, n,) Absolute embryo and thymus volumes (μl) were measured from the MRI datasets from 5 wild-type (wt), 3 heterozygote (h), and 8 Ptdsr-/- (m) embryos at 15.5 dpc. There was no significant difference in the wild-type and heterozygote data, which were therefore pooled together (wt/h). (o) Relative thymus volumes (% of embryo volume) were calculated as Ptdsr-/- embryos were slightly smaller than littermate wild-type embryos. The data are represented as mean ± S.D. The probability of a type I error (P) is indicated. Scale bars = 317 μm; axes: r – right; l – left; d – dorsal; v – ventral; a – anterior, p – posterior. Individual embryos are indicated by number.
Figure 7 Cardiac malformations in Ptdsr-/- embryos: analysis using histology Embryos analyzed by MRI (Figure 3) were sectioned transversely and stained with hematoxylin and eosin. (a–c) Serial caudal to cranial sections of the wild-type embryo showing normal cardiac and vascular anatomy. The left and right ventricles (lv, rv) are separated by the interventricular septum (ivs). The ascending aorta (a-ao) arises from the left ventricle, continues on as the aortic arch (ao-a), which joins the descending aorta (d-ao). The pulmonary artery (pa) arises from the right ventricle via the pulmonary valve (pv) and continues as the arterial duct (ad), which joins the descending aorta. The left and right atria (la, ra), trachea (tr), right main bronchus (rmb) and esophagus (es) are indicated. (d) Section through embryo 33 indicating the ventricular septal defect (VSD). (e, f) Sections through embryo 55 showing that both aorta and pulmonary artery arise from the right ventricle (double outlet right ventricle), and that the arterial duct of the pulmonary artery is narrow in comparison to the aorta – indicating pulmonary artery hypoplasia. The aortic valve (aov) is indicated. (g–i) Serial caudal to cranial sections through embryo 35 showing a VSD, aorta arising from the right ventricle (double outlet right ventricle), and a severely narrowed arterial duct. Scale bars = 500 μm; axes: r – right; l – left; d – dorsal; v – ventral. Individual embryos are indicated by number.
We also observed a modest degree of thymic hypoplasia in Ptdsr-/- embryos (Figure 6k). To confirm this, thymus and embryo volumes were measured in 8 Ptdsr-/- embryos and 8 wild-type or heterozygous control embryos at 15.5 dpc. Embryo volume measured from the MRI datasets correlated very strongly with embryo weight (Figure 6l), and was modestly reduced in Ptdsr-/- embryos (Figure 6m). The volume of the thymus was significantly reduced in Ptdsr-/- embryos, even after correction for embryo volume, to 58% of the control value (Figure 6n,o). To correlate the identified cardiopulmonary malformations in Ptdsr-/- embryos with expression of the Ptdsr gene during heart development, we made use of our Ptdsr gene-trap reporter mouse line [21]. Using X-Gal staining in heterozygous embryos staged between 9.5 dpc and 12.5 dpc we found specific Ptdsr expression in the heart starting at 10.5 dpc and getting more defined to the compact zone and the trabeculi from 11.5 dpc onwards (Figure 8). Furthermore, when we analyzed Ptdsr-/- hearts by histopathology at 16.5 dpc we observed a severe differentiation defect in the compact zone as well as in the trabeculi (Figure 9), thus demonstrating that Ptdsr is in addition required for heart muscle differentiation at later stages of development. Taken together these results indicate that Ptdsr plays a hitherto unsuspected role in cardiovascular development as well as in cardiac muscle differentiation.
Figure 8 Analysis of Ptdsr expression in the embryonic heart (a, b) Staining of heterozygous Ptdsr-βgeo-embryos [21] using X-Gal at 10.5 dpc (a) and 11.5 dpc (b). (a) At 10.5 dpc Ptdsr expression can be seen throughout the heart. (b) Transverse sections of X-Gal stained embryos at 11.5 dpc showed an increased expression of Ptdsr in the myocardial wall and a beginning decrease of the expression in the trabeculation. Scale bar = 100 μm in (b).
Figure 9 Myocardial wall malformations in Ptdsr-/- embryos (a, b) Sagital sections of wild-type (a) and homozygous mutant (b) embryos at 16.5 dpc revealed a thinning of the myocardial wall (compact zone) and an increased myocardial trabeculation (b) in the mutant heart. Scale bar = 100 μm.
Discussion
Utility of MRI in identifying mouse models of human malformations
Our results show that it is possible to efficiently identify and quantitate relatively subtle cardiac and visceral malformations in late gestation mouse embryos using multi-embryo MRI. We have deliberately optimized our technique at late gestation in order to identify those congenital defects that allow survival through most of gestation. Importantly, these defects resemble human congenital malformations, and would provide mouse models for the study of these diseases. Our method represents a simpler alternative to the multi-coil approach published recently [27,28] in which up to eight fixed mouse embryos were imaged simultaneously, with a resolution of 200 μm. In comparison, the multi-embryo method described here has a higher experimental resolution of 43 μm. Notably, it requires substantial developmental and financial effort to equip an experimental MR-system with multiple coil and receive capability.
Role of MRI in investigating murine embryonal or perinatal lethality
Many mouse gene knockouts display late gestational lethality, but incomplete analysis and loss of 3D information consequent to histological sectioning, results in major developmental malformations being frequently missed. As shown here, Ptdsr-/- embryos on a C57BL/6J background develop heart defects. This was not noted in recent reports [22,24], and emphasizes the need not only for completely examining several mutant embryos, but also repeating these examinations in different genetic backgrounds. A major advantage of MRI is the ability to easily ship fixed embryos from referring laboratories to the laboratory that performs the MRI analysis. This minimizes the expense of animal relocation, re-derivation, and breeding required to generate the embryos, and significantly reduces animal experimentation.
Role of MRI in high-throughput phenotype driven screens
MRI screens performed at late gestation would be expected to identify genes that affect later aspects of development, and identify hypomorphic and haploinsufficient alleles of genes that affect earlier steps of development. Published data from genome-wide ENU mutagenesis screens in the mouse indicate that ~30% progeny carry a heritable recessive phenotype, making 3-generation recessive screens the method of choice for identifying developmental malformations [18]. At least 24 3rd generation progeny per 1st generation mutant are typically screened, resulting in a >78% probability of identifying at least one fully penetrant recessive homozygous mutant. A typical recessive screen (e.g. 50 – 100 first generation mutants per year) would require the analysis of ~1200 – 2400 embryos per year. Our results show that multi-embryo MRI is eminently suitable for such throughput. Although, screening of 32 embryos overnight requires typically about 16 hours of data analysis, multi-embryo MRI mutagenesis screens can be easily performed at a reasonable scale if multiple operators analyze the data in parallel. As the data are permanently stored on DVDs, and can be analyzed easily by commercially available software, they can be without difficulty disseminated to specialists for further and more detailed analysis. Another powerful application of multi-embryo MRI will likely be the investigation and screening of potentially teratogenic drugs.
Functional role of Ptdsr in heart development
Our results presented here indicate a new, and hitherto unsuspected role for the phosphatidylserine receptor in controlling ventricular septal, outflow tract, pulmonary artery, and thymus development. This finding suggests that a novel Ptdsr-mediated pathway is required for cardiac and thymus development. Recently, we have demonstrated that in contrast to previously reported hypothetical Ptdsr functions, the Ptdsr protein is not required for the clearance of apoptotic cells [21]. Moreover, detailed analysis of apoptosis induction and apoptotic cell clearance in Ptdsr+/+ and Ptdsr-/- embryos during heart development did not reveal any difference in the number and location of apoptotic cells between the genotypes (J.B., A.D.G. and A.L. unpublished observations). This further excludes that Ptdsr has any function in apoptotic cell clearance and points to other developmental mechanisms that are affected by Ptdsr ablation. The neural crest plays an important role in the development of the cardiac outflow tract, aortic arches, and the thymus [29]. As Ptdsr-deficient embryos lack intestinal ganglia [21] which are also derived from the neural crest, these results suggest that Ptdsr-/- mice may have an underlying neural crest defect. Importantly, dysfunction of these Ptdsr-mediated pathways during development could also potentially result in heart defects in humans.
Conclusions
Our results validate the utility of multi-embryo MRI for high-throughput identification of murine models for human congenital cardiac malformations, and using this technique we have shown that Ptdsr is essential for normal cardiac development. Further experiments are needed to define exactly in which pathways Ptdsr is involved during heart development. We expect that multi-embryo MRI will be an important technology for future phenotype-driven mouse mutagenesis screens. The technology can be easily implemented at standard MRI imaging centers, thus allowing by collaboration with individual researchers or mouse mutagenesis centers, a high-throughput functional genetic dissection of mechanisms underlying cardiac development and congenital heart diseases.
Methods
Mice
Cited2-/- [19], Trp53-/- [30], and Ptdsr-/- mice [21] have been described previously. All embryos were harvested at 15 days after detection of the vaginal plug.
Embryo preparation
Embryos were fixed in 4% paraformaldehyde at 4°C for ~1 week, and then embedded in 1% agarose (Seakem) containing 2 mM gadolinium-diethylenetriamine pentaacetic anhydride (Gd-DTPA, Magnevist, Schering UK) in 28 mm nuclear magnetic resonance tubes (Figure 1). The left forelimb was removed from each embryo to facilitate the identification of the left side. In addition, embryos had other limbs and/or tails removed before embedding so that each embryo in a given layer (of four embryos) could be unequivocally identified.
Magnetic resonance imaging
Single embryo imaging was performed as described previously [15-17]. For multi-embryo imaging, we used the same 11.7 Tesla (500 MHz) vertical magnet (Magnex Scientific, Oxon, UK). This was interfaced to a Bruker Avance console (Bruker Medical, Ettlingen, Germany) equipped with a shielded gradient system with a maximal gradient strength of 548 mTesla/m (Magnex Scientific, Oxon, UK), and quadrature-driven birdcage type coils with an inner diameter of 28 mm (Rapid Biomedical, Würzburg, Germany). Compressed air at room temperature was used to reduce the heating induced by the gradients. A 3D spoiled gradient echo sequence (echo time 10 ms), a π/2 excitation pulse with rectangular pulse shape, (π/2 = 100 μs), was used with a short repetition time (30 ms) to obtain strong T1 contrast. A matrix size of 512 × 512 × 768 (bandwidth: 130 Hz/pixel) at a field of view of 26 × 26 × 30 mm achieved an experimental resolution of 51 × 51 × 39 μm when imaging up to 16 specimens. In case of 32 embryos, a matrix size of 608 × 608 × 1408 at field of view of 26 × 26 × 50 mm, yielded an experimental resolution of 43 × 43 × 36 μm. The total experimental time was ~8.75 hours for 16 embryos, and ~12.3 hours for 32 embryos (typically overnight runs) whereby each phase encoding step was averaged four times.
Data reconstruction and analysis
The raw MR data were reconstructed into a stack of 1024 (for 16 embryos), or 2048 (for 32 embryos) 2D TIFF files (16 bit pixel resolution, 2 or 4 GB total size) using purpose-written software as described previously [16]. The TIFF files were analyzed using Amira 3.1(TGS Europe, Mérignac Cedex, France). 3D reconstructions were performed using the Image Segmentation Editor, and tissue volumes for morphometric analysis were measured using the Measure Tissue Statistics tool available in Amira 3.1. The probability (p) of a Type I error was calculated using a 2-sample equal variance 2-tailed t-test in Microsoft Excel.
Histology
Embryos were dehydrated in ethanol, embedded in paraffin wax, and sections were stained with hematoxylin and eosin.
X-Gal staining of embryos
Embryos were dissected free of extraembryonic membranes and then fixed in 4% paraformaldehyde at 4°C. Expression of Ptdsr was detected by staining the embryos overnight in X-Gal according to standard protocols. The embryos were postfixed in 4% paraformaldehyde and processed for documentation or histology.
Authors' contribution
J.E.S. developed the multi-embryo MRI technique, J.B. generated both Ptdsr knockout lines and harvested embryos for MRI and histopathological analysis, S.D.B developed the sample preparation for embryonic MRI, A.D.G. carried out the histopathological analysis of Ptdsr-/- mutants, C.B. prepared the embryos for MRI, K.C. and S.N. assisted the experimental development, S.B. analyzed the MRI and histopathological data, A.L. and S.B. were responsible for the co-ordination of the study and the drafting of the paper.
Acknowledgments
We thank Tim Bardsley and Neil Hoggarth for help with information technology, and Titus Lanz (Rapid Biomedical) for developing the MR probe. These studies were funded by the Wellcome Trust, British Heart Foundation, and the EU project EUMORPHIA (QLG2-CT-2002-00930). S.B. is a Wellcome Trust Senior Fellow in Clinical Science.
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| 15615595 | PMC545075 | CC BY | 2021-01-04 16:40:09 | no | BMC Dev Biol. 2004 Dec 22; 4:16 | utf-8 | BMC Dev Biol | 2,004 | 10.1186/1471-213X-4-16 | oa_comm |
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BMC Fam PractBMC Family Practice1471-2296BioMed Central London 1471-2296-5-301560691410.1186/1471-2296-5-30Research ArticleDevelopment of a questionnaire weighted scoring system to target diagnostic examinations for asthma in adults: a modelling study Hirsch Sybil [email protected] Timothy L [email protected] Jonathan L [email protected] Michelle L [email protected] Peter I [email protected] General Practice Research Unit, North West Lung Research Centre, Wythenshawe Hospital, Manchester, M23 9LT, UK2 Department of Computer Science, University of Manchester, Oxford Road, Manchester M13 9PL, UK2004 17 12 2004 5 30 30 14 7 2004 17 12 2004 Copyright © 2004 Hirsch et al; licensee BioMed Central Ltd.2004Hirsch 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
Identification and treatment of unrecognised asthmatics in the community is important for improving the health of the individual and minimising cost and quality of life burden. It is not practical to offer clinical diagnostic assessment to whole communities, and a simple tool such as a questionnaire is required to identify a smaller target group. Conventional questionnaire screening methods which separate individuals into positive and negative categories have resulted in large numbers of individuals requiring clinical assessment. This study has therefore developed and tested a weighted scoring system that prioritises those most urgently in need, based on their questionnaire responses.
Methods
A stratified random sample of adult respondents to a general practice postal questionnaire survey were categorised 'asthmatic' or 'non-asthmatic' according to three expert physicians' opinions. Based on this categorisation, logistic regression was used to derive weights reflecting the relative importance of each question in predicting asthma, allowing calculation of weighted scores reflecting likelihood of asthma. Respondents scoring higher than a chosen threshold would be offered diagnostic examination.
Results
Age and presence of wheeze were most influential (weight 3) and overall weighted scores ranged from -1 to 13. Positive predictive values (PPV) were estimated. For example, setting the threshold score at nine gave an estimated PPV for asthma diagnosis of 93.5%, a threshold score of seven corresponded to PPV 78.8%. PPV estimates were supported by examining 145 individuals from a new survey.
Conclusion
Weighted scoring of questionnaire responses provides a method for evaluating the priority level of an individual 'at a glance', minimising the resource wastage of examining false positives.
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Background
There are individuals in the community who are asthmatic but are not receiving treatment because they are unknown to the medical services [1-5]. Detecting these unrecognised asthmatics may be important for short-term health, prevention of long term airway remodelling and minimisation of cost and quality of life burden [6]. The reliable diagnosis of asthma, however, requires full clinical assessment [7]. Since it is clearly not practical in terms of resource allocation to offer this to whole communities, a simple tool such as a questionnaire is needed to identify a smaller target group. Conventional targeting approaches which separate individuals into 'positive' and 'negative' categories have failed to reduce the number of potential examinations to a manageable level. For example, in the Wythenshawe Community Asthma Project (WYCAP) [8-10], respondents who had four or more 'yes' answers to six key questions on a postal respiratory questionnaire were deemed possible asthmatics in need of clinical assessment. However, this method identified approximately 350 individuals for examination per practice. A more refined screening method, which could identify more than two categories, would allow the screening threshold to be adjusted to take into account available resources.
Defining additional categories according to the possible numbers (0 to 6) of 'yes' answers to the 6 key questions is too simplistic as it assumes equal weights for each question and takes no account of variables such as age and gender. The present paper describes the development of a more refined method, which includes additional questions and uses question weights to calculate a score for each respondent. This weighted score reflects probability of asthma, allowing clinical resources to be targeted to those most likely to have the disease. A score is chosen as a threshold and the questionnaire is given in an opportunistic fashion to patients consulting a medical practice for any reason. The questionnaire score is calculated 'at a glance' and those whose score is higher than the chosen threshold, and who are not already receiving asthma treatment, are offered diagnostic examination. The threshold can be adjusted to take resource limitations into account.
Methods
The study described in this paper was based on data collected as part of the WYCAP investigation. WYCAP is a long term prospective investigation of the natural history of asthma in two general practice populations. It is based on a postal respiratory questionnaire adapted from the European Community Respiratory Health Questionnaire (ECRHQ) [11]. Four postal questionnaire surveys of all adults (16 years and over) registered to the two practices were conducted in 1993, 1995, 1999 and 2001. Additional data were obtained in 1995 and 2001 by invitation of selected respondents for clinical review. Ethical approval for the present study was obtained from South Manchester Local Research Ethics Committee and statistical analysis was performed using SPSS for Windows [12]. The study uses questionnaire responses and additional data obtained as part of the 1995 and 2001 adult surveys and the investigations were conducted in three phases. First, a weighted scoring system was developed based on a subset of adult questionnaires and clinical review data collected in 1995. Next, this scoring system was applied to the questionnaires from the whole population of respondents to the 1995 survey in order to estimate its positive predictive value (PPV) in finding undiagnosed asthmatics in the community. Finally, the validity of the scoring system was tested in a new data set in the form of a subset of questionnaires and clinical review data selected from the 2001 WYCAP survey.
Phase 1: Development of the weighted scoring system
It was necessary to select an asthma-enriched sample in order to determine response patterns of both asthmatic and non-asthmatic respondents [13]. Respondents from the 1995 survey were stratified based on the number of 'yes' answers (0, 1–3, 4, 5 and 6) to six key questions on the postal questionnaire. These were questions which were believed to be important in the detection of an asthmatic, namely questions relating to symptoms in the last 12 months (wheezing, chest tightness, shortness of breath, night cough), family history of asthma and associated conditions of hayfever or eczema [see Additional file 1]. The target numbers selected from each stratum were based on clinical intuition and aimed to provide a stratified random sample with approximately equal numbers of asthmatics and non-asthmatics. Individuals were selected from one of the practices and invited for clinical review by the research clinician, including full history, physical examination, spirometry, a test for reversibility to beta-2 agonists, bronchial challenge with histamine, electronic peak flow diaries and skin prick testing to five common allergens. The clinical review results were sent separately to three expert respiratory physicians who had no knowledge of the questionnaire responses. Each physician was asked to rate the probability of asthma as <50%, 50–90%, or >90%. Bayesian methods were used to combine the physicians' opinions into a consensus estimate of probability of asthma for each individual [14]. In order to ensure the independence of the expert opinions required by these methods, the physicians did not confer with each other and were not given any diagnostic criteria or guidelines. Those reviewed individuals in whom the consensus estimate of probability of asthma was 50% or more were designated clinically asthmatic and the remainder were designated clinically non-asthmatic. By modelling the relationship between questionnaire responses and clinical asthma status, a weight was determined for each question, reflecting its importance in predicting asthma status of the respondent. For each questionnaire, the weights for the relevant responses ('yes' answers) were summed to produce an asthma score reflecting the probability of asthma in that individual.
The weights were derived based on a linear logistic regression model, designed to take as input the questionnaire responses 1 to 11 and predict the probability of asthma for any individual from the wider community. The model associates each question on the questionnaire with a coefficient reflecting the relative importance of that question in determining probability of asthma. A positive value for a coefficient influences towards a decision that the individual is classified asthmatic, the higher the value the greater the effect. Negative coefficients influence away from a decision that the individual is classified asthmatic. In order to provide a model representative of the wider community, a cross-validation technique was used [15] in which several candidate logistic regression models were produced and their respective coefficients averaged. These average values were rounded to the nearest whole number to give the question weights prior to summing to produce the weighted scores.
Phase 2: estimating positive predictive value
The full set of postal responses to the 1995 questionnaire survey was used to assess effectiveness by estimating PPV of the scoring system in detecting undiagnosed asthmatics in the community. Weighted scores were calculated for all 1995 survey respondents and evidence of either 'asthma diagnosis ever' or 'prescription for asthma medication in the last 12 months' was obtained from their medical records. The percentage of undiagnosed asthmatics with a threshold score or higher, who could be expected to be confirmed clinically asthmatic, was estimated using a technique which referenced the probabilities of asthma of sample respondents, (based on the combined expert opinions), their population strata (based on number of 'yes' answers to the six key questions) and weighted scores (based on questionnaire responses) [see Additional file 2]. This estimation was repeated for various score thresholds. These expected percentages provided estimates of the positive predictive values of the score thresholds [16].
Phase 3: testing the validity of the weighted scoring system in a new data set
The estimates of PPV for the various score thresholds depend on the fact that higher scores correspond to higher probability of asthma. The weighted scoring system is valid if it can be shown to retain this property when applied to a new data set, and this was tested by comparing scores with clinical diagnosis of asthma in a subset of data collected in 2001. Sophisticated modelling techniques had been used to rank the 2001 survey questionnaires in order of probability of asthma as part of another study [17,18]. The test subset for this study consisted of questionnaires from the top 10% of this ranked list (most likely asthmatics) and a random sample (approximately 1 in 5) of low probability individuals selected at random from the end 200 of the population list. As in 1995, each selected individual was invited for clinical review and the results were sent separately to another three respiratory physicians. The purpose of the low probability individuals was simply to illustrate the diagnostic discrimination of the physicians.
The method of combining the three independent physician opinions differed from that used in phase 1. This time, the aim was not to produce diagnoses as the basis of a mathematical model, but rather to emulate as far as possible diagnosis in a clinical setting. Each physician was therefore asked, on the basis of the clinical review information, to classify each individual as 50% or greater probability of obstructive airways disease (OAD), or less than 50% probability. For those 50% or greater probability (more likely than not to have OAD), the physician was asked to state whether the disease was asthma, chronic obstructive pulmonary disease (COPD) or mixed disease (asthma and COPD). Individuals in whom at least two physicians diagnosed asthma or mixed disease were designated clinically asthmatic.
As part of the 2001 population survey, each reviewed individual had already completed a postal questionnaire, and a weighted score was calculated based on questionnaire responses. For each possible score, the proportion of reviewed individuals with that score or higher, who had been designated clinically asthmatic was calculated. These percentages reflect the association between score and probability of asthma and were used as an indication of the validity of the postal questionnaire and scoring system.
For comparison, unweighted scores (number of 'yes' answers to the six key questions) were also calculated for respondents from the top 10% of the ranked population list, and the percentage of asthmatics associated with each of the unweighted score thresholds was similarly calculated.
Results
In the 1995 WYCAP survey, 10429 individuals were sent a postal respiratory questionnaire with a 72.7% response rate, leaving 6825 after exclusion of incomplete questionnaires. Four hundred and twenty individuals were selected for the stratified random sample and invited for clinical review. Of these, 201 (48%) attended, leaving 180 after exclusion of those whose screening questionnaire was incomplete. The ages of the 180 respondents with fully completed questionnaires ranged from 16 to 83 years, with a median age of 50.5 years, and 41.7% were male. Eighty four individuals were designated clinically asthmatic and 96 non-asthmatic based on the combined opinions of the three physicians.
Phase 1
The weights derived for each question are shown in table 1. The features most influential towards classifying an individual as asthmatic (weight 3) were reporting of wheeze in the last 12 months and being in age group 1 (16–34 years). Reporting an asthma attack in the last 12 months and being in age group 2 (35 – 54 years) were next most influential with weights of 2. Cigarette smoking (weight -1) was a small influence away from a classification as asthmatic. Under this scoring system, the responses to questions 5 and 6 (woken by an attack of shortness of breath or coughing) were found to be zero weighted and therefore, when used in combination with this set of questions, did not contribute further to the overall weighted score. The questions relating to breathlessness whilst wheezing and wheeze in the absence of a cold (questions 3.1 and 3.2) are conditional on the reporting of wheeze at any time (question 3) and therefore although the model for the prediction of asthma probability requires them, it is difficult to place a clinical interpretation on their values.
Table 1 Weights associated with each question on the questionnaire
Question Number Variable based on question Scoring system weights
1 *Age (16 – 34 years) 3
1 *Age (35 – 54 years) 2
1 *Age (55 – 74 years) 1
2 Sex male 1
3 **Wheeze in last 12 months 3
3.1 Breathless whilst wheezing 0
3.2 Wheeze in absence of cold -1
4 **Woken by chest tightness in last 12 months 1
5 **Woken by shortness of breath in last 12 months 0
6 **Woken by night cough in last 12 months 0
7 Asthma attack last 12 months 2
8 Currently taking asthma medication 1
9 **Family history of asthma 1
10 **Hayfever/eczema ever 1
11 Smoker -1
* Age was aggregated into 4 categories. The 4th age category (>= 75) years is excluded from the model because it is dependent on the other 3
** Key variables used in simple scoring of 'yes' answers to key questions
Phase 2
Weighted scores were calculated for all postal questionnaires (n = 6825) in the 1995 WYCAP population and ranged from -1 to 13. Table 2 shows the percentage of previously undiagnosed asthmatics expected to be found when using the various scores as thresholds, that is, recommending a clinical review for all individuals with that score or higher. A score of nine or more was found in 335 individuals. Of these, 80 had no evidence of asthma diagnosis in their medical records and it would be expected that 75 (93.5%) would be diagnosed clinically asthmatic. Lowering the score threshold below nine progressively decreased the expected efficiency, so that examining those scoring eight or more increases the number of false positives with only 82.5% expected to be confirmed asthmatic. The 4537 individuals scoring four or less are much less likely to be asthmatic (expected percentage of asthmatics in this group was 5%).
Table 2 Positive predictive value estimates for weighted scoring applied to all respondents to 1995 postal survey (n = 6825)
Threshold Score Number of individuals above threshold Number eligible for review (no asthma diagnosis) Undiagnosed individuals expected to be diagnosed clinically asthmatic
Number Percent (Positive predictive value)
11 76 13 12 90.5
10 178 33 31 94.2
9 335 80 75 93.5
8 577 195 161 82.5
7 891 394 310 78.8
6 1289 715 437 61.2
5 2098 1427 678 47.5
4 3332 2592 769 29.7
3 4667 3874 894 23.1
2 5769 4938 929 18.8
1 6524 5670 933 16.5
All respondents 6825 5964 934 15.7
Phase 3
The third phase confirmed that higher scores correspond to higher probabilities of asthma in the independent test subset selected from the 2001 survey respondents. Two hundred and eighty three individuals comprised the top 10% of the probability ranked list and were invited for review along with 39 low probability individuals selected from the end 200. In total, 145 individuals attended for review, their ages ranged from 16 to 91 years with a median age of 50 years, and 40.7% were male.
One hundred and twenty six high probability and 19 low probability individuals attended. For the 126 high probability asthmatics from the top 10% of the ranked list, scores ranged from six to 12, compared with a maximum score of eight in those respondents not in the top 10%. The age range and median age associated with each score threshold is reported in table 3, which shows that lower score thresholds are associated with increasing age.
Table 3 Percentage of individuals with threshold scores or higher who were found clinically asthmatic in the 2001 validation set (n = 126)
Threshold Score Number of individuals above threshold Median age (years) [range] Majority verdict asthma Majority verdict mixed disease Majority verdict asthma or mixed disease
Number Percent
11 11 33 [21–54] 9 1 10 90.9
10 32 40.5 [21–74] 27 1 28 87.5
9 72 44.5 [18–75] 53 10 63 87.5
8 114 47 [17–88] 76 19 95 83.3
7 121 47 [17–88] 77 19 96 79.3
6 126 47 [17–88] 81 20 101 80.1
In general, agreement between the experts was good with pair-wise kappa statistics of 0.62, 0.64 and 0.65 respectively. All three experts agreed the same diagnostic category for 99 out of the 145 reviewed individuals (82 from the high probability group and 17 from the low probability group) and two out of three experts agreed for a further 38 (37 high probability and one low probability). The percentages of individuals diagnosed as having asthma or mixed disease associated with each score threshold are shown in table 3. The higher the questionnaire score the higher the probability of asthma in the respondent. Of the 72 individuals scoring nine or more, 63 (88%) were clinically diagnosed as having asthma or mixed disease (53 asthma, 10 mixed). Lowering the threshold reduces the percentage of asthmatics diagnosed, such that of the 121 individuals scoring seven or more only 96 (79%) were clinically diagnosed as having asthma or mixed disease (77 asthma, 19 mixed). The 19 low probability individuals selected from the end 200 of the ranked list all achieved scores of zero or one and 18 were diagnosed clinically non-asthmatic based on the physician opinions.
Table 4 shows the percentage of clinical asthmatics associated with each of the non-weighted score thresholds, illustrating that ranking according to non-weighted scores does not demonstrate the ranking in order of probability of asthma that is achieved by the weighted scoring system.
Table 4 Percentage of individuals with non-weighted threshold scores or higher who were found clinically asthmatic in the 2001 validation set (n = 126)
Number 'yes' answers out of 6 key questions (screening threshold) No. individuals above threshold Number (percentage) diagnosed asthma or mixed disease
6 30 25 (83.3%)
5 74 64 (86.5%)
4 103 86 (83.5%)
3 119 98 (82.4%)
2 124 100 (80.6%)
1 126 101 (80.1%)
Discussion
In general, questionnaire surveys for detecting individuals likely to have a disease partition respondents into 'positive' and 'negative' categories. This is appropriate when a community-wide survey can be undertaken and the medical services are able to provide clinical assessment for all those in the 'positive' category. This study has developed a method for optimising limited resources by targeting expensive clinical examinations to those most likely to be at risk, minimising the chance of needlessly examining a healthy individual and allowing resource availability to be taken into account.
Developing a model which uses a questionnaire to identify likely asthmatics in the general population crucially depends on having a subset of questionnaires from respondents reliably classified as 'asthmatic' or 'non-asthmatic' on which to build the model. In a condition such as asthma there is inherent uncertainty in the diagnosis, and even detailed clinical review information inevitably produces disagreement between experts as to the diagnostic category for some of the reviewed individuals. Other studies have produced questionnaire models for predicting high risk asthmatics [4,9,10,19] and these studies have either used a single expert or resolved the problem of disagreement between experts using methods such as 'majority verdict' or designated diagnostic rules. However, where more than one expert is involved, even permitting the experts to discuss problem cases did not always result in complete agreement [4]. The importance of this present study lies in the rigorous methods used to capture the uncertainty inherent in asthma diagnosis by combining the opinions of three experts using standard statistical techniques. These aimed to produce a reliable diagnosis for each individual in the phase 1 subset of questionnaires on which the weighted scoring model was built. These methods are described in detail elsewhere [14] but briefly, the opinions of the three experts were combined using probabilistic techniques which took into account not only differences between responders but also differences in diagnostic judgement thresholds between the experts. The result of applying these techniques was a number between 0 and 1 for each questionnaire in the reviewed phase 1 subset which reflected probability of asthma of the respondent. Those with probability greater than 0.5 were designated 'asthmatic'.
Since the aim of a screening system is generally to identify individuals with a high probability of the condition being tested for, the performance of the screening system on high probability individuals is most important. For this reason it was necessary in this study to identify a set of high probability asthmatics for clinical review in the phase 3 validation stage. It was also necessary that these high probability asthmatics were identified by a system which was independent of the weighted scoring system being tested. For this reason, a neural network was used to rank the 2001 survey questionnaires in order of probability of asthma. The neural network is a sophisticated statistical technique that was used to model complex relationships between questions on the questionnaire to predict probability of asthma of a respondent based on questionnaire responses. The neural network model was validated [20] and applied to the questionnaires from the 2001 population survey to produce a population ranking based on individual predicted probability of asthma according to the neural network model. The top 10% most likely asthmatics from this population ranking, along with some low probability individuals, comprised the independent subset used to test the weighted scoring system in phase 3.
The question weights were consistent with clinical explanations. Wheeze in the last twelve months was the most important symptom and the weights for each age band decreased with increasing age which is consistent with the explanation that wheezing in older people can be explained by reasons other than asthma, for example, heart disease, malignant lung disease or one of the chronic obstructive lung conditions collectively referred to as COPD. The interpretation of the low weighting given to respiratory symptoms other than wheeze, for example, night cough or shortness of breath, is that these symptoms are highly correlated with wheezing, and as such provide no additional information over an above the high 'wheeze' weighting. Whereas any of these symptoms viewed in isolation would be highly correlated with a diagnosis of asthma, when viewed in the context of a multivariate model such as this weighted scoring system, it is the interaction between the variables which defines the final model. The small negative coefficient for smoking is consistent with the explanation that those with asthma tend to stop smoking or never to begin. Alternatively, asthma-like symptoms in some smokers may be caused by conditions other than asthma, for example malignant lung disease, COPD, or heart disease, or smoking itself may cause symptoms.
The intended application of weighted scoring is to find individuals who are likely to have asthma but are not already receiving appropriate treatment. Superficially, it may appear that the questions relating to an attack of asthma in the last twelve months and current medication for asthma should be excluded from the model as they may produce higher scores in asthmatics who are already diagnosed and receiving treatment, effectively disadvantaging the undiagnosed individuals. However, the relationship between these two questions and practice records of diagnosis is far from definitive. For example in the 1995 survey, of the 6570 respondents who had no record of either diagnosis of asthma nor medication prescription in the last year, 175 individuals answered 'Yes' to the current medication question on the questionnaire and 128 reported an asthma attack in the last twelve months. The scoring system reflects an accurate ranking of the whole population and the questions relating to attack of asthma in the last 12 months and current medication for asthma were found to be important in the whole population model. In general, higher scores reflected higher PPV estimates, but the final column of table 2 illustrates that this is not a simple linear relationship, rather the scores can be considered associated into four priority levels, that is, 9 or more, 7–8, 5–6, and 4 or lower.
One possible source of error in the weighted scoring system is under or over self-reporting of symptoms, such as the individual ranked as low probability who was found clinically asthmatic in the absence of reported symptoms on the questionnaire. Another possible source of error is differences between the development data sample and the intended target population due to non-response in the postal survey, exclusion of incomplete questionnaires and non-attendance at clinical review. Analyses revealed no evidence of sample bias from these sources in terms of gender or numbers of 'yes' answers to the six key questions on the questionnaire. Those who attended for review had a higher median age (50 years) than those invited but not attending (32 years). It is also important to acknowledge that the system here was tested using postal questionnaires, whereas in practice the patient may well complete the questionnaire in a surgery or clinic environment.
The question weights were derived from a logistic regression model and commonly, scarcity of data means that this modelling technique is applied as a single model fitted to the entire data subset [21] with none reserved for independent validation and no allowance made for differences in prevalence between training data and the general population. However, this gives limited information about how well the model will generalise to the wider community. In this study three steps were taken to gain a realistic estimate of model performance. First, the regression coefficients were not based on a single model but were the average of several representative models [22]. Second, there was an independent validation set comprising examples from the higher scores likely to be of interest when targeting diagnostic examinations, along with a small number of respondents believed to have a low probability of asthma. Third, the consensus probability of asthma information available for the phase 1 reviewed individuals allowed the positive predictive values estimated in phase 2 to take account of differences in prevalence of asthma between the questionnaires used in developing the scoring system and the general population. However, the intended target population is practice attendees rather than a population-wide survey and prevalence of all diseases including asthma may be higher in practice attendees than in the general population. In this case the positive predictive values reported in phase 2 may be under-estimates.
Assessing likelihood of asthma and ranking by simply counting the number of 'yes' answers to key questions is a simple but relatively crude method since it assumes equal weights for the questions. However, some responses may exert a stronger influence than others, there may be interactions between responses and features such as age and gender cannot be included at all. For example, an elderly person with four 'yes' answers to the key questions may be considered less likely to be asthmatic than a young person with three, since respiratory symptoms in the elderly may sometimes be explained by conditions other than asthma. Hence, ranking according to the number of 'yes' answers may not produce the required probability ordering. This is illustrated by the small differences in proportions of clinical asthmatics associated with the various simple score thresholds shown in table 4.
Where the availability of resources is a limiting factor, the relevant measure of effectiveness in targeting clinical review is the 'true positive' rate, that is, the percentage of individuals scoring above the threshold in whom the diagnosis is confirmed. The other commonly reported test measures of sensitivity, specificity and negative predictive value have no equivalent in a technique based on population ranking where the aim is to target expensive clinical examinations to those most at risk and minimise the 'wasted' examination of false positives.
Conclusions
When resources for diagnostic examination are limited prioritisation may be necessary. The use of weighted scoring and priority levels rather than the more conventional binary separation into positive and negative allows the score threshold to be adjusted to balance patient need with available resources
Abbreviations
COPD Chronic obstructive pulmonary disease
OAD Obstructive airways disease
PPV Positive predictive value
WYCAP Wythenshawe Community Asthma Project
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
SH performed the statistical analysis and developed the weights. TF and PF performed the clinical reviews. JS advised on statistical aspects of combining expert opinions. MH managed the data collection. All authors participated in study design and read and approved the final manuscript.
Table 5 Information for estimating PPV for those scoring 9 or more in the 2001 population survey
Stratum (i) (number of 'yes' answers to key questions) Score (j) Number of respondents ωij proportion of population postal questionnaires in stratum i, ∏'ij prevalence of asthma for score j in stratum i ωij × ∏'ij
6 12 2 0.0250 0.9914 0.0248
6 11 6 0.0750 0.9914 0.0744
6 10 5 0.0625 0.9867 0.0616
6 9 7 0.0875 0.7996 0.0700
5 12 1 0.0125 0.7181 0.0090
5 11 3 0.0375 0.7181 0.0269
5 10 9 0.1125 0.9777 0.1100
5 9 16 0.2000 0.8938 0.1788
4 10 3 0.0375 0.8908 0.0334
4 9 11 0.1375 0.9975 0.1371
1 – 3 11 1 0.0125 0.9633 0.0120
1 – 3 10 3 0.0375 0.9757 0.0366
1 – 3 9 13 0.1625 0.9880 0.1606
Total 80 1
Estimated PPV for those scoring 9 or more:
Pre-publication history
The pre-publication history for this paper can be accessed here:
Supplementary Material
Additional File 1
The Respiratory Questionnaire. The respiratory questionnaire used in the postal survey
Click here for file
Additional File 2
Estimating PPV for weighted scores. Details of method used for estimating PPV based on postal questionnaire responses
Click here for file
Acknowledgements
Doctors, staff and patients at the two practices. Dr. Roseanne McNamee for help with selection of the phase 1 stratified random sample. Clinicians and nurses for assistance with the clinical examinations. The expert physicians who classified the patients into diagnostic categories. Colleagues for critically reading the manuscript.
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| 15606914 | PMC545076 | CC BY | 2021-01-04 16:28:59 | no | BMC Fam Pract. 2004 Dec 17; 5:30 | utf-8 | BMC Fam Pract | 2,004 | 10.1186/1471-2296-5-30 | oa_comm |
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BMC OphthalmolBMC Ophthalmology1471-2415BioMed Central London 1471-2415-4-161561757710.1186/1471-2415-4-16Research ArticleKeratocyte loss in corneal infection through apoptosis: a histologic study of 59 cases Vemuganti Geeta K [email protected] Kishore [email protected] Ghazala [email protected] Prashant [email protected] Savitri [email protected] Ophthalmic Pathology Service, Brien Holden Eye Research centre, Hyderabad Eye Research Foundation, L.V. Prasad Eye Institute, Hyderabad2 Cornea Service, Brien Holden Eye Research centre, Hyderabad Eye Research Foundation, L.V. Prasad Eye Institute, Hyderabad3 Jhaveri Micrbiology Centre, Brien Holden Eye Research centre, Hyderabad Eye Research Foundation, L.V. Prasad Eye Institute, Hyderabad2004 24 12 2004 4 16 16 26 4 2004 24 12 2004 Copyright © 2004 Vemuganti 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
Keratocyte loss by apoptosis following epithelial debridement is a well-recognized entity. In a study of corneal buttons obtained from patients of corneal ulcer undergoing therapeutic keratoplasty, we observed loss of keratocytes in the normal appearing corneal stroma, surrounding the zone of inflammation. Based on these observations, we hypothesized that the cell loss in the inflammatory free zone of corneal stroma is by apoptosis that could possibly be a non-specific host response, independent of the nature of infectious agent.
Methods
To test our hypothesis, in this study, we performed Terminal deoxyribonucleotidyl transferase-mediated d-Uridine 5" triphosphate Nick End Labelling (TUNEL) staining on 59 corneal buttons from patients diagnosed as bacterial, fungal, viral and Acanthamoeba keratitis. The corneal sections were reviewed for morphologic changes in the epithelium, stroma, type, degree and depth of inflammation, loss of keratocytes in the surrounding stroma (posterior or peripheral). TUNEL positivity was evaluated in the corneal sections, both in the zone of inflammation as well as the surrounding stroma. A correlation was attempted between the keratocyte loss, histologic, microbiologic and clinical features.
Results
The corneal tissues were from 59 patients aged between 16 years and 85 years (mean 46 years) and included fungal (22), viral (15), bacterial (14) and Acanthamoeba (8) keratitis. The morphological changes in corneal tissues noted were: epithelial ulceration (52, 88.1%), destruction of Bowman's layer (58, 99%), mild to moderate (28; 47.5%) to severe inflammation (31; 52.5%). Morphologic evidence of disappearance or reduced number of keratocytic nuclei in the corneal stroma was noted in 49 (83%) cases; while the TUNEL positive brown cells were identified in all cases 53/54 (98%), including cases of fungal (19), bacterial (14), viral (13), and Acanthamoeba keratitis. TUNEL staining was located mostly in the deeper stroma and in few cases the peripheral stroma. TUNEL positivity was also noted with the polymorphonuclear infiltrates and in few epithelial cells (10 of 59, 17%) cases, more with viral infections (6/10; 60%).
Conclusions
We report apoptotic cell death of keratocytes in the corneal stroma in infectious keratitis, a phenomenon independent of type of infectious agent. The inflammatory cells in the zone of inflammation also show evidence of apoptotic cell death. It could be speculated that the infective process possibly triggers keratocyte loss of the surrounding stroma by apoptosis, which could possibly be a protective phenomenon. It also suggests that necrotic cell death and apoptotic cell deaths could occur simultaneously in infective conditions of the cornea.
infectious keratitisapoptosiskeratocytesTUNEL stain
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Background
Infections of the cornea are potentially blinding diseases and may be caused by fungi, bacteria, viruses and a few protozoans. Despite best efforts with early diagnosis and specific antimicrobial treatment about one-third of cases require surgical intervention [1]. Those which respond to medical treatment result in scarring which may lead to varying degree of visual disability. The tissue destruction in infectious keratitis, irrespective of the etiologic agent, is the compound effect of cytokines and inflammatory mediators released by the microorganism, host inflammatory cells and the metalloproteases that act on the collagen [2-5]. The aim of treating corneal infections is not only to control the infection but also to restrict the tissue damage so that the corneal stroma maintains the transparency thus retaining its visual function. Though wound healing following surgical procedures including routine keratoplasty and refractive surgeries is well documented [6-11], healing after an infectious etiology is not well documented. We had observed the loss of keratocytes in deeper stroma in Acanthamoeba keratitis and had proposed that one of the mechanisms for this loss was apoptosis of keratocytes [12]. We have since then observed the paucity of stromal keratocytes in the zone surrounding the infected area in all types of corneal infections, prompting us to speculate that it is possibly a host mediated response to an infectious process.
Apoptosis is an active process orchestrated by a series of events starting from activation of genes, release of enzymes, specific morphologic changes and ultimately leading to "an environmental-friendly cell death" whereby the dead cells are removed without affecting the bystander cells [13-15]. This is in contrast to necrotic cell death whereby the cell dies with release of various molecules that destroy the surrounding cells and stroma. Though tissue necrosis is an accepted fate of tissues in an infective process, it is associated with unwanted loss of visual function in infections of cornea. Apoptotic cell loss may be an attempt by the host stroma to escape necrosis, thereby minimizing tissue damage.
The corneal buttons removed during therapeutic keratoplasty for uncontrolled infections could possibly throw some light on these events. This study was designed to look for the evidence of apoptotic cell death of keratocytes, along with the morphological changes in corneal tissues in infective conditions.
Methods
Patients and samples
Corneal tissues from patients diagnosed as infectious keratitis undergoing therapeutic keratoplasty between 1998 and 2002 were reviewed. The corneal sections of those cases, in which the normal stroma surrounding the zone of inflammation and necrosis was visible, were included in the study. We included 59 patients diagnosed for fungal (22), viral (15), bacterial (14) and Acanthamoeba (8) keratitis in the study. The diagnosis of fungal, bacterial and Acanthamoeba keratitis was based on the microbiological investigations as described below. The etiologic diagnosis was consistent with histopathology of the corneal buttons excised during therapeutic penetrating keratoplasty. Clinical information was available in 43 cases from the ulcer database of our institute. The cases with missing clinical data on a specific parameter were excluded while calculating the statistical significance of that parameter. The clinical data was analyzed for duration of symptoms, size of ulcer, location, depth of infiltrates, presence of perforation, thinning, anterior chamber infiltration and duration of treatment.
Microbiology
All patients had undergone microbiological investigations by processing of their corneal scrapings for bacteria, fungus, Acanthamoeba. Corneal scrapings were subjected to Grams, Giemsa and Calcofluor white stain for direct smear examination and culture on blood agar, chocolate agar, brain heart infusion broth, thioglycollate broth, Sabouraud's dextrose agar and non-nutrient agar with Escherichia coli overlay. Immunofluorescent assay for HSV-1 was done on cold acetone fixed corneal scraping smears using anti-rabbit HSV-1 polyclonal antibody and FITC conjugated swine antirabbit immunoglobulin (DAKO, Denmark). In addition, polymerase chain reaction using primers specific for HSV-1 glycoprotein D gene was done on DNA extracted from corneal scraping with commercially available DNA zol solution (Helena Bio Sciences, UK)
Histopathology
The excised corneal buttons of the 59 patients were subjected to routine histopathology processing. The paraffin embedded sections were stained with hematoxylin eosin, periodic acid Schiff's and special stains like Gomori's methenamine silver stain and Grams stains. The sections were studied for histologic changes viz. ulceration, inflammation, vascular channels, necrosis, keratocyte loss and other details. Inflammation was graded in a semi-quantitative way as mild, moderate or severe based on the density of the inflammatory cells and the visibility of the stromal collagen in the background. We looked for the absence or the reduced number of the keratocytic nuclei in the corneal stroma surrounding the zone of inflammation.
TUNEL staining
Terminal deoxyribonucleotidyl transferase-mediated d-Uridine 5" triphosphate Nick End Labeling (TUNEL) staining was performed on all the 59 sections, as per the manufacture's instructions using In-Situ Cell Death Detection Kit, POD (Roche Diagnostics, Manheim, Germany). In brief, the sections were de-paraffinized, re-hydrated and incubated with proteinase K (20 ug/ml) for 10 minutes at room temperature. After rinsing with phosphate buffered saline (PBS) the sections were blocked with 3% hydrogen peroxide in methanol for 10 minutes. The sections were then incubated in the permeabilization solution consisting of 0.1% triton × 100 in 0.1% sodium citrate for 20 minutes. After washing the sections were incubated with TUNEL reaction mix at 37°C in a humidified chamber for 2 hours followed by washing and incubation with POD converter for 2 hours, the sections were stained with diaminobenzidine and counter stained with hematoxylin and observed under the microscope. Sections from the normal corneal tissues obtained from the eyebank corneas were used as control slides. Statistical analysis was done using Chi-Square test and Fisher Exact test.
Results
We studied the excised corneal buttons of 59 patients who underwent therapeutic penetrating keratoplasty for fungal, bacterial, viral and Acanthamoeba keratitis. The mean age of the patients was 46 years (range 15–85 years) with male to female ratio of 1 (22): 1.7 (37). Based on microbiologic and histologic findings, the buttons were diagnosed as fungal keratitis in 22; bacterial in 14, viral in 15 and Acanthamoeba keratitis in 8 cases.
Clinical profile
The clinical features of 43 patients at the time of presentation are given in Table 1. The size and location of the ulcer with depth of infiltrates was noted. Based on the microbiology report, these patients were treated with specific antimicrobial therapy. The cases with advanced disease at presentation or not responding to treatment were subjected to penetrating keratoplasty. The patients were treated for a median duration of 8 days (1–164 days) with specific anti-microbial therapy respectively.
Histopathology
The histologic changes were observed in the sections of the excised corneal tissues are described in Table 2. The common observations were ulceration (52, 88.1%) (figure 1), destruction of Bowman's layer (58,98%), mild to moderate (28; 47.5%) to severe inflammation (31; 52.5%). Vascular channels were seen in 13.6% of the cases, while necrosis was noted in 48 (81.4%) cases. The morphologic evidence of disappearance or reduced number of keratocytic nuclei (figure 2) in the corneal stroma was noted in 49 (83%) cases. Necrosis was more in cases of fungal keratitis as compared to other infections (22 of 22), while it was minimal in viral infections (7 of 15). Similarly severe inflammation was more in fungal keratitis (16 of 22 cases).
TUNEL assay
TUNEL staining could be assessed in 53 cases, rest 6 cases, it could not be evaluated due to either a total loss of keratocytic nuclei in the stroma, or absence of inflammation-free zone of corneal stroma. The TUNEL positive brown cells in the stromal region were noted in the region of inflammation with necrosis as well as in the surrounding stroma (figure 3), away from the zone of inflammation. In the zone of inflammation, the TUNEL positivity was seen as diffuse staining of the background as well as within the neurtrophils seen in that region. In the surrounding stroma, the TUNEL positivity was seen within the keratocytic nuclei as linear or granular nuclear staining (figure 4). This was seen in 53 (90%) cases, located mostly in the deep stroma (29; 49%) and in few cases the peripheral stroma (20; 34%). It was seen in 19 of 22 cases of fungal, 14/14 cases of bacterial, 14 of 15 cases viral and 6 of 8 cases of Acanthamoeba etiology. In addition, TUNEL positive cells were seen in the epithelium in 10 cases, (figure 5) located in the basal layer, middle layers as well as the superficial cells. Normal nuclei were also noted. The control slides showed no positive staining within the corneal stroma. Occasional positive cells were noted in the superficial cells of the corneal epithelium.
The keratocyte changes and the TUNEL staining was compared to the size of ulcer, thinning, degree of inflammation, necrosis and etiologic agent, as shown in Table 3. The correlation between apoptotic cell death (TUNEL positive cells) and the various types of infections could not be evaluated in view of high prevalence of positive staining in all types of infection. The epithelial cell apoptosis was seen in 10 cases, of which 6 were of viral etiology.
Discussion
Inflammation of the avascular transparent cornea is unique and different from other tissues in the body. Breakdown of the physiological barrier of epithelium and the tear film leads to the entry of microorganisms, which proliferate in the stroma. This is followed by a sequence of events including edema, inflammatory cell influx from tears, limbus, abscess formation and necrosis of the stroma [2]. When healing initiates there is epithelial migration of epithelial cells into the crater of the ulcer, along with growth of blood vessels into the stroma, proliferation and migration of stromal fibroblasts and influx of macrophages for scavenger activity, finally resulting in the scar formation. The degree of opacification and visual dysfunction appears to be related to the number and density of inflammatory cells and their location, and to the presence or absence of associated enzymatically induced stromal damage, vascularization, and other secondary changes. The visual recovery would therefore depend on the elimination of the inflammatory cell infiltrates, regression of edema, scarring and the newly developed vessels.
Though the observation that the stromal keratocytes disappear after epithelial injury was published more than 30 years ago, the nature of cell death was attributed to apoptosis by Campos et al and subsequently by Wilson SE et al [16-18]. Wilson et al also reported keratocyte apoptosis in association with viral infection of the overlying corneal epithelium by the herpes simplex virus [19]. They had hypothesized that one of the primary functions of keratocyte apoptotic response is to inhibit the virus that infects the corneal epithelium from spreading into the stroma and to deeper areas by temporarily eliminating the cells that are needed for viral replication and extension. This is in accordance to the Samurai principle in biology " it is better to die than do a wrong", where the damaged cells commit suicide or the programmed cell death by apoptosis [20,21].
Apoptotic cell death can be evaluated by many tests like DNA laddering as documented by gel electrophoresis, detection of enzymes like caspases, expression of mRNA of the corresponding proteins and enzymes involved in the process and by TUNEL staining [22-26]. The latter involves labeling the nick ends of the DNA fragments by labeled digitoxin molecules mediated by tdt transferase enzyme. The advantage of this technique is it identifies the apoptotic cell death in the formalin-fixed, paraffin embedded tissues and thus can be interpreted in the light of other morphological changes that take place in the stromal tissues in infections of cornea.
Ours is the first study demonstrating apoptosis in corneal tissue obtained from patients diagnosed as fungal, bacterial, viral and Acanthamoeba infections. A few animal experiments document the phenomenon of apoptosis in inflammatory conditions. In an animal experiment using rabbits, Moreau et al demonstrated that intrastromal injection of purified alpha-toxin mediates cell death of epithelial cells both by necrosis and apoptosis [27]. Similar observations have been made in our study where the infected corneal tissues showed both necrosis and apoptotic cell death. The diffuse TUNEL positive staining in the region of inflammation and necrosis also supports the hypothesis that the inflammatory cells also undergo apoptotic cell death. This process could be self-induced or due to the inflammatory cytokines which are capable of inducing the apoptosis of the host cells and also triggering apoptosis in themselves. Similar kind of observation has been made by Ozaki et al in artificially induced Arthus reaction, wherein they demonstrated that the inflammatory cells and the new blood vessels formed in the cornea regress by the process of apoptosis thereby reducing the haze and leading to corneal transparency [28]. A few other reports show that PRK produces more haze than lasik, not only because of the lack of the epithelial covering above but because of the polymorphonuclear infiltrates [29]. Therefore it could be speculated that apoptotic cell death is more of a protective phenomenon and reduces the degree of scarring that could result from necrosis alone. How this would affect the healing process and the scarring following the control of infection in early stages of corneal infection in humans in another aspect which requires further studies. It is not the scope of this study to look for the mediators of apoptosis, but evidence in literature points to the role of Fas-Fas ligand, interleukin 1, enzymes and free radicals in this process [30,31].
Loss of keratocytic nuclei was detected in 73% of cases on light microscopy, while TUNEL stain detected apoptotic cell death in 83% of cases included in the study. In 6 cases, it could not be assessed due to either the total loss of keratocytes, or due to presence of inflammatory infiltrates in all regions of corneal stromal, without an inflammatory-free zone for proper interpretation. This makes us suspect that probably it is seen in all cases of infectious keratitis. The keratocyte loss was noted mostly in the deep posterior stroma. This is similar to our study on Acanthamoeba keratitis where the remaining keratocytes in the posterior stroma showed TUNEL positivity [12]. The keratocyte loss following epithelial scraping is noted in the superificial stroma, which is the first region to be exposed following the insult to the epithelium [32]. In tissues from corneal ulcer as used in this study, the injurious stimulus is present both in the epithelium and the superficial stroma, the deeper keratocytes are possibly are continuously exposed to the insult, thus initiating the suicidal cell death. Since this study includes only those cases of infectious keratitis which underwent therapeutic keratoplasty and whose buttons histologically showed evidence of preserved stroma to comment upon, it is difficult to extrapolate the sequence of events in different phases of the infective process. Planned animal experiments could possibly throw more light on these events. But with the knowledge that the apoptotic bodies are rapidly removed form the tissues by the neighboring cells and the scavenger cells, it is possible for us to presume that apoptotic cell death does occur in late phases of the infectious process. Though TUNEL assay is an accepted method of morphological detection of apoptosis in cells and tissues, assessment of apoptosis by an additional method would have been more informative and could have overcome the limitation of this study.
Conclusion
In summary, the corneal tissues involved by various types of infection including fungal, viral and bacterial infections show evidence of inflammation, necrosis as well as apoptotic cell death of keratocytes and inflammatory cells. We speculate that the keratocyte loss adjacent to the infected tissue could be a protective phenomenon, which requires evaluation by further studies.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contribution
This work was conceived and planned by Geeta K Vemuganti. Clinical and microbiological support was provided by Prashant Garg and Savitri Sharma respectively. The bench work and analysis was done by Kishore Reddy and Ghazala Iftekhar.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This work was funded by the Indian Council of Medical Research (No.5/4/6/2/99-NCD-II) and Hyderabad Eye Research Foundation.
Figures and Tables
Figure 1 Section of the corneal button from a case of bacterial keratitis showing epithelial ulceration. Note the dense the inflammatory infiltrates in the anterior two-thirds of corneal stroma and the quiet deep corneal stroma. (Hematoxylin and eosin, ×125)
Figure 2 The section from a case of Acanthamoeba keratitis showing cysts of Acanthamoeba in the deep stroma. Note the complete absence of inflammatory cells in this area as well as the loss of keratocytic nuclei. (Hematoxylin and eosin, ×500)
Figure 3 A low power picture of corneal button with Acanthamoeba keratitis showing inflammation in the superficial layers with few keratocytes in the deeper stroma. (Hematoxylin and Eosin, ×100) [Left]. TUNEL stained sections of the same case show dense diffuse brown staining in the inflammatory region in the anterior stroma while the deeper stroma shows brown positivity in the few remaining keratocytic nuclei. (TUNEL ×100) [Right]
Figure 4 The corneal stroma in a case of fungal keratitis shows diffuse TUNEL positivity in the zone of inflammation (*) as well as discrete positive staining in the peripheral keratocytes away from zone of inflammation (arrow head) (TUNEL, ×250) [Left]. The higher magnification of the same shows TUNEL positive nuclei of the keratocyte (Arrow) in a clean background, free of inflammatory cells. (TUNEL staining, ×500) (TUNEL ×400) [Right]
Figure 5 Section from a case of viral keratitis showing TUNEL staining in the epithelial cells. The underlying stroma shows inflammatory cells and irregular keratocytic nuclei. (TUNEL, ×500)
Table 1 Clinical features of cases at presentation.
Clinical features Frequency(%)
Size of ulcer * (38) Small 6 (15.8%)
Medium 15 (39.5%)
Large 17 (44.7 %)
Location of ulcer* (42) Central 28 (66.7%)
Peripheral 6 (14.3%)
Total 8 (19.0%)
Depth of infiltrates* (38) Anterior 4 (10.5 %)
Middle 15 (39.5%)
Posterior 19 (50.0%)
Perforation (43) Absent 40 (93.0%)
Present 3 (7.0%)
Thinning (43) Absent 24 (55.8%)
Present 19 (44.2%)
Infiltrates in Anterior chamber*(34) Absent 4 (11.8%)
Present 30 (88.2%)
• Due to missing data valid percentage has been considered
Table 2 Histologic features of excised corneal tissue of patients with infectious keratitis
Histologic changes Frequency of histologic changes in 59 cases
Epithelial Ulceration Absent 7 (11.9%)
Present 52 (88.1%)
Bowmans membrane Absent 58 (98.3%)
Present 1 (1.7%)
Inflammation Mild/Mod 28 (47.5%)
Severe 31 (52.5%)
Vascular channels Absent 51 (86.4%)
Present 8 (13.6%)
Stromal necrosis Absent 11 (18.6%)
Present 48 (81.4%)
Keratocyte loss* Absent 8 (13.6%)
Reduced 47 (79.7%)
• could not assessed in two cases
Table 3 Comparison of clinical, histologic features and degree of keratocyte loss in various types of corneal infections
Features Etiology Correlation of apoptosis with clin/hist features
Fungus (22) Virus (15) Bacteria (14) Acanth (8)
Size of ulcer (38) Small (≤2 mm2) 1 (6.7%) 2 (28.6%) 1 (9.1%) 2 (40.0%) P = 0.224
Medium and Large (≥25 mm2) 14 (63.63%) 5 (33.33%) 10 (71.4%) 3 (37.5%)
Stromal thinning (43) Absent 10 (62.5%) 3 (42.9%) 6 (42.8%) 5 (83.3%) P = 0.313
Present 6 (37.5%) 4 (57.1%) 8 (57.1%) 1 (16.7%)
Necrosis (59) Absent 0 8 (53.3%) 2 (14.3%) 1 (12.5%) NA
Present 22 (100%) 7 (46.7%) 12 (85.7%) 7 (87.5%) NA
Inflammation (59) Mild/Moderate 6 (27.3%) 10 (66.7%) 7 (50.0%) 5 (62.5%) P = 0.087
Severe 16 (72.7%) 5 (33.3%) 7 (50.0%) 3 (37.5%)
Keratocytes (57) Absent 4 (19.1%) 4 (28.6%) 0 0 NA
Reduced 15 (71.4%) 10 (71.4%) 14 (100%) 8 (100%)
Normal 2(9.5%) 0 0 0
Apoptosis (54) Absent 0 1 (6.7%) 0 0 NA
Present 19 (100%) 14 (93.3%) 14 (100%) 6 (100%)
NA = Not applicable
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| 15617577 | PMC545077 | CC BY | 2021-01-04 16:03:49 | no | BMC Ophthalmol. 2004 Dec 24; 4:16 | utf-8 | BMC Ophthalmol | 2,004 | 10.1186/1471-2415-4-16 | oa_comm |
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BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-6-21563435210.1186/1471-2105-6-2SoftwareVisualization of comparative genomic analyses by BLAST score ratio Rasko David A [email protected] Garry SA [email protected] Jacques [email protected] The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850 USA2005 5 1 2005 6 2 2 18 10 2004 5 1 2005 Copyright © 2005 Rasko 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 first microbial genome sequence, Haemophilus influenzae, was published in 1995. Since then, more than 400 microbial genome sequences have been completed or commenced. This massive influx of data provides the opportunity to obtain biological insights through comparative genomics. However few tools are available for this scale of comparative analysis.
Results
The BLAST Score Ratio (BSR) approach, implemented in a Perl script, classifies all putative peptides within three genomes using a measure of similarity based on the ratio of BLAST scores. The output of the BSR analysis enables global visualization of the degree of proteome similarity between all three genomes. Additional output enables the genomic synteny (conserved gene order) between each genome pair to be assessed. Furthermore, we extend this synteny analysis by overlaying BSR data as a color dimension, enabling visualization of the degree of similarity of the peptides being compared.
Conclusions
Combining the degree of similarity, synteny and annotation will allow rapid identification of conserved genomic regions as well as a number of common genomic rearrangements such as insertions, deletions and inversions. The script and example visualizations are available at: .
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Background
In the decade since the publication of the Haemophilus influenzae genome sequence in 1995 [1], 191 microbial genomes have been completed, with another 276 in progress [2]; as of October 14, 2004). Multiple strains of the same organism, or multiple species of the same genus are being sequenced or have been completed, making comparative genomic analysis possible on an unprecedented scale. As the technology continues to improve, the number of completed microbial genome sequences will further increase – a major challenge of the comparative genomic era is to fully exploit this data. However, the development of tools for analysis of such data sets has not kept pace.
BLAST analysis has become a ubiquitous method of interrogating new sequence data, but there are limitations to using BLAST alone as a discriminating tool. Many other methods and individuals use BLAST output E-values as a criterion for data parsing. While this measure may be efficient, the output is often skewed by both the database used for comparison and the length of the match [3]. Small regions of high similarity can generate an artificially low E-value and negate the global level of similarity exhibited by the sequence. This bias is eliminated when using the BLAST raw score as it is directly derived from the similarity of the match. However the value of the BLAST score varies with the length of the peptide queried, and hence is not suitable alone for comparative analysis using universal cutoffs [4].
Several other tools utilize the BLAST algorithms to compare nucleotide or peptide sequences from genome projects. The Wellcome Trust Sanger Institute ACT software [5] can display nucleotide similarity between two genomes based on BLASTN E-value. ACT builds upon Artemis and displays regions of high similarity mapped on the genome annotation. The GenomeComp tool [6] displays a similar analysis also based on BLASTN E-values to compare genome sequences. NCBI Taxplot, a three-way genome comparison tool based on precomputed protein BLASTP E-values displays a point for each protein in the Reference genome based on the best alignment with proteins in each of the two genomes being compared [7]. On the other hand, the SimiTri program utilizes BLASTP comparisons of three proteomes and uses the raw BLAST score, not E-values. However, only protein similarity data is represented and no information on the comparative structure of the genomes is provided [8]. Moreover, the SimiTri program does not address BLAST artifacts derived from the size of the database or the length of the match. This paper describes the BLAST score ratio (BSR) algorithm that enables comparative analysis of multiple proteomes, together with visualization of genome structure (synteny).
BSR analysis is a departure from traditional genome scale analyses as it overcomes the limitations of BLAST E-values in comparative studies by normalizing the BLAST raw scores. BSR analysis is a tool for the rapid comparison of complete proteomes of any three genomes, and enables a visual evaluation of the overall degree of similarity of these proteomes and their genomic structure.
Implementation
We have implemented the BSR algorithm using Perl. The inputs are the predicted proteomes of each of the three genomes under analysis, formatted as multi-FASTA files. An additional file for each of the proteomes is required. This file must contain a unique identifier, matching the FASTA header of the corresponding peptide in the multi-FASTA file, the relative genomic location of the start and stop of the coding region as well as the annotation for each peptide. The user selects one proteome as the "Reference"; the other two proteomes are termed "Query1" and "Query2" respectively. Initiation of the script results in each of the putative peptides in the Reference proteome being compared to all of the other peptides in the Reference and Query proteomes using NCBI BLASTP.
The BSR is then computed as follows. The BLAST raw score for each Reference peptide against itself is stored as the Reference score. Each Reference peptide is then compared to each peptide in the Query1 and Query2 proteomes with each best BLAST raw score recorded as Query1 and Query2, respectively (Figure 1). The BSR is calculated by dividing the Query score by the Reference score for each Reference peptide (Figure 1A). Thus, for each peptide in the Reference genome, two numbers are generated, one from each from the best matches in Query1 and Query2, thus normalizing all scores in the range of 0 to 1. A score of 1 indicates a perfect match of the Reference peptide to a Query peptide and score of 0 indicates no BLAST match of the Reference peptide in the Query proteome. The BLAST raw score is used rather than the E-value for the BLASTP results as it more accurately accounts for the length of the similarity between the Reference and Query peptides [4,9]. This normalized pair of numbers can be plotted as coordinates in Cartesian space for each peptide in the Reference proteome, enabling the visualization of the entire Reference proteome in comparison to the two Query proteomes (Figure 1B).
Outputs
Following calculation of the BSRs, a number of output files are generated, including both text and graphical formats. The text files are tab-delimited for ease of parsing; filenames are derived from the named proteome files used as input into the script. The R_Q1_Q2.txt (Reference_Query1_Query2.txt) output contains an ordered list including the Reference peptide unique identifier, annotation, and Reference BLAST raw score, in addition to the unique identifier of the best hits in the Query proteomes, corresponding BLAST raw scores and the calculated BSR. Additionally, four unique files are generated corresponding to the peptides within the four quadrants delineated in Figure 1B. The four quadrants are derived from a BSR threshold value of 0.4, which was empirically determined to represent approximately 30% amino acid identity over approximately 30% of the peptide length, a commonly used threshold for peptide similarity [10]. This threshold value can be adjusted using the "-C" option (see help file).
The graphical output files are viewed with Gnuplot [11] to reveal the global similarity of the compared genomes as well as the level of conserved genome structure. PostScript and xfig [12] graphic files are subsequently generated by Gnuplot. The scatter or similarity plot provides an overall view of the level and number of similar and dissimilar proteins in the Reference proteome when compared to the Query proteomes (Figure 1C). The regions of the graph are color-coded depending on the level of similarity between the three genomes (Figure 1B). Quadrant A (BSR < 0.4), colored in orange, contains peptides unique to the Reference proteome with little similarity in either of the Query proteomes. Quadrant C (BSR > 0.4), colored Red, contains peptides that have significant similarity in all three compared proteomes. Quadrant B, colored green, contains Reference peptides with similarity to only Query proteome 2, whereas Quadrant D, colored blue, contains Reference peptides that have similarity to only Query proteome 1.
Two additional plots, termed synteny plots, are generated, one for comparison of the Reference proteome to each Query proteome, by plotting the genomic location of the Reference peptide on the X-axis and the genomic location of the most similar Query peptide on the Y-axis. This plot alone would demonstrate the level of synteny (conservation of gene order) between the two genomes [13], however, an additional level of information is included by coloring each point based on the BSR (see legend Figure 2). The color provides an additional visual clue to the global level of similarities of the proteomes. For example genomes can be highly syntenic with relatively low levels of proteomic similarity as is shown in Figure 2A and 2B or they may have a high degree of protein similarity andconserved genome structure (Figure 2C).
The Gnuplot, PostScript and xfig outputs allow publication-quality, global visualization of the similarity and synteny of the selected genomes. However these formats do not allow the annotation associated with individual peptides to be viewed interactively. To overcome this limitation, additional XML files for the similarity and synteny plots described above are generated. These files are the input for the freely available GGobi software. GGobi is a data visualization system for viewing high-dimensional data [14]. The tools provided in the GGobi software package allow the annotation associated with individual points within the similarity and synteny plots to be viewed interactively (Figure 3).
The GGobi package also allows the expansion of the BSR approach to include more than three genomes or other additional parameters associated with proteomic or genomic data, enabling interactive, user-driven exploration of these complex datasets. The current BSR implementation uses three genomes as input; however, additional genomes can readily be added as new dimensions simply by repeating the analysis with the same Reference genome and varying the Query genomes. Additional non-BSR dimensions are readily included, such as pI or %GC, or factors such as surface localization or some other feature of the peptides of interest.
Results
Genome structure is often altered during the evolution of species [13]. Visualization of this structure often lends insight into genome evolution and examination of the various BSR outputs rapidly reveal alterations of the genome structure as well as the overall similarity of the two Query proteomes to the Reference proteome. The genomes of the Order Chlamydiales (Figures 1, 2 A and 2B) provide an example of this insight. In Figure 1a large proportion of the peptides are conserved, with 71.7% of the proteins shared between all three proteomes. If the Query proteomes are further used as the Reference proteome and vice versa we still see a similar trend (data not shown). Additionally, the proteome of C. pneumoniae AR39 (GenBank Accession Number AE002161) is more similar to C. caviae GPIC (GenBank Accession Number AE015925) than C. muridarum strain Nigg (GenBank Accession Number AE002160) as 7.3 % of the proteome is shared between only C. caviae GPIC and C. pneumoniae AR39 compared to only 1.6% between C. caviae GPIC and C. muridarum strain Nigg. Finally, Figure 1 demonstrates that 19.4% of the C. caviae proteome has no significant hit to any of the peptides in the Query proteomes, although many of these peptides (78.2%) are currently annotated as hypothetical.
From the analysis in Figure 1 we could conclude that the chlamydial proteomes are extremely similar and suggest that the genome structure will also be similar. However, the synteny plots in Figure 2A and 2B demonstrate that while the chlamydial proteomes exhibit a high degree of similarity, there is significant alteration in the genomic structure. The comparison of the proteomically similar organisms, C. caviae GPIC and C. pneumoniae AR39 reveals that the genomes contain two points of inversion (arrows in Figure 2A). One of these points of inversion is centered on the terminus of replication. There are more extensive genomic rearrangements between the C. caviae GPIC and C. muridarum strain Nigg genomes (Figure 2B). The additional color information extends the utility of these synteny plots. While the chlamydial genomes show regions of conserved synteny, as demonstrated by the peptides in the same genomic location forming a line with a slope of 1 or -1, the absolute degree of similarity between the peptides, demonstrated by color indicates divergence. By contrast the synteny plot of two Escherichia coli genomes (Figure 2C) demonstrates a high level of synteny with a number of unique insertions, however no inversions are present. Moreover the color dimension on this plot reveals that unlike the chlamydial proteome comparisons the E. coli proteomes have a high level of similarity andsynteny.
In the analysis of the Chlamydial proteomes using BSR score and BLAST E-values approximately 1% of peptides examined have a BSR score > 0.4 and BLAST E-value > 1 × 10-15. These peptides were all very small in size (< 70 amino acids) and greater than 50% amino acid identity. This group of peptides is more readily identified by BSR analysis than BLAST E-value, which is artificially low due to the small peptide size. Additionally, peptides that have a BSR score < 0.4 but a BLAST E-value < 1 × 10-15 correspond 7.8% of the proteome. These represent divergent peptides with an artificially high BLAST E-value score resulting from limited regions of identity. The BSR analysis more accurately classifies these peptides based on the amino acid identity over the entire peptide. As the BSR comparison utilizes a single genome as a reference, the BSR score is calculated using a unidirectional best BLAST hit. However, when the Chlamydial proteomes were compared only one case in over 1000 could be found with a BSR score > 0.4 that was not also a bidirectional best BLAST hit.
Conclusions
The BSR approach allows rapid evaluation of the level of conservation of any three proteomes and the degree to which the genome structure between the three genomes is similar. While in this report we discuss the applications of this approach to whole genomes, the analysis has been performed on portions of genomes such as genomic or pathogenicity islands, plasmids and phage to identify peptide similarity and regional structure.
More genome sequences are being generated from closely related organisms – a trend which shows no sign of abating. The BSR approach has become a crucial tool in our comparative genomics armamentarium and has been utilized in a number of genomic comparisons, revealing regions of similarity and difference between both closely and distantly related organisms [10,15,16].
Availability and requirements
Project name: BSR.pl
Project homepage:
Operating System: Unix and MacOS X
Programming language: Perl
Other requirements: Perl Statistics::Descriptive module
License: None
Any restrictions to use by non-academics: None
List of abbreviations
BSR – BLAST score ratio; BLAST – basic local alignment search tool.
Authors' contributions
DAR, GSAM and JR conceived and implemented the first versions of BSR and prepared the manuscript. All authors have read and approved the final manuscript.
Acknowledgments
The authors would like to thank Timothy D. Read for initial consultation on development of the BSR algorithm. JR and DAR are supported by Federal funds from the National Institute of Allergy and Infectious Disease, National Institutes of Health, under Contract No. N01-AI15447. GSAM is supported by NIAID R01 AI051472.
Figures and Tables
Figure 1 BSR rationale and scatter plot example. A. BLAST score ratio analysis (BSR) calculation demonstrating how the two coordinates for plotting in figures B and C are calculated. B. Locations of the peptide spot revels the similarity that the peptide has to the two Query genomes. Use of a 0.4 separator is based on ~30% amino acid identity over 30% of the length of the peptide [10]. C. Sample data obtained from comparison of Chlaymidia caviae GPIC (GenBank Accession Number AE015925) to the proteomes of Chlamydia muridarum strain Nigg (GenBank Accession Number AE002160) and Chlamydia pneumoniae AR39 (GenBank Accession Number AE002161) [17]. Each point in the figure represents a single peptide in Chlaymidia caviae GPIC This analysis reveals that while these organisms are very similar, C. caviae is more similar to C. pneumoniae AR39 than C. muridarum strain Nigg due the skew of peptides with a slope of greater than 1.
Figure 2 Genome structure visualization. Direct comparison of two genomes at a time demonstrating some examples of large-scale genomic rearrangements. Each protein is plotted by the genomic location of the coding region and is color-coded by the degree of similarity based on the BSR as is demonstrated in the legend. A. Comparison of C. caviae GPIC and C. pneumoniae AR39. This comparison contains two genomic rearrangements of different sizes as indicated by the arrows. B. C. caviae GPIC and C. muridarum strain Nigg comparison reveals a more extensive genomic rearrangements suggesting that while proteomically these organisms are similar the genomes have diverged significantly. C. E. coli CFT073 (GenBank Accession Number AE014075) vs. E. coli K12 (GenBank Accession Number U00096). E. coli CFT073 contains a number of unique insertions that are represented as breakpoints in the plot and highlighted with arrows. The high level of synteny and similarity are exhibited by these genomes.
Figure 3 Visualization with GGobi. GGobi screenshots of the graphical outputs from the BSR. The proteins for tryptophan synthase alpha and beta subunits are highlighted as they were unique in the C. caviae genome and represented a significant metabolic adaptation of this species in comparison to the other species compared [17]. A. The scatter plot represents the same figure as shown in Figure 1C, however the interactive nature of GGobi allows visualization of the annotation associated with any of the peptides. B. Synteny plots as seen in GGobi. These same genes from Figure 3A can be highlighted in the in the synteny plots and the genomic location can be observed. To take advantage of the usefulness of the interactive mouseover the BSR is included with the annotation.
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| 15634352 | PMC545078 | CC BY | 2021-01-04 16:02:51 | no | BMC Bioinformatics. 2005 Jan 5; 6:2 | utf-8 | BMC Bioinformatics | 2,005 | 10.1186/1471-2105-6-2 | oa_comm |
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BMC Evol BiolBMC Evolutionary Biology1471-2148BioMed Central London 1471-2148-5-31563435810.1186/1471-2148-5-3Research ArticleThe genetics of ray pattern variation in Caenorhabditis briggsae Baird Scott Everet [email protected] Cynthia R [email protected] Justin C [email protected] Department of Biological Sciences, Wright State University, Wright State University, Dayton OH 45435, USA2 Department of Pulmonary Medicine, Children's Hospital Medical Center, Cincinnatti OH 45229-3039, USA3 College of Medicine, Cleveland Clinic Foundation NA24, 9500 Euclid Avenue, Cleveland, OH 44195, USA2005 5 1 2005 5 3 3 20 10 2004 5 1 2005 Copyright © 2005 Baird et al; licensee BioMed Central Ltd.2005Baird 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
How does intraspecific variation relate to macroevolutionary change in morphology? This question can be addressed in species in which derived characters are present but not fixed. In rhabditid nematodes, the arrangement of the nine bilateral pairs of peripheral sense organs (rays) in tails of males is often the most highly divergent character between species. The development of ray pattern involves inputs from hometic gene expression patterns, TGFβ signalling, Wnt signalling, and other genetic pathways. In Caenorhabditis briggsae, strain-specific variation in ray pattern has provided an entrée into the evolution of ray pattern. Some strains were fixed for a derived pattern. Other strains were more plastic and exhibited derived and ancestral patterns at equal frequencies.
Results
Recombinant inbred lines (RILs) constructed from crosses between the variant C. briggsae AF16 and HK104 strains exhibited a wide range of phenotypes including some that were more extreme than either parental strain. Transgressive segregation was significantly associated with allelic variation in the C. briggsae homolog of abdominal B, Cb-egl-5. At least two genes that affected different elements of ray pattern, ray position and ray fusion, were linked to a second gene, mip-1. Consistent with this, the segregation of ray position and ray fusion phenotypes were only partially correlated in the RILs.
Conclusions
The evolution of ray pattern has involved allelic variation at multiple loci. Some of these loci impact the specification of ray identities and simultaneously affect multiple ray pattern elements. Others impact individual characters and are not constrained by covariance with other ray pattern elements. Among the genetic pathways that may be involved in ray pattern evolution is specification of anteroposterior positional information by homeotic genes.
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Background
A fundamental problem of morphological evolution is how does microevolution relate to macroevolution [1]. Are variation and selection within species sufficient to account for genetic divergence between species? One approach to this problem has been comparative developmental genetic studies in closely related species. This approach is complicated by developmental redundancy and cryptic variation [2-4]. Developmental redundancy occurs in species pairs in which similar adult morphologies are attained through distinct developmental processes. For example, in the nematodes Caenorhabditis elegans and Oscheius tipulae, cells in the vulval equivalence group that do not participate in vulval development are eliminated through fusion with the multinucleated syncytial epidermis [5-8]. In Pristionchus pacificus, that same outcome is achieved through apoptosis [9]. Cryptic variation occurs in species pairs in which the same developmental process is regulated through divergent genetic mechanisms. For example, Drosophila melanogaster and D. simulans have similar numbers and patterns of bristles. Hybrids have fewer bristles than either parental species [10,11]. Thus, the conserved bristle phenotypes of D. melanogaster and D. simulans mask cryptic variation in the genetic regulation of bristle pattern.
Another approach has been the study of morphological variation within species. In the butterfly Bicyclus anynana, expression patterns of distalless (dll), engrailed (en), and spalt (sal) coincide with eyespot coloration patterns [12,13]. Moreover, allelic variation in distalless has been associated with size variation in eyespots [12]. Thus, evolution of eyespot patterns may have resulted from selection for variant alleles of dll, en, and sal. Consistent with this model, conservation of the expression patterns of these genes in developing eyespots has been demonstrated in multiple butterfly species [13]. Developmental variation also has been characterized in several nematode species. For example, variant patterns of cell division in the ventral epidermis have been described in C. elegans, C. briggsae, and O. tipulae [14].
Another promising model for microevolutionary studies of morphological evolution is ray pattern variation in rhabditid nematodes. The rays are male-specific peripheral sense organs that mediate mating behavior [15-17]. In most rhabditid species there are nine bilateral pairs of rays embedded within the copulatory bursa of the tail [17]. The pattern of rays; their anteroposterior placement and the dorsoventral position of their sensory endings, generally are constant within species but often are the most divergent character between species [18]. Ray pattern in adult males is determined by cell contacts that form between ray structural cells and the surrounding cells of the lateral epidermis in L4 larvae [19,20]. These contacts in turn are determined by the specification of 'ray identities' [21]. In Caenorhabditis elegans, ray identities are specified through the integration of several regulatory inputs including: positional information from homeotic genes [22-26]; TGF-beta-like signalling [27-29]; Wnt signalling [25]; and ephrin/semaphorin signalling [30].
The ray pattern of Caenorhabditis briggsae differs from that of C. elegans in the placement of ray 3 [31,32]. In C. elegans males, ray 3 is located at the cloaca and is separate from from all other rays. In the canonical C. briggsae ray pattern, ray 3 is located in a posterior cluster of rays 3–6 and frequently is fused with ray 4. The C. elegans ray pattern is shared with several other Caenorhabditis species and is ancestral to the Elegans-group [33], a monophyletic clade that includes C. briggsae [34-36]. The C. briggsae ray pattern is mimicked by several C. elegans mutations including mutations in homeotic genes or in genes that regulate homeotic gene expression patterns [21,22,26,37,38]. Thus, the derived C. briggsae ray pattern may have arisen through changes in the specification of anteroposterior positional identities. It now is possible to address this hypothesis as variant C. briggsae strains that express an ancestral ray pattern at high frequencies have been identified [39]. In this paper, the segregation of ray pattern phenotypes in crosses between C. briggsae strains is described.
Results
The elements of ray pattern
The male-specific rays of rhabditid nematodes are embedded within a lateral fold of cuticle called the bursa or tail fan [19] (Figure 1). The sensory endings of most rays are attached to and open through the surface of the bursa. Ray pattern refers collectively to the anteroposterior positions of each ray, the surface (dorsal, ventral or medial) through which their sensory endings are exposed, and whether or not individual rays are fused with each other. Rays also differ in regard to neurotransmitter usage [5,21,40]. Mutations that affect ray pattern also affect neurotransmitter usage indicating that multiple properties of rays arise through the specification of 'ray identities' [21,24].
Figure 1 Ray patterns of C. briggsae and C. elegans. Ventral views of a) C. elegans and b) C. briggsae male tails. Anterior to the left. Left side up. Bilateral pairs of rays are numbered from anterior to posterior. a) C. elegans pattern in which ray 3 is separate from all other rays. This pattern is referred as a 2(1)3+3 pattern. b) C. briggsae pattern in which ray 3 is clustered with rays 4 – 6. This pattern is referred to as a 2/4+3 pattern. In this pattern ray 3 may be either free (right side) or fused with ray 4 (left side). The 2(1)3+3 pattern is ancestral to the Elegans-group, a monophyletic clade that includes C. elegans and C. briggsae [34].
The derived ray pattern of C. briggsae entails the posterior displacement of ray 3 from a position level with the cloaca to the post-cloacal cluster of rays 4 – 6 and the frequent fusion of ray 3 with ray 4 [31,32,34]. This ray pattern is exhibited in nearly all males of C. briggsae strains AF16 and VT847 [39]. Ray pattern in C. briggsae strains HK104, HK105, and PB800 is more variable. These strains exhibit the derived and ancestral ray patterns at approximately equal frequencies [39].
Segregation of C. briggsae ray pattern phenotypes
The genetic basis of ray pattern variation in C. briggsae was characterized through the segregation of ray pattern phenotypes in a set of recombinant inbred lines (RILs). These RILs were constructed from a cross between strains AF16 and HK104. Individual RILs were established from F2 progeny of this cross. RILs were inbred, one hermaphrodite per generation, through F11. Based on this degree of inbreeding, 99.9% of loci in each RIL should be homozygous. RILs were scored for two aspects of ray pattern; the position of ray 3 and the fusion of rays 3 and 4 (Figure 2).
Figure 2 Segregation of ray pattern phenotypes in C. briggsae RILs. a) Scatter plot comparing frequencies of derived character states for the position of ray 3 (Y-axis) and fusion of rays 3 and 4 (X-axis) in RILs. Each point represents an individual strain for which a minimum of 100 sides were scored for ray pattern. Parental strains, AF16 and HK104 represented by red square and red diamond, respectively. RILs represented by closed circles. Correlation coefficient, r, for ray position and fusion equaled 0.80. b) Frequency distributions for derived character states of ray 3 position (black bars) and ray 3–4 fusion (red bars) among RILs.
Several RILs exhibited ancestral or derived phenotypes at frequencies more extreme than either parental strain (Figure 2a). This was true for both the position of ray 3 and for fusions of rays 3 and 4. For ray 3 position, one quarter of the RILs exhibited phenotypes more extreme than either parental strain. For ray fusions, half of the RILs were more extreme than the parental strains. Such transgressive segregation is best explained through the presence, in both AF16 and HK104, of alleles at some loci that promoted ancestral character states and alleles at other loci that promoted derived character states. The high frequency of RILs with extreme phenotypes indicated that both parental strains likely were fixed for antagonistic alleles at multiple loci.
A relatively strong correlation was observed between variation in the position of ray 3 and its fusion with ray 4 (r = 0.797; Figure 2a). This correlation was highly significant (p = 2.5 × 10-23). This was expected as rays 3 and 4 could not have fused unless they were adjacent to each other [19] and because the placement of rays and fusion between rays have been considered to be pleiotropic phenotypes derivative of the specification of ray identities [19,22-25,27-30]. If anything, it was surprising that the correlation was not stronger. Based on the observed correlation coefficient, approximately one third of the variation in ray fusion frequency was independent of variation in the position of ray 3. This was readily apparent when the frequency distributions of these two ray pattern elements were compared (Figure 2b). The frequency distribution for the placement of ray 3 was highly skewed toward the derived phenotype. The frequency distribution for fusions of rays 3 and 4 was much broader and was only slightly skewed. The differences in the shapes of these distributions were significant (Kolmgorov-Smirnov, D = 0.4396, p < 0.001).
Neither the frequency distribution for the placement of ray 3 nor that for the fusion of ray3 to ray 4 were normal (p = 0.00 and p = 0.03, respectively; Figure 2b). The frequency distribution for the placement of ray 3 was highly skewed toward the derived phenotype. This result was inconsistent with the placement of ray 3 being governed strictly by additive affects. More likely, the placement of ray 3 was regulated by epistasis. Twentynine per cent of RILs exhibited the most extreme phenotype (Figure 2b). Fixation of derived alleles at as few as two major-effect genes could have been sufficient to ensure a posterior localization of ray 3 at this frequency. The frequency distribution for fusions of rays 3 and 4 was very broad and and only slightly skewed. This was consistent with ray fusion being a complex character with inputs from multiple developmental processes. At least one of these processes was shared with ray positioning; rays 3 and 4 could not have fused if they were not adjacent.
Association of ray pattern with allelic variation in homeotic genes
The derived C. briggsae ray pattern may have evolved through changes in homeotic gene expression patterns [33,39]. The homeotic genes most likely to affect ray pattern are mab-5 and egl-5. These genes are expressed in the posterior body and tail regions of C. elegans [40] and mutations in them can alter ray pattern [22]. Allelic variants that affect the size of the first intron of Cb-egl-5 have been identified (Figure 3). The AF16::HK104 RILs were genotyped for these variants to test Cb-egl-5 for association with ray pattern variation [see Additional File 1].
Figure 3 Allelic variation of Cb-egl-5. Cb-egl-5 amplification products of 1) AF16; 2) PB800; 3) HK104; 4–17) selected RILs; 18) an HK104/AF16 heterozygote. The expected amplification product size based on the C. briggsae AF16 genome sequence was 1,723 bp. m) Marker DNA, sizes of selected markers as indicated.
Significant associations were observed between Cb-egl-5 allelic variants and both the position of ray 3 and the fusion of ray 3 to ray 4 (Table 1). These associations were transgressive; the HK104 allele of Cb-egl-5 cosegregated with derived phenotypes, the AF16 allele with ancestral phenotypes. Allelic variants in Cb-mab-5 have not been identified. However, the Cb-mab-5 (=CBG00029) and Cb-egl-5 (=CBG00023) are located within 60 kb of each other [41,42]. Because of this close physical linkage, allelic variants in these genes very likely cosegregated. Thus, allelic variation in Cb-mab-5, Cb-egl-5, and/or other linked loci may have been responsible for observed transgressive associations.
Table 1 Cosegregation of Cb-egl-5 with ray pattern phenotypes.
allele1
phenotype AF16 HK104 p value
ray position 0.81 0.90 6.2 × 10-4
ray fusion 0.42 0.68 4.2 × 10-5
1 Mean frequency of derived character states exhibited in RILs homozygous for either parental Cb-egl-5 allele.
mip-1 marker-assisted introgression
The segregation of phenotypes in the RILs indicated that multiple major-effect and additive loci were involved in ray pattern variation in C. briggsae. Because a high density recombination map for C. briggsae has not yet been constructed, it is not possible to identify these loci through QTL analyses. However, a limited number of mutations with visible phenotypes have been mapped to C. briggsae chromosomes [B. Gupta, pers. comm., [43]]. These mutations all were generated in an AF16 background. One such mutation defines the gene mip-1. mip-1 very likely is the C. briggsae homolog of C. elegans unc-22 [D. Baillie, pers. comm.]. In C. elegans, unc-22 is located on chromosome IV and is linked to several genes known to regulate homeotic gene expression patterns, e.g. lin-49 which encodes a bromodomain protein thought to be involved in chromatin remodeling [26]. mip-1 marker-assisted introgression was used to determine if any genes linked to it were involved in ray pattern variation in C. briggsae.
Four C. briggsae strains were constructed in which HK104 DNA, in the region of mip-1, has been introgressed into an otherwise AF16 background. This was accomplished through a series of backcrosses that were initiated by mating HK104 males to mip-1 (AF16) hermaphrodites. For each introgressed strain, F1 through F6 males were crossed to mip-1 (AF16) hermaphrodites. Wild-type F7 hermaphrodites were selected and propagated by self-fertilization. From each F7 hermaphrodite, multiple wild-type F8 hermaphrodites were picked. Homozygous strains were established from F8 hermaphrodites that segregated only wild-type progeny. From each set of backcrosses, a single introgressed strain was retained. These introgressed strains were scored for the position of ray 3 and for ray 3–4 fusions (Figure 4). The ray patterns of all four introgressed strains differed significantly from AF16 both in the placement of ray 3 and in the frequency with which ray 3 fused to ray 4 (Table 2). Significant differences also were apparent between some pairs of introgressed strains, most notably between PB1060 and PB1065. PB1060 and PB1065 did not differ in the placement of ray 3 but only in the frequency with which ray 3 fused to ray 4 (Figure 4; Table 2). Thus, it appears that allelic variation in at least two genes linked to mip-1 is involved in ray pattern variation. One of these genes affects the position of ray 3 and possibly the frequency of ray fusion. The HK104 allele of this gene was present in all mip-1 introgressed strains. The other gene affects only the frequency of ray fusion. The HK104 allele of this gene was present in PB1065 but not in PB1060. As more C. briggsae mutant strains become available, it should be possible to identify these genes through additional introgression studies coupled with genotypic characterizations of introgressed DNA.
Table 2 Comparison of mip-1 introgressed lines.
p values for reciprocal chi-squared tests1
AF16 PB1060 PB1061 PB1062 PB1065
ray pattern2
AF16 --- 9.6 × 10-30 3.3 × 10-54 6.5 × 10-32 5.3 × 10-46
PB1060 7.9 × 10-4 --- 0.031 0.093 1.2 × 10-5
PB1061 2.2 × 10-7 0.026 --- 0.367 0.011
PB1062 2.8 × 10-6 0.092 0.293 --- 0.091
PB1065 2.3 × 10-9 1.3 × 10-4 0.038 0.151 ---
ray position3
AF16 --- 6.9 × 10-4 1.5 × 10-5 3.6 × 10-4 3.2 × 10-4
PB1060 1.32 × 10-29 --- 0.068 0.72 0.67
PB1061 6.4 × 10-53 0.053 --- 0.12 0.14
PB1062 8.3 × 10-30 0.73 0.16 --- 0.95
PB1065 1.65 × 10-36 0.66 0.14 0.95 ---
ray fusion4
AF16 --- 0.13 5.3 × 10-3 3.1 × 10-3 9.8 × 10-6
PB1060 0.013 --- 0.061 0.033 3.1 × 10-5
PB1061 4.74 × 10-6 0.057 --- 0.80 0.029
PB1062 9.37 × 10-7 0.030 0.80 --- 0.052
PB1065 1.33 × 10-16 1.59 × 10-6 0.013 0.028 ---
1 Probabilities in each column were determined using frequencies of different ray patterns in each strain (column head) as the null hypothesis.
2 p values for the complete ray pattern (3 posterior and fused, 3 posterior not fused, 3 anterior).
3 p values for ray placement (anterior vs posterior).
4 p values for fusion of rays 3 and 4 when ray 3 is in a posterior position.
Figure 4 Comparisons of ray pattern phenotypes in mip-1 introgressed strains. Frequencies of ray pattern phenotypes exhibited in AF16 and four mip-1 introgressed strains. Black bars represents ray 3 in a posterior position and fused with ray 4. Red bars represents ray 3 in a posterior position but not fused with ray 4. White bars represents ray 3 in an anterior postion.
Discussion
Ray pattern in C. briggsae varies with respect to the placement of ray 3 and fusions of ray 3 to ray 4. Ancestral states for these characters in the Elegans-group of Caenorhabditis are an anterior location, level with the cloaca, and the absence of ray fusions [34]. Derived states are a posterior location, clustered with rays 4 through 6, and fusion of rays 3 and 4. An unexpected result was the incomplete correlation between the position or ray 3 and its fusion with ray 4. In C. elegans, mutations that alter ray position have been accompanied by ray fusion. For this reason, ray position and ray fusion were thought to be pleiotropic phenotypes that arose from the specification of ray identities [19,22-25,30]. Because of this, it was expected that ray pattern evolution would involve changes in suites of covariant characters rather than independent modification of individual ray pattern elements. However, only two-thirds of variation in ray fusion resulted from variation in ray position (r2 = 0.635). Moreover, evidence for a gene affecting only ray fusion was obtained from comparisons of ray patterns exhibited by the mip-1 introgressed strains. Hence, variation in ray pattern evolution is not wholey constrained by the specification of ray identities and other ray pattern elements, such as neurotransmitter usage, also may vary independently. A similar pattern of partial constraint and flexibility has been observed for variation in eyespots size in the butterfly Bicyclus anynana [12].
Two haplotypes of C. briggsae have been described [44] and variation in ray pattern follows haplotype structure [39]. Strains in haplotype 1, including AF16, exhibit almost exclusively a derived ray pattern. Strains in haplotype 2, including HK104, exhibit derived and ancestral ray patterns at equal frequencies. One interpretation of these results is that full expression of the derived ray pattern never became fixed within haplotype 2. However, a C. briggsae-like ray pattern also has been reported for C. clavopapillata [45], another species within the Elegans-group. C. clavopapillata has not been observed since its first description and some authors have considered it and C. briggsae to be synonymous [34]. If C. clavopapillata is distinct from C. briggsae, the posterior position of ray 3 and its frequent fusion with ray 4 may be ancestral for these two species. This would make the C. elegans-like ray pattern in haplotype 2 an atavistic character. It should be possible to discriminate between these two models either through the characterization of additional C. briggsae strains or through the redescription and molecular characterization of C. clavopapillata.
Regardless of their evolutionary history, haplotypes 1 and 2 provided an entrée to the genetics of morphological variation of ray pattern in C. briggsae. The relatively slight difference between the AF16 and HK104 phenotypes hid a wealth of genetic variation. This was evident in the nearly continuous variation exhibited in RILs for both the position of ray 3 and its fusion with ray 4. This variation included many RILs with phenotypes more extreme than either parental strain. Such transgressive segregation is common in both plants and animals and is thought to arise through fluctuating selection and/or through stabilizing selection at minor QTL when directional selection at major QTL overshoots a phenotypic optimum [46]. The transgressive segregation of ray pattern phenotypes may have resulted from selection on homeotic gene expression patterns. In C. elegans, ray 3 precursor cells are born at the junction of the mab-5 and egl-5 expression domains [23,47]. These homeotic genes are required for the specification of positional identities in the posterior body and tail regions, respectively [47,48] and some mutations in them cause the C. elegans ray pattern to phenocopy that of C. briggsae [22,26]. We have demonstrated a significant transgressive association between allelic variation at Cb-egl-5 and variation in ray pattern. As Cb-egl-5 and Cb-mab-5 are closely linked, variation in either or both of these genes may be responsible for the observed association. Alternatively, linkage to Cb-egl-5 may be coincidental, and ray pattern variation in C. briggsae may not result from variation in homeotic gene expression patterns. The best test of these alternative models will be the high resolution mapping of the allelic variants responsible for ray pattern variation.
Loci not linked to the homeotic gene cluster also must be involved in ray pattern variation. Direct evidence for this was obtained from the mip-1 marker assisted introgression studies. The C. elegans homolog of mip-1, unc-22, is not linked to the the homeotic gene cluster and linkage of mip-1 to Cb-egl-5 and Cb-mab-5 is unlikely. There appear to be at least two loci that affect ray pattern linked to mip-1. One of these affected both the placement of ray 3 and its fusion with ray 4. The other affected only ray fusion. These associations were not transgressive, i.e. the allelic variants linked to mip-1 antagonized the allelic variants linked to Cb-egl-5. If the transgressive segregation linked to Cb-egl-5 does result from allelic variation in one or more homeotic genes, then the antagonistic variation linked to mip-1 may be in genes that regulate homeotic gene expression patterns. A direct test of this hypothesis is possible. Several genes required for proper regulation of homeotic gene expression patterns have been identified in C. elegans [25,26,37,38,49]. C. briggsae homologs of these genes could be tested for association with ray pattern variation in the RILs. Ideally, these tests would be integrated into a genome wide screen for variant loci with effects on ray pattern. This will require the enhancement of genetic resources available for C. briggsae.
Conclusions
Ray pattern in Caenorhabditis provides a powerful model for the study of morphological evolution. Macroevolutionary comparisons between species and microevolutionary analyses of variation within species are possible. Augmenting these approaches is a detailed understanding of the genetic and cellular basis of ray pattern development in C. elegans. In C. briggsae, intraspecific variants have been characterized that affect the expression of ancestral and derived ray patterns. These variants have a complex genetic basis involving multiple genes. Some of these genes exhibit transgenic segregation, some affect all of elements of ray pattern, and some that affect only a subset of ray pattern elements. At least one gene that affects ray pattern variation in C. briggsae is linked to the homeotic gene cluster. Thus, ray pattern variation may result from altered expression patterns of homeotic genes. Further characterizations of the genetics of ray pattern variation will test this model and will address interactions between different genes that impact ray pattern in C. briggsae.
Methods
Strains and strain maintenance
C. briggsae strains AF16, and HK104 are available from the Caenorhabditis Genetics Center [50]. These strains were maintained on agar plates seeded with E. coli strain OP50. Recombinant inbred lines (RIL) were constructed starting with HK104 males mated to sperm-depleted AF16 hermaphrodites [15]. Each RIL was initiated with a single F2 hermaphrodite and propagated through F11, one hermaphrodite per generation.
Microscopy
Ray pattern phenotypes were scored using differential interference contrast optics at a magnification of 400× [5]. Right and left sides were scored independently [39]. Microscopic images of worms anethesized in 0.2% sodium azide were digitally captured using a Spot Camera and Software (Diagnostic Instruments, Inc., Sterling Heights MI).
PCR amplification
Forward and reverse primers for PCR amplifications Cb-egl-5, CAGGGAGCGGACAACTTCAAAGG and GGACACAGCCCAGGATTAGCGAC, respectively, were designed based on genome sequence data from C. briggsae strain AF16 [41]. Amplification products were size fractionated by electrophoresis through 1% agarose.
Statistical analyses
Pearson's product moment correlation cofficients between ray pattern elements in RILs and Wilcoxon nonparametric tests of the association between Cb-egl-5 allelic variation with ray pattern variation were determined online at [51]. Frequency distributions of the segregation of ray 3 positional and fusion phenotypes were compared using the Kolmogorov-Smirnov tests as implemented at [52]. Ray pattern phenotypes of mip-1 introgressed strains were compared using reciprocal chi-squared tests using Excel v10.1.0 (Microsoft, Inc., Redman WA).
Abbreviations
RIL = recombinant inbred line; QTL = quantitative trait loci; dll = distalless; en = engrailed; sal = spalt
Author's contributions
SEB: Planned and supervised all research activities. Participated in construction and genotyping of RILs. Reviewed all statistical analyses. Wrote manuscript.
CRD: Constructed, genotyped, and characterized ray pattern phenotypes of RILs. Statistical analyses of the segregation of ray pattern phenotypes and association of Cb-egl-5 variation with ray pattern variation. Reviewed manuscript.
JCB: Constructed and characterized phenotypes of mip-1-assisted introgressed lines. Statistical analyses of ray pattern phenotypes of mip-1 introgressed lines. Reviewed manuscript.
Supplementary Material
Additional File 1
AF16 × HK104 RIL ray pattern data. Cb-egl-5 genotypes and ray pattern phenotypes of RIL derived from AF16 × HK104.
Click here for file
Acknowledgements
We thank R. Miller for sharing with us his unpublished data and J. Puglise and the reivewers of this manuscript for many helpful comments. This work was supported by NIH R15GM65847.
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| 15634358 | PMC545079 | CC BY | 2021-01-04 16:37:16 | no | BMC Evol Biol. 2005 Jan 5; 5:3 | utf-8 | BMC Evol Biol | 2,005 | 10.1186/1471-2148-5-3 | oa_comm |
==== Front
BMC Med Res MethodolBMC Medical Research Methodology1471-2288BioMed Central London 1471-2288-5-21563894410.1186/1471-2288-5-2Research ArticleSystematic reviews of epidemiology in diabetes: finding the evidence Royle Pamela [email protected] Lynda [email protected] Norman [email protected] Department of Public Health, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland2005 8 1 2005 5 2 2 11 8 2004 8 1 2005 Copyright © 2005 Royle et al; licensee BioMed Central Ltd.2005Royle 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
Methodological research to support searching for those doing systematic reviews of epidemiological studies is a relatively neglected area. Our aim was to determine how many databases it is necessary to search to ensure a comprehensive coverage of the literature in diabetes epidemiology, with the aim of examining the efficiency of searching in support of systematic reviews of the epidemiology of diabetes
Methods
Three approaches were used. First, we defined a set of English language diabetes journals and examined their coverage in bibliographic databases. Second, we searched extensively for diabetes epidemiology articles (in all languages) to determine which are the most useful databases; and third, we analysed the scattering of these articles to determine the core journals in the area.
Results
The overlap between MEDLINE and Embase for diabetes journals was 59%. A search for diabetes epidemiology articles across both MEDLINE and Embase, showed that MEDLINE alone retrieved about 94% of the total articles. Searching for diabetes epidemiology studies beyond MEDLINE and Embase retrieved no additional English language journal articles. The only diabetes epidemiology studies found by searching beyond MEDLINE and Embase were found in LILACS, and were Spanish or Portuguese language studies from Latin America; no additional English language studies were found. Only 30% of the meeting abstracts were converted to full publication after three years. One third of journal articles were published in just six journals, with Diabetes Care contributing 14.3% of the articles, followed by Diabetic Medicine (5.0%); Diabetes Research & Clinical Practice (4.1%); Diabetologia (4.0%); Diabetes & Metabolism (2.4%) and Diabetes (2.0%).
Conclusions
Our results show that when searching for articles on diabetes epidemiology, MEDLINE and Embase would suffice for English language papers, with LILACS giving some additional non-English articles from Latin America. Although a MEDLINE-only search will retrieve the vast majority of the relevant literature, Embase and LILACs should also be searched to ensure the search is comprehensive. Searching for meeting abstracts is recommended to alert reviewers to unpublished work. The low rate of full publication of meeting abstracts has the danger of producing bias in reviews. Our findings on scattering show that the core literature in this field is concentrated in just six journals.
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Background
Review articles have a valuable role in the medical literature, because the volume of journals and articles is such that keeping up to date is very difficult. Reviews are much more valuable if they are systematic reviews done to internationally agreed standards, as non-systematic reviews are known to be subject to bias [1,2].
Dickersin noted that there was a shortage of systematic reviews in epidemiology, and called for more reviews, and more research into the methodology relating to reviews in epidemiology [3]. A study by Breslow found that more than 60% of epidemiology reviews were not methodologically systematic [4], and there has been little methodological research relating to their performance. Also, the methods have not been standardised, and the literature searching has not been supported, as done by the Cochrane Collaboration to support systematic reviews of clinical trials.
One of the key quality criteria for systematic reviews is the comprehensiveness of the searching, as failure to identify all relevant reports can result in selection bias[5]. The usual first source for identifying studies for reviews is MEDLINE, which currently indexes 4780 titles [6] of the estimated 14 000 biomedical titles currently published throughout the world [7]. In addition to searching MEDLINE, it is recommended that an extensive range of additional sources are searched [1].
A study, using Ulrich's International Periodicals Directory as a gold standard, found that a MEDLINE search in psychiatry would retrieve only about half the relevant journals [8]. Similarly, we were interested to investigate the coverage of diabetes journals and diabetes epidemiology articles in medical databases, in order to determine how many databases it is necessary to search to ensure a comprehensive coverage of the literature in diabetes epidemiology.
The scattering of the journal literature in a subject area can provide a useful insight into the number and range of journals needed to capture the key literature in a field. Bradford's Law of Scattering states that on any one subject, a small group of 'core' journals (Zone 1) will provide about one third of the articles on that subject, a medium number of less-core journals (Zone 2) will provide another third, and a large number of peripheral journals (Zone 3) will provide the final third of the articles [9,10].
The aim of this study was to examine the efficiency of searching in support of systematic reviews of the incidence and prevalence of diabetes by providing empirical data to answer the following questions:
1. Which databases index diabetes journals (restricted to English language)?
2. Which databases outside MEDLINE and Embase index diabetes epidemiology journal articles and grey literature?
3. How are diabetes epidemiology articles scattered across the journals, and what are the core journals in this area?
Accordingly, this study was divided into three parts.
(Note: This study is concerned only with searching for the epidemiology of diabetes itself, not with its complications).
Methods
For the purposes of this study, epidemiology articles were defined as studies of incidence or prevalence of diabetes, or of factors affecting those (thereby excluding studies looking at other epidemiological aspects such as mortality). Basic science studies, e.g. biological mechanisms of disease, were not included.
We started from a position that both MEDLINE and Embase should always both be searched, since the overlap between these databases has been estimated to range from 10% to 87% [5,11-17]. Also, it is recognised that many relevant studies will appear in non-diabetes journals, and in sources not indexed in MEDLINE or Embase.
Hence, a three-part approach was used to investigate literature searching to support systematic reviews of diabetes epidemiology, and address each of our aims in turn.
Part one: defining a set of 'diabetes' journals, and determining the databases in which theses journal were indexed
Diabetes journals were identified from the 'Medical Sciences – Endocrinology' section of Ulrich's Periodical Directory 2003 (a comprehensive bibliographic database providing detailed information on periodicals published throughout the world) [18]. This was supplemented with a search of PubList.com [19] for journals with 'diabet' in title.
The inclusion criteria for the journals were: i) the word stem 'diabet' in the title, ii) contains original scientific studies of an academic or scholarly nature, iii) currently in print, and iv) published in English. If inclusion could not be decided on the basis of the information provided in Ulrich's, the contents pages for the past five years, and where necessary, abstracts or the full journal articles, were examined by a diabetes epidemiologist.
Journals which fulfilled all the above criteria were then checked against the List of Serials Indexed for Online Users [6] to see if they were indexed in MEDLINE. To determine if a journal was indexed by Embase, a search was done of 'diabet$' in the Journal Word (JW) field using the Embase OVID search interface, and limiting to publication year 2003.
If any journals were not indexed in either MEDLINE or Embase, searches of BIOSIS, BNI, CINAHL, SCI were done to determine if the journals were indexed in any of these databases.
Part two: searching databases other than MEDLINE and Embase for journal articles and grey literature on diabetes epidemiology
Databases searched were: AMED, ASSIA, BIOSIS (abstracts only), BNI, CINAHL, Conference Papers Index, Dissertation Abstracts US, Health Management Information Consortium (HMIC), Index to Theses UK, ISI Proceedings, PsycINFO, NLM Gateway, LILACS (Latin American and Caribbean Health Sciences), National Research Register (NRR), SIGLE, SCI (abstracts only), SSCI, and Zetoc.
The search strategy used was 'diabetes and (epidemiology or incidence or prevalence)' in the Title (TI) field and restricted to publication years 1998 to 2003.
The titles (and abstracts when available) of all records were checked by an expert in diabetes epidemiology, in order to determine their potential usefulness for those doing systematic reviews.
Part three: investigating the scatter of diabetes epidemiology journal articles found in a search of MEDLINE and Embase, and determining the core journals in this area
MEDLINE and Embase were searched using the search strategy: 'Diabetes Mellitus as the major subject heading, with the sub-heading 'Epidemiology' assigned (which includes incidence and prevalence), and restricted to publication years 1998 to 2003'. All languages were included. Duplicates found in both MEDLINE and Embase were removed. The journal titles in which the articles were found were ranked according to the number of articles contributed by each journal. The cumulated numbers of articles and journals were calculated and plotted. This was used to identify Bradford zones; that is, the number of journals needed to cover about one third, two thirds or all the relevant articles in the field.
Results
Part one: defining a set of 'diabetes' journals, and determining the databases in which theses journal were indexed
Searches of Ulrich's Periodicals Directory and PubList.com initially identified four English language journals that were of potential interest but not indexed by MEDLINE or Embase. On closer inspection, three of these, Clinical Diabetes, Journal of Diabetes Nursing, and Diabetes and Primary Care were excluded as they did not appear to contain any primary research; the articles were mainly educational, professional news and views, opinions, or narrative reviews. The fourth journal, The Diabetic Foot, contained primary research, but did not appear to contain studies useful for epidemiological reviews of diabetes itself (as opposed to complications). It is indexed by CINAHL only.
As shown in Table 1, 27 English language diabetes were covered collectively by MEDLINE and Embase in 2003. Seventy-four percent (20) were indexed in MEDLINE, 85% (23) in Embase, and 59% (16) were in both MEDLINE and Embase.
Table 1 English language diabetes journals indexed in either MEDLINE or Embase in 2003
Indexed in
Acta Diabetologica MEDLINE & Embase
Diabetes MEDLINE & Embase
Diabetes & Metabolism MEDLINE & Embase
Diabetes Care MEDLINE & Embase
Diabetes Research MEDLINE & Embase
Diabetes Research & linical Practice MEDLINE & Embase
Diabetes Technology & Therapeutics MEDLINE & Embase
Diabetes, Nutrition & Metabolism – Clinical & Experimental MEDLINE & Embase
Diabetes, Obesity & Metabolism MEDLINE & Embase
Diabetes/Metabolism Research Reviews. MEDLINE & Embase
Diabetic Medicine MEDLINE & Embase
Diabetologia MEDLINE & Embase
Experimental & Clinical Endocrinology & Diabetes MEDLINE & Embase
Experimental Diabesity Research MEDLINE & Embase
Journal of Diabetes & its Complications MEDLINE & Embase
Pediatric Diabetes MEDLINE & Embase
Current Diabetes Reports MEDLINE
Diabetes Educator MEDLINE
Diabetes Forecast MEDLINE
Diabetes Self-Management MEDLINE
British Journal of Diabetes & Vascular Disease Embase
Canadian Journal of Diabetes Embase
Cme Bulletin Endocrinology & Diabetes. Embase
Current Opinion in Endocrinology & Diabetes Embase
Diabetologia Croatica Embase
Journal of Endocrinology, Metabolism & Diabetes of South Africa Embase
Practical Diabetes International. Embase
The four diabetes journals unique to MEDLINE were all published in the USA. By contrast, only one of the seven journals unique to Embase was published in the USA.
Part two results: searching databases other than MEDLINE and Embase for journal articles and grey literature on diabetes epidemiology
The results are summarised below:
Journal articles (English language)
No English language journals articles that were not also indexed in MEDLINE or Embase were identified.
Journal articles (non-English language)
There were 23 Spanish and Portuguese language articles identified in LILACs. On the basis of the English translation of the titles, they all reported studies done in Latin America.
Grey literature
We defined grey literature as any literature not published in a peer reviewed journal. After removing duplicates, there were 51 dissertations identified from searches of Dissertations Abstracts US, Index to Theses UK, and SIGLE. The research presented in the vast majority (92%) of the dissertations appeared to have been written up as articles in journals indexed in MEDLINE or Embase. No grey literature studies of any format other than dissertations were retrieved from SIGLE, so there was very little additional information gained by these searches.
Research in progress
The National Research Register (a database of ongoing and recently completed research projects funded by, or of interest to, the United Kingdom's National Health Service) gave brief details of 18 projects in progress that had not been otherwise identified. Searching the NRR might be useful if unpublished results could be included in the review, but its main value would be to indicate when the review was likely to need updating.
Meeting abstracts and conference proceedings
The search of the Conference Proceedings Index retrieved 25 articles, none of which appeared to have been published as journal articles after five years. The Zetoc Conference Search found eight articles, of which 50% had been published as full journal articles in MEDLINE or Embase.
The search of Science Citation Index (SCI), restricted to meeting abstracts only, found 171 relevant studies. The time to publication of the SCI abstracts was examined by checking how many had subsequently been published as journal articles indexed in MEDLINE or Embase. It was found that 30% had reached full publication after three years.
A search of BIOSIS, restricted to meeting abstracts only, retrieved 71 additional relevant abstracts that were not in SCI. Most (65%) of these 71 abstracts came from the supplements of Diabetes and Metabolism and Diabetes Research and Clinical Practice. Of these, 11 (12%) had been published in journals. The average time delay from the date of publication of the abstract to full publication was 1.4 years.
Databases searched where no articles not in MEDLINE or Embase were found
These included AMED, BNI, HMIC, NLM Gateway meeting abstracts, PsychINFO, and SSCI,
In summary, the data indicate that when searching for English language journal articles on diabetes epidemiology, searches of MEDLINE and Embase would suffice. The exception would be for studies from Latin America, where LILACS should also be searched. Searching for meeting abstracts may alert reviewers to forthcoming or unpublished work.
Part three: investigation of the scatter of diabetes epidemiology journal articles found in a search of MEDLINE and Embase, and determination of the core journals in this area
The searches for diabetes epidemiology articles in MEDLINE and Embase resulted in 2923 articles being found in 696 different journal titles; 39% were found to be in 'diabetes journals' and 14% were non-English language.
Figure 1 shows the distribution of all 696 articles retrieved across the journals.
Figure 1 Distribution of diabetes epidemiology articles across journals
Applying Bradford's Law of Scattering gives three zones, each providing one-third of the articles.
Zone 1
The first one-third of articles were from six journals. They are in rank order: Diabetes Care (contained 14.3% of the articles); Diabetic Medicine (5.0%); Diabetes Research & Clinical Practice (4.1%); Diabetologia (4.0%); Diabetes & Metabolism (2.4%), Diabetes (2.0%) These six journals represent 0.9% (of the 696) total journals, and all are indexed in MEDLINE.
Zone 2
The second one-third of articles were from 62 journals, representing 9.1% of the total journals. The four journals in Embase only were: Practical Diabetes International; Diabetologia Polska; Diabetes und Stoffwechsel and Journal of the Japan Diabetes Society. Hence, 94% of Zone 2 journals are covered by MEDLINE.
Zone 3
The final one-third of articles were in 628 journals, representing 90.2% of the total journals. MEDLINE indexed 88% of these journals.
Overall, for the three zones, the search of MEDLINE and Embase for diabetes epidemiology articles revealed that MEDLINE indexed 89% of the total journals, and these contained 94% of the articles.
Discussion
Our results showed that there was an overlap of only 59% in current English language 'diabetes journals' indexed by both MEDLINE and Embase. Also, a search for diabetes epidemiology articles across both MEDLINE and Embase showed that MEDLINE alone retrieved about 94% of the total articles; therefore, both databases should be searched. Embase appears to index more diabetes journals published outside the USA. Therefore, if searching is limited to MEDLINE only (Embase being less accessible and more expensive than MEDLINE, which is free via PubMED) this could potentially introduce a bias. Also, duplication of searching can be useful, as due to differences in indexing practices, a search of one database may retrieve something missed by the other.
We also found that despite a wide range of additional databases searched after MEDLINE and Embase, no additional English language journal articles on diabetes epidemiology were identified. The LILACS database was a useful source of Spanish and Portuguese language articles on the epidemiology of diabetes in Latin American countries.
Meeting abstracts appeared to be valuable sources of information on forthcoming studies, but their inclusion in systematic reviews is contentious. Some reviewers exclude abstracts on the grounds that the quality of the study cannot be judged because of the inevitably limited detail. However, others include them on the grounds that abstracts provide the most up-to-date information.
Nearly all the dissertations identified had been published as journal articles. However, it was found that only 30% of the meeting abstracts were converted to full publication after three years, which is considerably lower than the figure for RCTs, which is 56% [20]. This has the danger of producing bias in systematic reviews, if failure to publish is based on the size and direction of study results.
Scattering of the diabetes epidemiology articles revealed that the 'core' literature in this field is concentrated in just six journals, with Diabetes Care alone containing about 14% of the articles. A similar concentration effect in journals was also shown in a study of 3400 science journals in the SCI database, where just 100 journals accounted for 22% of the published articles and 100 journals also accounted for 44% of cited articles [21].
This study has a number of limitations. The search to identify diabetes journals was restricted to English language journals only, as we were unable to assess articles in other languages. We did not compare the quality of the articles identified from databases outside MEDLINE and Embase. Also, when searching for articles, we were necessarily limited to the range of databases available to us.
Finally, there may be databases inaccessible or unknown to us that cover foreign language and regional journals not indexed in MEDLINE and Embase. Such journals may carry studies of incidence which may seem of primarily local interest, but which may be useful contributions to the international body of evidence because they may show large variations in incidence, or in its relationship to possible aetiological risk factors.
It is often useful to study the epidemiology of a disease where it is rare, as well as where it is common. However it is likely that studies which report high incidence are more likely to be published than those which report low incidence. Similarly with risk factors; a study which finds no link between factor x and disease y may be less likely to be published than one which does show a correlation [22,23].
There is a need for further research to see whether our findings apply to searching for epidemiological reviews of other diseases, and on measuring the sensitivity and specificity of various search filters to retrieve epidemiological studies in MEDLINE and Embase.
We endorse Dickersin's suggestion of an international collaborative effort to establish an 'epidemiological Cochrane-like database' to identify all relevant studies and to begin systematically reviewing available data for important epidemiological questions [3].
Conclusions
Searching MEDLINE and Embase appears to provide comprehensive coverage of the English language journal literature in diabetes epidemiology. LILACs is a useful source of Spanish and Portuguese language articles on diabetes epidemiology done in Latin American countries and published in regional journals not indexed in MEDLINE and Embase. Searching for meeting abstracts is recommended to alert reviewers to unpublished work.
The volume of literature on diabetes epidemiology makes it impossible for one person to read everything. However the provision of systematic reviews makes keeping up with research manageable, and more reviews are needed. Our findings on scattering shows that the core literature in diabetes epidemiology is concentrated in a small number of core journals, and that in the absence of reviews, one can follow the field by reading these journals. It may also be reassuring that a good MEDLINE-only search will retrieve the vast majority of the relevant literature.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
PR and NW conceived the study and drafted the initial manuscript. PR and LB collected and analysed the data. All authors contributed to and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We wish to thank Elizabeth Payne for help with literature searching
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| 15638944 | PMC545080 | CC BY | 2021-01-04 16:32:52 | no | BMC Med Res Methodol. 2005 Jan 8; 5:2 | utf-8 | BMC Med Res Methodol | 2,005 | 10.1186/1471-2288-5-2 | oa_comm |
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BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-4-681562500410.1186/1471-2458-4-68Research ArticleAn assessment of factors contributing to treatment adherence and knowledge of TB transmission among patients on TB treatment Kaona Frederick AD [email protected] Mary [email protected] Seter [email protected] Lenganji [email protected] Mwengu Social and Health Research Centre, 12 Kafupi Road, Plot number 1430/130, Northrise P.O Box 73693, Ndola, Zambia2 University of Zambia School of Medicine, Department of Community Medicine, P.O Box 50110, Lusaka, Zambia3 Ministry of Agriculture Food and Fisheries Headquarters, Box RW 50291, Lusaka, Zambia2004 29 12 2004 4 68 68 11 6 2004 29 12 2004 Copyright © 2004 Kaona et al; licensee BioMed Central Ltd.2004Kaona 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 treatment guidelines for tuberculosis treatment under Directly Observed Treatment, Short-course (DOTS) have been a common strategy for TB treatment in Zambia. The study was carried out in Ndola, Zambia, to investigate factors contributing to treatment non-adherence and knowledge of TB transmission among patients on TB treatment, in order to design a community-based intervention, that would promote compliance.
Methods
A household-based survey was conducted in six randomly selected catchment areas of Ndola, where 400 out of 736 patients receiving TB treatment within the six months period, were recruited through the District's Health Management Board (DHMB) clinics. All patients were interviewed using a pre-tested structured questionnaire, consisting of i. Socio-demographic characteristics ii. Socio-economic factors iii. Knowledge about TB transmission and prevention iv. Patterns in health seeking behaviour and v. TB treatment practices at household level.
Results
Most male TB patient respondents tended to be older and more educated than the female TB patient respondents. Overall, 29.8% of the patients stopped taking their medication. There were 39.1% of the females and 33.9% of the males, who reported that TB patients stopped taking their medication within the first 2 months of commencing treatment. Age, marital status and educational levels were not significantly associated with compliance. The major factors leading to non-compliance included patients beginning to feel better (45.1% and 38.6%), lack of knowledge on the benefits of completing a course (25.7%), running out of drugs at home (25.4%) and TB drugs too strong (20.1% and 20.2%). There was a significant difference [OR = 1.66, 95% CI 1.23, 2.26] in TB knowledge, with more males than females reporting sharing of cups as a means for TB transmission, after adjusting for age, marital status and educational levels. Significantly [p = 0.016] more patients who had resided in the study for less than two years (59%) were more likely to report mother to child transmission of TB, compared to 41.2% of those who had been in the area for more than 2 years.
Conclusion
This study established that 29.8% of TB patients failed to comply with TB drug taking regimen once they started feeling better.
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Background
Tuberculosis is one of the major public health risk factors of high mortality among patients in Zambia. The prevalence of tuberculosis among adults has more than doubled since the onset of AIDS epidemic in Zambia [1], with most of the cases often related to HIV/AIDS conditions. Because of the advent of AIDS epidemic in the country, tuberculosis cases have increased to over 500/100,000 population [2]. Due to AIDS, it has been projected that TB cases would reach 38,000 by the year 2004 in Zambia [3]. A multisite study conducted in Ndola in 1997 [4] revealed that HIV prevalence was as high as 42%, and in a sub-sample of women attending antenatal clinics (ANC), HIV prevalence rate was 28.4% [5]. Data from Ndola and other Zambian communities revealed that the number of TB cases had risen five-fold since 1996 [6]. Because of the communities' health seeking behaviour and their perceived causes of TB among patients, those suffering from TB would transmit the disease to others and merging drug-resistance strains would make it even more difficult to limit the spread of the infection [1,3].
Although tuberculosis treatment guidelines had been circulated for the management of the disease at health facility down to household level, DOTS was the only best means of increasing compliance among TB drug-taking patients in Zambia [1,7]. As early as 1980s, the African countries had embraced and implemented the WHO recommended DOTS strategy, which was meant to standardize methods for case detection, case management and monitoring [8]. DOTS as a strategy, entails that medication is taken in while the care provider is observing the patient swallowing the drug [9]. Diagnosing TB patients at a health facility and sending them to continue taking their treatment at home, poses serious challenges for other members of the households who assist the patients with treatment.
Work carried out in Haiti [7], Zambia [10] and most other rural communities [11] present the challenges facing compliance with TB treatment. Tuberculosis like HIV/AIDS is often associated with stigmatization and thus may create resistance among patients to treatment. A study carried out in Nigeria [12], raised an important point of delays in care seeking behaviour due to stigma experienced by TB patients. Studies [13] have shown that stigmatization creates a lot of self-denial among those with diseases like TB and Sexually Transmitted Infections (STIs); hence most of them fail to comply with the treatment regime.
Issues of diverse nature to caring of patients at home are rampant in communities that have low literacy levels [13,14]. The possibility of being exposed to tuberculosis by merely associating with patients may create some resentment by those in the household to providing proper care of the patient and encourage non-compliance [15,16]. In their studies [4,15,18], researchers found that fear of catching the disease was a factor in household members' negative reactions to care of the TB patient. The high incidence of chronic TB-related problems among the patients increases the probability that care of TB patients becomes costly at home, in terms of food provision and other essentials [18,19]. While TB treatment lasts between 6 and 8 months, the effective treatment of multidrug resistant may be more pronounced in low-income communities and can even be more complex if not properly supervised.
Discrimination on the basis of disease at health facilities sometimes exacerbates problems with compliance with tuberculosis drug taking behaviour [19]. Unless there is privacy during drug collection schedules, patients may resist going to collect their drug supplies at the clinic because of discriminatory behavior by health care providers. Some studies that have been carried out in Africa and other parts of the world [15,19] found that health care providers discriminated against AIDS patients. The type of language used at both the health facility and the homes has a strong bearing on the reactions patients may have and their compliance with treatment. Where TB is perceived to be AIDS, a disease that is frightening and a lurking cause of premature death, those associated with it may sometimes withdraw themselves from the rest of the community because, they believe they have reached the end of their lives.
More often than not, when patients are diagnosed as having TB, communities immediately construct them as social and sexual misfits in the society, which is often followed by exclusion from social interactions and relationships [20]. In homes where care is given, a patient may experience feelings intense loneliness and abandonment. A study among the Nigerian medical students [21] revealed that the majority of the students considered AID a divine punishment for sexual excess. In order to try and improve community participation, organisations such as WHO have developed strategies to enhance TB/HIV/AIDS control [22]. This paper presents factors contributing to treatment adherence and knowledge of TB transmission among patients on TB treatment.
Methods
Study design and population
A cross sectional study was conducted among tuberculosis patients who were getting their drugs from the clinics operational in Ndola, Zambia by the Ndola District Health Management Board. In this study, compliance refers to patients who took their TB drugs daily for 8 months. On the other hand, any patient who stopped taking TB drugs during the treatment period was regarded as non-compliant patient. This study took into account an analytical approach of comparing two groups (the compliant against the non-compliant group). The study was limited to these patients because of the research focus on compliance of patients over time to prescribed TB drug regimens identified from medical records. In addition to medical records, TB patients were also directly asked if they had ever stopped taking the drugs since starting the treatment.
Sample
Using the medical records from the six sampled Ndola District Health Management Board Clinics, a list of all TB patients seeking treatment within the six months period was drawn. A total of 736 patients were listed from the six study sites. Using this figure, and a prevalence of 50% +_5% at 95% confidence level, the minimum sample size was determined to be 248. Although 400 TB patients were recruited in the study, 18 were excluded from the analysis because only demographic data was obtained. Reasons for incompleting questionnaires included some patients becoming restless and rushed to the hospital, while others were too sick to continue with the interview and had died by the re-appointment date.
TB patients were classified as follows: those who never attended the TB-clinic after diagnosis, those who attended 1–2 times after diagnosis and commencement on treatment, and those who attended regularly (i.e. 3 or more times) after diagnosis and continuation of treatment. Patients who attended the clinic regularly and continued taking the treatment were classified as controls, and the rest of the patients were classified as cases.
Out of 18 clinics that were run by the District Health Management Board in Ndola, six were randomly selected from the list of clinics so as to enable the team obtain a representative sample of the clinics in the district. In the sampled clinic catchment areas, a household-based survey was conducted and all patients that met the criteria were invited to take part in the study. In all, 114 non-compliant and 268 treatment-compliant TB patients participated in the study.
Ethical considerations
TB is strongly associated with HIV/AIDS because of the perceived mode of transmission, which stigmatises and discriminates patients. These are common in most African communities [24] and Zambia in particular [25]. TB like HIV/AIDS becomes difficult to discuss in public. Looking at the sensitive nature of the study, all patients were assured of confidentiality and anonymity. Information on patients, residential addresses, and health facilities to which patients were affiliated were collected for the purpose of follow-up. Respondents were informed that this information would not be made available to persons outside the study team. Respondents were further assured that no person-identifiers would be used for publication.
Data collection, management and analysis
Those who had agreed to participate were interviewed using a pre-tested structured survey instrument. Data collected were individual socio-demographic characteristics and economic characteristics, knowledge on TB transmission, prevention and treatment practices, patterns in health seeking behaviour, compliance and non-compliance to TB drug taking, and perceived community support during the drug taking period.
The data was entered in EPI6. It was edited using consistency and range checks after data entry. However, data analysis was done in the statistical package, SPSS. The Chi-squared Yates corrected test for 2 by 2 tables and Pearson's uncorrected Chi-squared test for higher contingency tables were used to determine associations between qualitative factors. A multivariate logistic regression model was used to adjust for differences in the distributions of age, marital status and education between males and females in the relationships between knowledge items and gender. The cut off point for statistical significance was set at the 5% level.
Results
The demographic characteristics of household-based patient population that participated in the survey are presented in Table 1. Information on gender was not recorded for two respondents. Most of the male patient respondents (44.0%) were in the age group 30–39 years, while the majority of female patient respondents (38.2%) were of age 20–29 years (p = 0.001). Among women, TB patients were 2.04 times more likely to be in the age group 10–19 years, compared to males in the same age group. Significantly more females than males were either divorced/separated or widowed (p < 0.001). In terms of educational attainment, males tended to be more educated than females (p < 0.001).
Table 1 Socio-demographic characteristics of Patients by Gender
Total Male Female
Demographic characteristics n % N % n %
AGE [p = 0.001] (years) Total = 379 Total = 193 Total = 186
< 10 22 5.8 10 5.2 12 6.5
10–19 20 5.3 5 2.5 15 8.1
20–29 116 30.6 45 23.2 71 38.2
30–39 145 38.3 85 44.0 60 32.3
40–49 47 12.4 31 16.1 16 8.6
50+ 29 7.7 17 8.8 12 6.5
MARITAL STATUS [p < 0.001] Total = 379 Total = 194 Total = 185
Married 168 44.3 108 55.7 60 32.4
Single 77 20.3 43 22.2 34 18.4
Divorce/Separated 69 18.2 25 12.9 44 23.8
Widowed 65 17.2 18 9.3 47 25.4
EDUCATION [p < 0.001] (Years in school) Total = 342 Total = 178 Total = 164
0–4 52 15.2 21 11.8 31 18.9
5–7 146 42.7 60 33.7 86 52.4
8–9 73 21.3 42 23.6 31 18.9
10+ 71 20.8 55 30.9 16 9.8
Table 2 shows only one significant difference in knowledge of TB transmission by gender. Significantly more males than females stated that sharing of cups as a means of transmitting TB (27.3% of males and 17.2% of females; p = 0.025). Males were still more likely to hold this belief even after adjusting for age, marital status and education.
Table 2 Knowledge on TB Transmission by Gender
Male Female
Total = 194 Total = 186
Sources of transmission n % n % p value OR (95% CI)*
Through sexual intercourse 24 12.4 25 13.4 0.875 0.91 (0.62, 1.31)
From mother to child 71 36.6 77 41.4 0.393 0.93 (0.73, 1.19)
Sleeping in same room with TB patient 10 5.2 7 3.8 0.684 1.03 (0.58, 1.82)
Sharing cups 53 27.3 32 17.2 0.025 1.66 (1.23, 2.26)
Patient coughing directly at others 15 7.7 21 11.3 0.313 0.65 (0.42, 1.02)
* OR (95%CI) [Odds Ratio and 95% confidence interval] adjusted for age, marital status and education
No significant differences by duration of residence in the community were observed in knowledge of TB transmission through sexual intercourse, sleeping in the same room with TB patient and patient coughing directly at others (Table 3). Significantly (p = 0.016) more patients with duration of stay of less than two years in the community (59.3%) reported that TB could be transmitted from mother to child compared to 41.2% of patients who had stayed for two or more years in the compounds. Furthermore, significantly (p = 0.019) more patients who had stayed for at least two years in the compounds (27.4%) stated that TB could be transmitted through sharing of cups compared to 11.9% of patients who had stayed in the compounds for less than two years.
Table 3 Associations between knowledge of TB transmission and duration of stay in the compound
Duration of stay in compound
Knowledge of TB transmission <2 years 2+ years p value
Total = 59 Total = 277
n % n %
Through sexual intercourse 9 15.3 40 14.4 0.966
From mother to child 35 59.3 114 41.2 0.016
Sleeping in the same room with TB patient 3 5.1 13 4.7 1.000
Sharing cups 7 11.9 76 27.4 0.019
Patient coughing directly at others 5 8.5 30 10.8 0.762
A total of 114 (29.8%) out of 382 patients stopped taking TB drugs at some point during the treatment regimen. Analyses for the factors associated with TB drug compliance revealed that sex, education, marital status, sharing a room with any one else, relationship with head of household, anyone suffered from TB in the house, close relative or friend that has suffered from TB, number of times suffered from TB, and use of traditional healers for treatment of TB were not significantly associated with compliance.
The common reason given for stopping treatment by both the compliant and the non-compliant patients were that they could not continue with the medication when they started feeling well (45.1% and 38.6%), respectively. Meanwhile, other reasons given by complaint patients were lack of knowledge on the benefits of completing TB course (25.7%), TB drugs too strong (20.1%) and lack of food in the home (11.4%). Similarly, the non-compliant patients mentioned running out of drugs at home (25.4%), TB drugs too strong (20.2%) and loss of hope to live (16.7%) as reasons for stopping.
Common reasons given for stopping treatment for male patients were that they ran out of drugs at home (29.1%) or had no food (23.6%). Meanwhile, most females reported that forgetting to take the medicine (32.0%), reaction to drugs (20.0%), and running out of drugs at home (20.0%) as the reasons for stopping taking drugs (Table 4).
Table 4 Perceived Reasons Given by Compliant and Non-compliant Patients Leading to Stoppage of TB Drug Taking
Reason Compliant Non-compliant
n = 268 % n = 114 %
Once they start feeling better 121 45.1 44 38.6
Lack of knowledge on the benefits of completing a course 69 25.7 14 12.3
Running out of drugs at home 15 5.6 29 25.4
TB drugs too strong to continue 54 20.1 23 20.2
Lack of food 40 14.9 13 11.4
Loss of hope to live 30 11.2 19 16.7
Lack of drugs at the clinic 5 1.9 5 4.4
Denial of suffering from TB 3 1.1 6 5.3
Doctors advice 0 0.0 2 2.0
Figures cannot add up because the question allowed for multiple responses
Results from the qualitative study reveal that 10 out of 17 non-compliant patients stressed running out of drugs as a reason for stopping. A young man who was a widower aged 32 years reported:
Okay, the drugs got finished but like I am not feeling well, that is why I don't go to collect, because here, there are no children you send. My nephew also has started what? The one who used to get it for me [referring to the nephew who had started work]
In another in-depth interview with a single non-compliant female patient aged 31 years, emphasised on running out of drugs at home and reported thus:
That one we drink two tablets, I..., we went on Monday, they said the Doctor was not there [referring to the clinical officer or nurse responsible for running the TB clinic]... he comes on Wednesday, so I have missed also.
The key determinant for stopping medication in both qualitative and quantitative was running out of drugs.
No significant gender differences existed at the stage at which most TB patients stopped taking drugs (p = 0.743). About a third of the respondents (33.9% males and 39.1% females) indicated that they stopped taking drugs within the first two months of starting treatment. However, by the 5th month of consistently taking medication, more men tended to stop taking their medicine (Figure 1). Most of the patients received the drugs from health facilities which were normally very close to their residences. Easy access to drugs created one more problem; where individuals failed to follow the drug schedules; they moved to the next health facility in the other neighbourhood and started their medication. In the new place, they gave false addresses as well as different names from those they were known in their neighbourhood. These are interesting findings that highlight why compliance continues to be difficult in most of the communities.
Figure 1 Months since starting treatment at which most TB patients stopped taking drugs Male Female
Discussion
The Ndola study showed that 29.8% of all TB patients on treatment did not comply. The majority of patients who participated in the study were between 20 and 39 years. This population is in agreement with other studies that had been conducted in Ndola [4,5,24]. It was striking to note that females in the age group 10–19 years were more likely to suffer from TB than their male counterparts in the same age category. Interestingly, this result is consistent with findings from some HIV/AIDS studies conducted in Zambia, which showed higher prevalence of HIV among females in this age group [1,3]. In terms of marital status, more males were identified as divorced/separated or widowed. The educational profile in this study was identical to that of the general population in Zambia [17,21,27]. Most patients had attended primary level of education, with more male patients in the majority. However, there was no relationship between the individuals' level of education and TB infection rates. TB is a serious public health issue that needs continued attention.
It has been demonstrated in other studies [22,26] that the TB epidemic may have changed the population's economic needs, resulting in forcing more young men and women to be exposed to situations that place them at higher risk for TB transmission, for instance, sharing overcrowded rooms even when the situation does exacerbate the risk of TB transmission.
Knowledge about TB transmission
No relationship has been found between marital status of an individual and the knowledge of TB. Lack of knowledge makes TB a serious public health problem, which needed an urgent attention. TB patients in Ndola could have continued sharing a room with an infected person, because of lack of accomodation, high rental costs and inadequate knowledge on TB transmission. Over 70% of TB patients easily mentioned the symptoms which included sweating at night, loss of weight, loss of appetite and prolonged coughing, which at times was accompanied by sign of blood in the sputum. Knowledge about the symptoms of the disease did not vary according to levels of education or age.
Knowledge modes of transmission and methods of prevention required revisiting, as community members seemed to have had knowledge that did not relate the disease to the environment (that TB could be transmitted through the air). They believed in physical contacts with objects, for instance, sharing of cups, having sexual intercourse with TB patient and from mother to child. This result is consistent with findings from a study in Botswana [19], which reported that TB was a result of pollution from breaking taboos.
It is not surprising therefore, to find no gender differences in terms of TB knowledge transmission among the age groups. The low knowledge in preventive measures in this group could be explained through our study sample itself. Although these age groups were of patients who could have received the information from the clinic during the time they were collecting drugs and their food supplies, which were often provided to patients, prevention did not seem to have been stressed by health care providers. The other minor reason could be that our questions were prompted; this could also explain why 13.3% reported that to prevent TB one should always keep to one partner. The care seeking behaviour remained the same as in other studies [25,26], where the majority of patients went to specialised health facilities once they were sick.
Duration of residence
The patients' duration of residence in the city of Ndola compared very well with another study [26], which found the median period for most women to be 10 years. It has been shown in this study that duration of residence was associated with the knowledge of tuberculosis transmission.
However, the study had identified some misconceptions on TB transmission, about a third of the respondents reported having got the diseases through sexual intercourse. This study suggested a relationship between community constructions of TB and HIV/AIDS campaigns. Patients could have related TB with HIV and selected sexual intercourse as a source of transmission for the disease, and therefore considered condom use as a means for preventing it. One explanation for this misconception could be attributed to the overstressing that TB is an opportunistic disease for HIV/AIDS. The study in Haiti [9], clearly showed that patients who were diagnosed as TB patients ended up being HIV positive. This misconception was critical in changing people's behaviour and therefore required an urgent intervention.
Non-compliance
Non-compliance to TB drug taking is reasonably lower than we originally thought. There were 29.8% of the patients who stopped taking their medicine at two months after commencing treatment. The defaulting progressed in the second month (16.8%), and then stabilized thereafter. Factors associated with defaulting in the current study, included patients beginning to feel better, lack of knowledge on the benefits of completing a course, running out of drugs at home and TB drugs being too strong to continue. However, the major and stricking determinant of non-complaince was the patient beginning to feel better.
While lack of food and other provisions were identified in other studies [17], as factors associated with defaulting, in this study lack of food did not come out as an issue. This could be due to the fact that the DHMB through the health facilities distributes food portions (herps, mealie meal, beans and cooking oil) as complimentary food availability at household level for TB patients. In the study at Lusaka University Teaching Hospital [28], it was found that over 45% of the TB patients could not comply with drug treatemnt instructions for various reasons, including poor transportation to the hospital and lack of family support.
Over 79% of the patients, had suffered from TB only once, while the rest had suffered more than twice. Results present a wide range in the percentage of opposing views for TB as an air borne disease. Within age groups, there was usually no relationship between the documented preventive measures against TB and air borne germs. The reasons were attributed to the fact that these were patients obtaining their drugs from a health facility, hence, received some of the information on disease transmission. The relationship between the awareness 'coughing blood' and 'TB' was explored during the health talks.
Conclusion
The findings in the Ndola study showed that approximately 39.8% of all TB patients on treatment did not adhere to their treatment schedules, when they started feeling better.
Competing interests
The author(s) declare that they have no competing interests.
Authors contributions
FADK was the principal investigator of the study and was responsible for the design, implementation and supervised data entry and cleaning. He worked closely with the biostatistician during data analysis. He is the principal author of this paper. MT was the co-investigator of the study. She contributed to the design of the study, coordinated data collection, entry and cleaning. She was part of the data analysis team. SS is a co-author of this puplication who carried out data analysis as a biostatistician. LS co-authored this publication and was responsible for data entry and cleaning. He was part of the data analysis team. All authors have read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgement
Authors wish to express their gratitude to all the doctors and nurses in the clinics where the study took place, for their support during the study period. Without their assistance, information would have been very difficult to collect. We wish to extend our gratitude to Dr. Ann Biddlecom, who willingly accepted to read through our manuscript. We are indebted to our research team: Chipila Smith Katewile, Harry Fileshimself Banda, Joyce Mulenga, and Sharon Chonya for hard work and commitment to duty. We would like to thank all the patients and their caretakers for participation in the study. This investigation received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), Geneva-Switzerland.
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| 15625004 | PMC545081 | CC BY | 2021-01-04 16:28:48 | no | BMC Public Health. 2004 Dec 29; 4:68 | utf-8 | BMC Public Health | 2,004 | 10.1186/1471-2458-4-68 | oa_comm |
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BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-5-2031560691510.1186/1471-2105-5-203Methodology ArticleA power law global error model for the identification of differentially expressed genes in microarray data Pavelka Norman [email protected] Mattia [email protected] Caterina [email protected] Monica [email protected] Andrea [email protected] Francesca [email protected] Paola [email protected] Department of Biotechnology and Bioscience, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy2004 17 12 2004 5 203 203 16 7 2004 17 12 2004 Copyright © 2004 Pavelka et al; licensee BioMed Central Ltd.2004Pavelka 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
High-density oligonucleotide microarray technology enables the discovery of genes that are transcriptionally modulated in different biological samples due to physiology, disease or intervention. Methods for the identification of these so-called "differentially expressed genes" (DEG) would largely benefit from a deeper knowledge of the intrinsic measurement variability. Though it is clear that variance of repeated measures is highly dependent on the average expression level of a given gene, there is still a lack of consensus on how signal reproducibility is linked to signal intensity. The aim of this study was to empirically model the variance versus mean dependence in microarray data to improve the performance of existing methods for identifying DEG.
Results
In the present work we used data generated by our lab as well as publicly available data sets to show that dispersion of repeated measures depends on location of the measures themselves following a power law. This enables us to construct a power law global error model (PLGEM) that is applicable to various Affymetrix GeneChip data sets. A new DEG identification method is therefore proposed, consisting of a statistic designed to make explicit use of model-derived measurement spread estimates and a resampling-based hypothesis testing algorithm.
Conclusions
The new method provides a control of the false positive rate, a good sensitivity vs. specificity trade-off and consistent results with varying number of replicates and even using single samples.
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Background
DNA microarrays have become common tools for monitoring genome-wide expression in biological samples harvested under different physiological, pathological or pharmacological conditions. One of the most challenging problems in microarray data analysis is probably the identification of differentially expressed genes (DEG) when comparing distinct experimental conditions. In spite of its biological relevance, there is still no commonly accepted way to answer this question.
An ideal DEG identification method should limit both false positives, i.e. genes wrongly called significant (type 1 errors), and false negatives, i.e. genes wrongly called not significant (type 2 errors). To this end, understanding how gene expression values measured in replicated experiments are spread around the true expression level of each gene, would help to distinguish biologically relevant gene expression changes from fluctuations due to different sources of variability that are unrelated to the biological phenomenon under investigation. Measurement error estimates can be obtained in two ways: either by empirically inferring noise from highly replicated data or by deducing noise from a theoretical error model [1]. Especially when the experimental design requires the investigation of a high number of conditions, the former strategy is not always feasible, because of the high cost of these experiments or due to the availability of biological material. In addition, there is still a lack of consensus on how gene expression values from replicated experiments should be theoretically distributed, which restricts the application also of the latter strategy.
The most widely used methods for identifying DEG range from purely empirical filtering techniques (e.g. selecting genes that show a fold change higher than a fixed threshold) to more sophisticated statistical tests such as the signal-to-noise ratio described by Golub et al. [2] or the Significance Analysis of Microarrays (SAM) method by Tusher et al. [3]. While empirical filtering techniques rely on arbitrarily chosen thresholds and are unable to provide any type of control on the significance of the results, the more sophisticated statistical tests usually need a high degree of replication in the data to accurately measure gene-specific variability.
In the past years various authors have proposed competing error models for microarray data from which discordant implications for the variance versus mean dependence can be deduced. Chen et al. [4] first proposed a simple Gaussian model, more recently Ideker et al. [5] and Li and Wong [6] introduced two-component models containing a multiplicative and an additive error term. All of these models implicitly or explicitly assume a constant coefficient of variation (CV), implying that standard deviation should vary proportionally with the mean. More recently, Rocke and Durbin [7] proposed a variation of the two-component model from which they derived that variance of repeated microarray measures is a quadratic function of the mean. Dealing specifically with spotted cDNA microarray technology Baggerly et al. [1] proposed a beta-binomial model, from which it can be derived that variance is a second-order polynomial function of the mean. Unfortunately, most of these models are based on theoretical assumptions that have been verified on simulated data or on data sets consisting of small numbers of replicates. More recently, Tu et al. [8] empirically modeled the variance versus mean dependence from a data set consisting of ten replicated oligonucleotide microarray experiments. According to the authors, the variance of the genes should decay exponentially with the mean, but only for moderately expression values. Taken together, all these aspects could limit the applicability of these error models.
Independently from the choice of the error model, another point that remains to be faced is on how to estimate residual error. A discussed by Wright et al. [9] the possibilities range between two extremes: either obtaining a single variance estimate across all genes or obtaining a gene-specific residual variance. In the same paper a hybrid approach is proposed in which information from all genes is used to fit a single linear model from which the gene-specific variance estimates can be deduced. In the present work we chose to follow an approach similar to the latter.
The aim of this study was to use highly replicated microarray data to empirically determine the true variance versus mean dependence that exists in this type of data. This knowledge enabled the proposal of PLGEM as a simple but powerful error model. We fitted the proposed model on various data sets without pre-filtering the data, deriving an improved test statistics and identifying DEG even in data sets with very little number of replicates.
Results
Variance versus mean dependence
The relationship between measurement variability and average expression values was investigated by means of scatter plots where different measures of spread were displayed against different measures of location. For each gene absolute or relative standard deviation was plotted against the mean expression value in either linear or log-log plots using data from the 16iDC data set (Figure 1). Independently from the choice of standard deviation or inter-quartile range as the estimate of spread and of mean or median as the location estimate we obtained qualitatively similar plots (data not shown). Log-log plots of both absolute and relative spread estimates revealed a strikingly linear dependency, indicating that measurement spread could depend on signal location following a power law.
Figure 1 Relationship between measurement variability and mean expression level. For each of the 12488 probe sets displayed on Affymetrix MG-U74Av2 chips the standard deviation (st. dev.) or the coefficient of variation (CV = st. dev. / mean) is plotted against the mean in either linear or log-log plots based on absolute expression values derived from the 16iDC data set.
The power law global error model
Based on the previous observation, we chose to empirically model measurement noise through linear regressions:
where s and respectively represent standard deviation and mean of repeated measures. Error term ε is the realization of a random variable E that we will show later to be normally distributed as assumed when fitting a linear model. Inspired by the previous experimental observations we propose the following power law global error model (PLGEM):
Model parameters α and β can be estimated from linear regression coefficients in 1 in a straightforward way:
α = ec eqn. 3
β = k eqn. 4
PLGEM fitting method
Instead of performing a simple linear fit through the whole set of points, we preferred to implement a method that could provide improved model robustness by partitioning the data to gain local estimates of spread as in Mutch et al. [9]. Most importantly, this method should also provide the possibility to choose different levels of confidence when modeling the spread of the data. Note that Mutch et al. [9] proposed to model within-replicates fold changes as a function of average expression using a model that was very different from PLGEM. Therefore, the following algorithm was applied:
• Rank genes according to their ln() value and subdivide the overall expression range into a given number p of partitions containing an equal number of ranked genes.
• Choose a "modeling quantile" q and determine for all the genes contained in each partition a single "modeling point" with median of ln() values as the x-coordinate and q-th quantile of ln(s) values as the y-coordinate.
• Finally, find a linear fit through the set of p modeling points using the least-squares method and obtain a slope k and an intercept c of the resulting regression function.
Thus, for all possible combinations of p and q a slope kp,q, an intercept cp,q and a correlation coefficient r2p,q can be obtained. Performance of this modeling method was tested also using different combinations of partitions, in the range between 5 and 500, and quantiles, ranging from 0.01 to 0.99 (Supplementary Table 1). For all 77 analyzed combinations of p and q regression lines gave good fit with the modeling points, with an adjusted r2 that was always very close to 0.99. In addition, all regression lines were strikingly parallel as judged by their slopes: 0.81 ± 0.068 (mean ± sd). The reason for not considering p > 500 was that above this number we empirically revealed a poorer modeling quality in terms of correlation coefficients (data not shown), most likely due to the decrease of the number of data points contained in each partition.
Table 1 Effect of the presence of DEG when applying the classic permutation strategy to the PLGEM-STN statistic.
FPR vs. significance level estimated vs. observed FDR
including DEG excluding DEG including DEG excluding DEG
# of genes in data set slope intercept adj. R^2 slope intercept adj. R^2 slope intercept adj. R^2 slope intercept adj. R^2
22300 1.690 3.070 0.856 0.871 -0.114 0.938 0.187 -0.383 0.314 1.090 -0.692 0.826
10000 1.710 2.470 0.881 0.888 -0.083 0.931 0.224 -0.247 0.433 1.040 -0.555 0.824
5000 1.460 1.270 0.880 0.877 -0.147 0.934 0.093 -0.228 0.067 1.080 -0.425 0.836
2500 1.590 1.180 0.888 0.864 -0.155 0.939 0.092 -0.176 0.135 1.110 -0.348 0.853
1500 1.670 0.935 0.908 0.876 -0.166 0.948 0.078 -0.122 0.170 1.130 -0.182 0.880
1000 1.720 0.689 0.874 0.944 0.002 0.956 0.038 -0.122 0.082 0.991 -0.226 0.897
500 1.900 0.263 0.864 0.857 -0.255 0.956 0.062 -0.030 0.307 1.160 0.038 0.909
200 2.490 -0.059 0.875 0.905 -0.378 0.946 0.064 -0.012 0.426 1.050 0.233 0.914
Eight data sets with different percentages of DEG were constructed from the Latin Square data set by keeping the 62 known spiked-in probe sets, but randomly removing increasing amounts of the remaining probe sets reaching the total indicated in the first column. The null distributions of the PLGEM-STN statistics were evaluated through the classic permutation strategy either including or excluding DEG. A wide range of significance levels was used to select DEG and correlation between the FPR and the significance level or between estimated and observed FDR was evaluated through linear regressions in log-log plots. Table reports slopes, intercepts and adjusted r2 of linear models. See text for details on estimation of FDR.
As a straightforward application of this modeling method, PLGEM could be fitted at the 50th-percentile to obtain a central tendency of standard deviation to be used for improving test statistics (see next section). Another application of this method could be to fit PLGEM at the 5th- and at the 95th-percentile of standard deviation, to consequently find the limits of the corresponding 90% empirical confidence interval of standard deviation.
In order to verify the feasibility of the former application, fitting of PLGEM on real-life data as well as distribution properties of the random variable E were investigated by analyzing the residuals of the model, i.e. differences between observed and expected values:
Figure 2A–D shows distribution of residuals εg computed from the 16iDC data set and its dependency on the rank of mean expression values. Figure 2E–L summarizes model validation on other two completely unrelated high-density oligonucleotide microarray data sets, the HG-U133A Leigh syndrome data set (Figure 2E–H) and the HG-U95Av2 Muscle biopsies data set (Figure 2I–L). For each tested data set an individual model was fitted and a distinct set of parameters α and β was determined. In all of the three independent data sets measurement variability could be accurately modeled through equation 2, with a power coefficient β that was always between 0 and 1 and a random variable E that appeared to be normally distributed with zero-mean and constant standard deviation over the whole range of expression values. Of course, these findings were eventually expected only for q = 0.5, and their occurrence demonstrated a goodness of fit of PLGEM on a series of unrelated real-life data sets.
Figure 2 Analysis of residuals of PLGEM fitted on three different real-life data sets. (A) PLGEM is fitted on the 16iDC data set (MG-U74Av2) following the method described in the text, setting p = 10 and q = 0.5. (B) Model residuals are plotted as a function of the rank of the mean absolute expression level. (C) Distribution of pooled residuals. (D) The quantiles of the distribution of pooled residuals are plotted against the quantiles of a standard normal distribution. The same procedure is applied to the Leigh syndrome data set (HG-U133A, panels E-H) and to the Muscle biopsies data set (HG-U95Av2, panels I-L).
Improved test-statistics for detecting differential expression
In order to identify DEG, we implemented the following general algorithm derived from the framework of statistical hypothesis testing, in which we test against the null hypothesis of non-differential expression. First of all, we chose to implement as the test statistic the signal-to-noise ratio (STN) already used by Golub et al. [2], because it explicitly takes unequal variances into account and because it penalizes genes that have higher variance in each class more than those genes that have a high variance in one class and a low variance in another [11]:
where in the original version and represent, respectively, the mean of the replicated expression measures of gene g in condition 1 and 2, whereas and are the corresponding standard deviations. Instead, we propose to use model-derived standard deviation estimates predicted by PLGEM in equation 2 for the corresponding signal mean, rather than data-derived standard deviation values calculated independently from the few data points that are usually available for every single gene.
The improvement of the test statistic in ranking DEG was evaluated as done by Broberg [12] through receiver operator characteristic (ROC) plots on the HG-U133A Latin Square data set, where there is an a priori knowledge on the truly differentially expressed transcripts. ROC plots investigate the relationship between false positive rates (FPR) and false negative rates (FNR) at different significance levels; in this way the performance of the PLGEM-derived STN statistic (PLGEM-STN) has been compared with the original STN statistic (CLASSIC-STN) and the statistic implemented in the commonly accepted Significance Analysis of Microarrays (SAM) DEG identification method (SAM-STAT). To this purpose Exp01 of the Latin Square was taken as the baseline to which the remaining 13 experiments were compared. For each comparison absolute values of each statistic were ranked in decreasing order and first n genes selected (where n ranged from 5 to 200). Figure 3 summarizes results only for the most informative comparisons, but in each tested comparison analysis PLGEM-STN was at least as good as the other two statistics for each tested value of n (data not shown). In addition, the ROC curve of PLGEM-STN always had the shortest distance from origin, indicating that it resulted in the best trade-off between sensitivity and specificity. Interestingly, improved sensitivity was observed especially when the nominal fold change was particularly low (see Exp02 vs. Exp01 and Exp14 vs. Exp01).
Figure 3 Performance of PLGEM-STN in ranking differentially expressed genes. ROC plots were used to compare the sensitivity vs. specificity trade-off of the following three statistics: PLGEM-STN (black), CLASSIC-STN (blue) and SAM-STAT (red). SAM-STAT values were obtained using the R package "siggenes" [21]. Absolute values of the corresponding statistics were sorted in decreasing order, first n genes were selected (where n ranged from 5 to 200) and false positive and false negative rates were evaluated on the HG-U133A Latin Square dataset. Note that, while the transcripts in Exp02 (Exp14) are spiked-in at twice (half) the concentration than in Exp01, in both Exp06 vs. Exp01 and in Exp10 vs. Exp01 comparisons the nominal fold-change of spiked-in transcripts ranged from 32 to 512.
Apart from discriminating between significant and not significant gene expression changes, an optimal test-statistic should additionally provide an accurate quantification of the actual degree of differential expression. Figure 4 shows that PLGEM-STN outperforms the competing statistics in correlating the value of the statistic with the nominal concentration variation of the known Latin Square DEG; this was particularly true for the most extreme variations.
Figure 4 Correlation between the value of PLGEM-STN and the nominal concentration variation in comparison to competing statistics. Exp01 of the Latin Square data set was taken as the baseline to which the remaining 13 experiments were compared. The observed values of the indicated statistics are plotted against the nominal log ratio, deduced from the known spiked-in concentrations (left panels). A nominal log ratio of 0 is assumed for the remaining transcripts and a box-plot of their corresponding values of the indicated statistics is superimposed to the plot. For those cases where one of the two known spiked-in concentrations is 0, the value of the statistic is instead plotted against the non-null concentration (right panels). Red and black dots represent transcripts that are present in Exp01 or in the remaining 13 experiments, respectively.
Identification of differentially expressed genes
A resampling-based method for estimating the null distribution
Though ranking of genes based on the absolute value of their test-statistic has been proven to be an effective method for selecting DEG, an even more useful way would be to compare the observed statistic with its null distribution (the distribution of values of the statistic that are expected by chance for a not differentially expressed gene), in order to control the FPR.
A classic approach to empirically obtain the null distribution of a test-statistic is running a series of random permutations of the chip indexes of the full data set and re-computing the test-statistics at each permutation. Permutated test-statistics can then be pooled and significance thresholds (i.e. expected false positive rates) are found as specific quantiles of the null distribution.
Nevertheless, we can foresee that the classic permutation strategy may not be optimal for estimating the actual FPR when the test-statistic makes use of a global error model such as PLGEM. We can in fact hypothesize that measurement spread of DEG may not be accurately described by means of a global error model that was designed to describe signal variability in absence of differential expression. To test this hypothesis we compared the correlation between the expected significance level and the observed FPR using PLGEM-STN and the classic permutation strategy either including or excluding DEG during the permutation step. To this end, data sets containing different percentages of DEG were obtained by merging the 62 known DEG of the Latin Square data set with differently sized random samples of not DEG extracted from the same data set. As predicted, the presence of DEG during the permutation step caused the significance level to be less correlated with the observed FPR and this correlation worsened with increasing percentages of DEG (Table 1). This lack of correlation was dramatically amplified when expected and observed numbers of false positives were divided by the number of selected genes to obtain an oversimplified estimate of the false discovery rate (FDR) and the observed FDR. Conversely, when DEG were omitted during the permutation step the correlation between estimated and observed FPR or estimated and observed FDR was sensibly higher for each tested percentage of DEG. We hereby by no means claim that this FDR estimate is the most accurate. A more appropriate relationship between FPR and FDR can be found in the paper by Storey and Tibshirani [13]. Nevertheless, the explicit control of the FDR goes beyond the scope of the present paper.
Since in real-life data sets true DEG are unknown in advance, we propose the following resampling-based method to obtain the null distribution of not DEG when comparing n1 replicates of condition A with n2 replicates of condition B:
• Artificial condition A* is obtained by randomly sampling with replacement n1 indexes corresponding to the replicates of only one experimental condition. If available, chose the condition with the highest number of replicates;
• Similarly sample n2 values from the same set to obtain indexes of artificial condition B*;
• Compute resampled test-statistics between A* and B* at each cycle.
The previous resampling should be repeated a sufficiently large number of times – as large as possible compared to the total number of possible combinations and compatibly with available computational resources – and the resampled test-statistics finally pooled. In our opinion resampling the expression values from only one experimental condition, rather than permutating indexes of both conditions, makes more sense with this particular statistic, because in this way we avoid merging true and false null hypothesis. Note that when more than one condition (all with the same number of replicates) are to be compared to a common baseline, the distribution of resampled test-statistics needs to be determined only once, obviously providing a computational advantage. As a test of substantial equivalence between this resampling method and the classic permutation strategy (excluding DEG), we compared the distribution of the permutated and of the resampled PLGEM-STN test-statistics in Q-Q plots. The distribution of the PLGEM-STN resampled from Exp01 of the Latin Square data set was almost identical with the distributions of permutated PLGEM-STN obtained with the classic strategy from each comparison with the remaining 13 experimental conditions (data not shown). Figure 5 shows that the quantiles of the resampled PLGEM-STN values have a good concordance with the mean quantiles of the classically permutated statistics averaged over the 13 comparisons, implying that no differences are expected also in the gene selection step.
Figure 5 Comparison of two methods for inferring the null distribution of the PLGEM-STN statistic. The classic permutation strategy (excluding DEG) was performed for each comparison in the Latin Square data set and the quantiles of the distribution of PLGEM-STN values were averaged over the 13 comparisons. The mean quantiles of the permutated statistics are plotted against the quantiles of the distribution of PLGEM-STN values obtained through the proposed resampling approach performed on the same data set but including DEG.
In accordance with the previous observations, the ROC curve of the resampling method applied to the PLGEM-STN statistic was not significantly different from the ROC curve of the classic permutation strategy (excluding DEG) applied to the same statistic on the Latin Square data set (data not shown). Conversely, ROC curves of the classic permutation strategy (including DEG) applied to the CLASSIC-STN statistic and of the SAM method gave poorer performance similarly to the results in Figure 3 (data not shown).
Increased robustness to varying number of replicates
Another appealing feature of an optimal DEG identification method is that it should provide consistent results when different replicates of a same data set or different numbers thereof are analyzed. We therefore compared the performance of our resampling approach applied to the PLGEM-STN statistic (method 1) with SAM (method 2) and with the classic permutation strategy applied to the CLASSIC-STN statistic (method 3). The number of available replicates for each experimental condition in the Latin Square data set was unfortunately too small to investigate this particular task. We therefore took advantage of the 16iDC+LPS data set, where the first sixteen columns can be considered as the baseline condition for the remaining four experimental replicates. We then constructed a series of reduced data sets in which the baseline columns were kept constant while all possible combinations of 1, 2 or 3 replicates of LPS-stimulated DC were systematically deleted from the 16iDC+LPS data set, reaching a total of fifteen distinct data sets including the original one. Since methods 2 and 3 are not applicable on the four reduced data sets containing single samples for the LPS experimental condition, only the eleven data sets with at least two replicates were used for comparison purposes. Since the sixteen baseline columns are identical in each reduced data set, PLGEM parameters were determined only once on this common baseline condition. Significance levels used by each method in all eleven data sets were empirically selected in order to achieve a similar number of significant genes (ca. 500 probe sets) in the full data set, i.e. the one containing all available replicates. Thus, for each method eleven lists of identified DEG were obtained and the consistency between these lists was evaluated by counting the number of times each probe set was selected, giving a probes set count between 1 and 11. In Figure 6 we compared the three distinct cumulative frequency curves for each method, which show the percentage of identified DEG that were selected at least a given number times. While method 2 and 3 gave similar results, the method proposed in the present work identified a larger number of probe sets in a larger number of lists.
Figure 6 Consistency of findings when different replicates or numbers thereof are analyzed. Ten reduced data sets were constructed by removing all possible combinations of 1 or 2 replicates of LPS-stimulated DC from the data set. The plot shows the cumulative frequency at which the probe sets are consistently selected in the 16iDC+LPS and in the ten reduced data sets by the following methods: the resampling approach applied to PLGEM-STN (filled squares), the permutation strategy applied to CLASSIC-STN (filled circles) and the SAM method (open triangles). In case of PLGEM, a single model was fitted on the common 16iDC baseline data set. The cumulative frequencies are normalized with respect to the total number of probe sets identified by the corresponding method.
We finally evaluated the possibility of applying our method also to data sets where one of the experimental conditions was investigated only with a single sample without replication. To this end, we used the remaining four reduced data sets that could not be used in the previous comparison. In this case, the same PLGEM parameters derived from the sixteen baseline columns were applied to each of the single LPS-treated DC sample to obtain an estimate of standard deviation associated to each gene expression value, treated here as if it was a mean value from a larger group of values. Interestingly, when results obtained through this procedure were compared to the previously described results a comparable number of DEG was identified and only one probe set was newly detected in comparison to the previously identified ones (data not shown), arguing for a good consistency of results.
Discussion
PLGEM accurately describes GeneChip data variability
In the present work we described a new global error model for microarray gene expression data that describes measurement variability with the same degree of accuracy over the whole dynamic range of values and that can be fitted at any desired quantile of spread. PLGEM has proven to correctly model signal standard deviation, in spite of the presence of different sources of variability, e.g. biological variability as well as the use of different target preparation protocols or of different chips. Moreover, PLGEM has shown to be able to deal with the great variability that exists at low expression levels while at the same time considering the significant relative reproducibility of highly expressed genes. Previously proposed error models assumed that measurement spread depended on signal location following different mathematical relationships, but none of them was based on a power law thus far. Analysis of the residuals showed a good fit of PLGEM to a number of high-density oligonucleotide microarray data sets, with model parameters being very similar to each other even when dealing with RNA samples coming from completely different biological sources and analyzed on different array layouts. This suggests that PLGEM could represent a general Affymetrix GeneChip measurement noise model. Even though scaled MAS5 Signals gave satisfactory modeling results, a further improvement could be achieved by using other emerging gene expression indices [6,14] or more sophisticated normalization techniques, e.g. quantile normalization [15]. Interestingly, if the same evaluation of sensitivity vs. specificity using ROC plots on the Latin Square data set was done using GCRMA expression values [16], the results were even more striking than using MAS5 Signals (data not shown). Further studies will be needed to assess if PLGEM is also able to deal with data coming from microarray technologies others than Affymetrix GeneChips.
Interestingly, model parameter β was found to be quite stable and comprised between 0 and 1 in all analyzed data sets. It is noteworthy that for β ∈ (0:1) absolute variability increases with growing expression values, while relative variability decreases (compare panel B with panel D of Figure 1). On the other hand, none of the models mentioned in the background section seem to agree with these experimental observations. Formal statistical reasoning could unravel the underlying theoretical error model that leads to the power law relationship that was observed to be at the basis of the variance versus mean dependence in replicated microarray data.
A PLGEM-based method successfully detects differential expression
In spite of the lack of a theoretical statistical model, the empirical model presented here has proven its applicability in the identification of DEG, providing improved results under a wide range of different testing conditions. In comparison to other commonly used DEG identification methods, the proposed approach demonstrated improved specificity and sensitivity on the Latin Square data set and robustness to decreasing number of replicates on the 16iDC+LPS data set. The good performance of our proposed method is reasonably due to the fact that it relies on a global error model. As an example, when the classic permutation strategy is applied to the CLASSIC-STN statistic or when the SAM method is used, the selected genes are apparently more dependent on the number and identity of the replicates than when our proposed approach is used. We hypothesize that, when no error model is assumed and a small number of replicates is present in the data set, the probability of observing for some genes coincidently very similar (or very dissimilar) values increases, thus leading to an underestimation (or overestimation) of the standard deviation and a consequent overestimation (or underestimation) of the test statistic, finally leading to false positives (or false negatives).
Interestingly, when the performance of our method was compared on a data set of DC stimulated for 24 hours with LPS, SAM showed a decreased sensitivity in identifying down-regulated genes when the number of LPS replicates was low (data not shown). Under these experimental conditions DC undergo a process known as maturation, which is a specialized form of cellular differentiation, for which both up- and down-regulation of gene expression is expected [17,18]. We speculate that SAM did not select these genes, because of the combination of two effects. First of all, down-regulated genes are expected to have lower and therefore intrinsically more variable expression values in the four LPS replicates than in the sixteen replicates of immature DC. When, in addition, the number of LPS replicates becomes too low, SAM filters these genes out to control the FDR. In agreement with this hypothesis SAM was perfectly able to identify down-regulation when the full data set was used (data not shown).
The gene selection method proposed in the present work does not provide a direct control on the FDR, but the significance level has been proven to be a direct estimate of the FPR. Thus, if a significance level of 0.001 is used and 12488 probe sets are displayed on the MG-U74Av2 chip, 12–13 genes are expected to be selected by chance in cases where all genes are in fact not differentially expressed. Therefore, a researcher can test how many genes would be selected over a range of different significance levels and chose the one that results in the most acceptable compromise between number of selected genes and estimated FPR.
Conclusions
The proposed DEG identification method provides a direct control of the FPR and an indirect control of the FDR. Moreover, as tested on the Latin Square data set, our method improved the specificity vs. sensitivity trade-off in comparison to other commonly applied DEG selection techniques. It finally showed an increased robustness when different replicates or numbers thereof are analyzed, giving consistent results even in data sets containing single samples. In conclusion, the global error model presented here may facilitate the analysis of microarray gene expression data by discriminating information from noise, and thus possibly helping the formulation of new hypothesis concerning gene functions.
Methods
Data sets
16iDC
RNA was harvested from ten biological samples of unstimulated immature mouse dendritic cells (DC), each extracted from an independent batch of cells. One operator prepared the biotin-labeled cRNA for hybridization from three of the ten RNA samples, a second operator prepared the remaining seven. While operator 1 applied the total RNA protocol to all of its three samples, operator 2 applied the purified mRNA protocol to five of its seven samples and the total RNA protocol to the remaining two. Two of the three cRNA samples prepared by operator 1 and four of the seven cRNA samples prepared by operator 2 have been hybridized twice; therefore, a total of 16 MG-U74Av2 GeneChips (Affymetrix, Santa Clara, CA) have been employed.
Leigh syndrome
Eight RNA samples were harvested from human fibroblast cell lines each deriving from a distinct Leigh syndrome patient [19,20] and individually hybridized on HG-U133A GeneChips (Affymetrix).
Muscle biopsies
Four individual and two pooled RNA samples from human muscle biopsies of sixteen healthy young male donors were hybridized on six HG-U95Av2 GeneChips (Affymetrix). This data set was downloaded from [21], experiment code: GSE80 [22].
Latin Square
This data set consists of 3 technical replicates of 14 separate hybridizations (named Exp01–14) of 42 spiked transcripts in a complex human background at concentrations ranging from 0.125 pM to 512 pM. Thirty of the spikes are isolated from a human cell line, four spikes are bacterial controls, and eight spikes are artificially engineered sequences believed to be unique in the human genome. Further details on the design of the Latin Square data set can be found at [23]. Considering the redundancy of some probe sets, there are a total of 62 distinct probe sets designed to match the 42 spiked transcripts.
16iDC+LPS
This data set consists of the same samples of the 16iDC data set, but includes additional four samples as a second experimental condition. To this end dendritic cells were stimulated to mature with lipopolysaccharide (LPS) for 24 hours. Two independent biological samples were harvested and individually processed by the same two operators that prepared the samples for the 16iDC data set: one applied the total RNA protocol, the other one applied the purified mRNA protocol. Each cRNA sample was hybridized twice, thus using a total of four Affymetrix MG-U74Av2 chips.
Software
All chips mentioned in the present study were hybridized and scanned following Affymetrix recommendations and MicroArray Suite 5.0 (MAS5) was used as the image acquisition and analysis software. All data sets used passed quality control tests and probe set signals were scaled so that the 4%-trimmed mean of all expression values of each chip was equal to a predefined reference intensity (called TGT) following manufacturer's recommendations:
TGT = 100 for MG-U74Av2 and HG-U133A chips and TGT = 500 for HG-U95Av2 chips.
All procedures for fitting PLGEM, for calculating observed PLGEM-based signal-to-noise ratios (STN), for obtaining expected PLGEM-STN through the resampling-based approach and for comparing observed with expected STN values have been implemented as R functions [24] and will be soon submitted for integration into the Bioconductor project [25].
Authors' contributions
NP conceived the study and drafted the manuscript. MP wrote the software, participated in the design of the study and in the editing of the manuscript. CV performed the microarray experiments, participated in the design of the study and the editing of the manuscript. MC participated in the microarray experiments, AS participated in the design of the algorithms, FG and PRC coordinated the study. All authors read and approved the final manuscript.
Supplementary Material
Additional File 1
Performance of modeling method using different combinations of parameters p and q. The modeling method described in this study was tested on the 16iDC data set using different combinations of partitions (5, 10, 20, 50, 100, 200 and 500), and quantiles (0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 0.8, 0.9, 0.95, 0.98 and 0.99). For all 77 analyzed combinations of p and q regression lines were fitted to the data as described in the text. Goodness of fit was evaluated from the resulting slope (panel A), intercept (panel B) and adjusted r2 (panel C).
Click here for file
Acknowledgements
This research was supported by AIRC (Italian Association for Cancer Research) and by a grant from the CARIPLO Foundation ("Development of Functional Genomics and Bioinformatics platforms aimed to foster novel approaches in immunotherapy and molecolar diagnostics"). Authors would like to kindly acknowledge Stefano Monti (Broad Institute, Cambridge, MA, USA) and Peter J. Park (Harvard Medical School, Boston, MA, USA) for helpful discussion and Ottavio Beretta, Gianpiero Cattaneo, Maria Foti, Giorgio Moro and Ettore Virzi (University of Milano-Bicocca, Milan, Italy) for critical reading of the manuscript. We are also grateful to Valeria Tiranti, Rossana Mineri and Massimo Zeviani (Unit of Molecular Neurogenetics, National Neurological Institute "Carlo Besta", Milano, Italy) for kindly providing the HG-U133A Leigh syndrome data set.
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| 15606915 | PMC545082 | CC BY | 2021-01-04 16:36:38 | no | BMC Bioinformatics. 2004 Dec 17; 5:203 | utf-8 | BMC Bioinformatics | 2,004 | 10.1186/1471-2105-5-203 | oa_comm |
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BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-5-2011560358510.1186/1471-2105-5-201Methodology ArticleA novel Mixture Model Method for identification of differentially expressed genes from DNA microarray data Najarian Kayvan [email protected] Maryam [email protected] Rad Ali [email protected] Siamak [email protected] Javad [email protected] Computer Science Department, University of North Carolina Charlotte, University City Blvd, Charlotte, NC, USA2 Computer Engineering and IT Department, Amirkabir University of Technology, Tehran, Iran3 Mechanical and Industrial Engineering Department, Concordia University, CONCAVE Research Centre, CR-200, Concordia University, Quebec, Canada2004 16 12 2004 5 201 201 24 3 2004 16 12 2004 Copyright © 2004 Najarian et al; licensee BioMed Central Ltd.2004Najarian 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 main goal in analyzing microarray data is to determine the genes that are differentially expressed across two types of tissue samples or samples obtained under two experimental conditions. Mixture model method (MMM hereafter) is a nonparametric statistical method often used for microarray processing applications, but is known to over-fit the data if the number of replicates is small. In addition, the results of the MMM may not be repeatable when dealing with a small number of replicates. In this paper, we propose a new version of MMM to ensure the repeatability of the results in different runs, and reduce the sensitivity of the results on the parameters.
Results
The proposed technique is applied to the two different data sets: Leukaemia data set and a data set that examines the effects of low phosphate diet on regular and Hyp mice. In each study, the proposed algorithm successfully selects genes closely related to the disease state that are verified by biological information.
Conclusion
The results indicate 100% repeatability in all runs, and exhibit very little sensitivity on the choice of parameters. In addition, the evaluation of the applied method on the Leukaemia data set shows 12% improvement compared to the MMM in detecting the biologically-identified 50 expressed genes by Thomas et al. The results witness to the successful performance of the proposed algorithm in quantitative pathogenesis of diseases and comparative evaluation of treatment methods.
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Background
Recently, microarray technology has provided the means for simultaneous screening and analysis of thousands of genes. Although an enormous volume of data is being produced by microarray technologies, the full potential of such technologies cannot be accessed without the ability to sift through the noisy signals to obtain useful information. The large data sets produced by microarray technology have resulted in the need for reliable, accurate, and robust methods for microarray data analysis. A major challenge is to detect genes with differentially expression profile across two experimental conditions. In many studies, the two sample sets are drawn from two types of tissues, tumours or cell lines, or at two time points during the course of a biological processes.
The computationally simple methods used for such analysis, including the methods of identifying genes with fold changes (such as the popular Log-ratio graphs) [1], are known to be unreliable due to the fact that in such methods the statistical variability of the data is not properly addressed. While various parametric methods and tests such as two-sample t-test [2] have been applied for microarray data analysis, strong parametric assumptions made in these methods as well as their strong dependency on large sample sets restrict the reliability of such techniques in microarray problems. The nonparametric statistical methods, including the Empirical Bayes (EB) method [3], the significance analysis specialized for microarray data (such as SAM [4]) and the mixture model method (MMM) [5] have been specialized and applied for microarray data analysis. It is claimed and argued that the new extensions of the MMM are among the best methods producing biologically-meaningful results [5,6]. In this paper, without ignoring the potential applicability of non-parametric methods in microarray processing applications, due to the claims made in [6], we have restricted the comparison of our methods only to the MMM based methods.
The major disadvantages of the above-mentioned methods, especially the MMM, include the lack of repeatability of the results under different runs of the algorithm, and the sensitivity of the algorithm on parameter initialization. A reliable microarray analysis method must be reproducible and applicable to different data sets under different experimental conditions. More specifically, an accurate microarray processing method is expected to pinpoint, with a relatively high degree of accuracy and robustness, genes with elevated expression levels that are related to the experimental condition in all runs. The main objective of this paper is to design and test an extension of the MMM whose results are reproducible, more biologically meaningful, and significantly less sensitive to the models' parameters.
The paper is organized as follows. In Algorithms section, a review of the MMM and its recent extensions, Mod2MMM, together with the detailed description of the proposed method are given. In Results and Discussion section, the K5M algorithm is first applied to the well-studied Leukaemia data set [7] that is often treated as a benchmark problem to compare different algorithms with each other. Once the desirable performance of the proposed algorithm is verified against the Leukaemia data set, the algorithm is applied to a new data set [[8-14] and [15]] that deals with the pathogenesis of Hypophosphatemia, which is a common X-linked metabolic bone disorder in human and mouse. Finally, the Conclusion section is in the end.
Algorithms
MMM & its recent extensions
We start this section with a brief review of the existing MMM based techniques. Consider Yij as the expression level of gene in array i (i = 1, ..., n; j = 1, ..., j1, j1 + 1, ..., j1 + j2), where the first j1 and last j2 arrays are obtained under two conditions. A general statistical model for the resulting data is:
Yij = ai + bixj + εij (1)
Where xj = 1 for 1 ≤ j ≤ j1 and xj = 0 for j1 + 1 ≤ j ≤ j1 + j2. In addition, εij is a random error with mean 0. From the above formulation, it can be seen that ai + bi is the mean expression level of the first condition, and ai is the mean expression level of gene i in the second condition. The method requires that both j1 and j2, the number of data sets for each experiment condition, be even.
The t-test statistic type scores (2) and (3) are calculated on the pre-processed data. Here, ai is a random permutation of a column vector that contains j1/2 1's and j1/2 -1's and bi contains j2/2 1's and j2/2 -1's.
Since the data are not assumed to be normally distributed, the distribution functions f0 and f are estimated as in (4) and (5), respectively. The null distributions, f0 and f, are estimated directly in a nonparametric model for gene expression data.
Where φ(z; μi, Vi) symbolizes the normal density function with mean μi, variance Vi, and the mixing proportions πi define the linear combination of the normal basis function. We use Φg0 to represent all unknown parameters {(πi, μi, Vi): i = 1, ..., g0 } in a g0-component mixture model. The number of normal basis functions, i.e. g0 can be estimated by the EM algorithm, which maximizes the log-likelihood function of (6) to obtain the maximum likelihood estimation of .
Within K iterations, the EM algorithm is expected to find the local maxima for all unknown parameters. It is recommended to run the EM algorithm several times with various random starting parameters and choose the final estimate as the one resulting the largest log-likelihood [6]. As mentioned above, using random starting points causes the result of the MMM instable and avoids reproducibility of the results. More specifically, in each run the MMM algorithm may give different number of expressed genes, which is not desirable in biological studies. This issue will be addressed in our proposed method.
After finding the optimized for different g0 's, the algorithm selects the sub-optimal g0 corresponding to the first local minimum of BIC or AIC [16].
where vg0 is the number of independent parameters in Φg0. Then, the algorithm uses the resulting g0 as the number of normal functions to fit f0. The same procedure is performed to estimate the sub-optimal number of normal functions to estimate f. As mentioned above, with the fixed number of normal functions, the parameters of functions f and f0 are iteratively updated for a number of iterations. When the iterations are terminated, the likelihood ratio is estimated based on the final estimations of f0 and f:
LR(Z) = f0(Z) / f(Z) (9)
A bisection method [17] with a Bonferroni adjustment is used to determine the cut-off points [18] for decision-making. This means that for a threshold value s, if LR(Z) <s, then the gene is identified to have significantly altered expression in two experiments. It is possible to determine the rejection region numerically, i.e. for any false positive rate α, the threshold value s = s(α) can be calculated from the following integral:
In literature of microarray processing, α = 0.01 is often used as the genome wide significant level, so the gene-specific significance level is: α* = α/(2n) Recently a new modification of the MMM algorithm, Mod2MMM hereafter, was introduced [6]. This method points out a problem in constructing the test and null statistics and indicates that the true distribution of z may be different from the null distribution of Z, which can lead to invalid inference. The modified algorithm starts with the assumption that j1 ≥ 2 j2 [6], and constructs the new z and Z as you can follow in appendix1.
For the cases where j1 ≥ j2 but j1 < 2 j2, j1 observations drawn under condition one are split into two equally-sized parts to calculate , vi(1a) and , vi(1b) respectively. To calculate and vi(2) about j1/2 observations are drawn under condition two. While this modification can address the differences in the distributions of f and f0, the stability of the parameter estimation step still remains a major problem.
The main difference between the conventional MMM and its recent extensions are that the conventional MMM disregards the fact that the true distribution of z (the statistical variable under study) may be different from the null distribution of the statistics Z (as defined below). This assumption can potentially lead to invalid inference. A modified version of the MMM (Mod2MMM hereafter), introduced in [6], assumes that the denominator and the numerator of one of t-statistic-type score zi may not be independent. This method addresses the issue by constructing new zi and Zi variables as will be discussed later.
A concern over all existing MMM based methods (including Mod2MMM) that greatly affects the results is associated with the way mixed distributions are estimated. In the MMM, Expectation Maximization (EM) algorithm [19] is often used to optimize the parameters of fitted mixture distribution functions of two t-statistic-type scores related with genes expression level. Starting the EM algorithm with random values as the parameters of the normal basis functions to estimate distributions makes the results depend highly on the exact initialization, and always makes variations in the results. On the other hand, if all parameters of the normal functions in the mixture model distributions are set without iterative optimization, the set values may never result to an accurate model of the data set in hand. We propose a modified version of MMM to address this problem. Our modified MMM (K5M hereafter) combines K-mean clustering and the EM estimation to not only optimize most of the parameters with the EM iteratively but also apply K-means to optimize other sensitive parameters to ensure complete reproducibility of the algorithm. The experimental results indicate superior robustness of the proposed algorithm compared to the conventional MMM and other recently introduced extensions of the MMM [6].
Proposed method (K5M)
In order to address the stability and reproducibility of the MMM, we propose a new modified approach for the MMM that estimates the distribution function of z by using mixture of normal distributions in a stable and reliable way. The following observations made in the experimental study of the MMM for gene expression analysis were the main motivations for the proposed changes to the MMM:
1 The observed variations in the parameter estimation process in some versions of the MMM can be attributed to the algorithm's attempt to iteratively update the means and variances of the normal distributions using often noisy data. In experimental studies, often the direct observation of the data reveals specific points where centers (means) can be positioned and the scattering patterns that can give reliable estimates on the variance of each cluster. However, the iterative updating of model parameters with noisy data and based on some random starting points often misses the true optimal points and even creates variations and fluctuations in parameter estimation in many runs.
2 Even when variations do not occur, two runs of the algorithm can result to significantly different estimations of f and f0. This in turns results to lists of differentially expressed genes in different runs. More specifically, a set of two typical runs of the algorithm on the same data set can result to two lists that are very different both in number of the genes as well as the exact genes picked up by the algorithm. In our study of the conventional MMM and Mod2MMM, two runs with the same algorithm (on the same data) resulted to lists whose size vary between 50 and 200.
3 The literature of other areas of research utilizing normal basis function for estimation including neural networks indicates that in order to have more robustness in different runs and have reproducible results, the means and variances of the basis functions must be estimated and fixed during the iteration on the coefficients [20]. This is due to the fact that updating means and variances makes the estimation process a nonlinear one that is highly sensitive and very likely to become unstable. However, when updating the values of coefficients only, the problem is reduced to a reliable linear estimation that is much more robust and stable.
4 Based on the observations mentioned above, in our proposed method, finding the distribution of z is regarded partially as a clustering problem, i.e. the means and variances of the normal distributions are estimated as the prototypes of a clustering step. Specifically, if z is distributed in a one-dimensional space, wherever there is a mass of z, there is a cluster with mean μi and variance Vi, which are identified by the members of that cluster.
Hence applying a clustering method is capable of estimating the means and variances of each normal distribution. The key is to use a simple clustering technique such as K-mean to estimate the mixture distributions f0 and f based on K normal distributions. While the algorithm can use K-means to find the optimal values of means and variances, the coefficients πi 's need to be optimized using an optimization process such as the EM.
Based on the above discussion, the proposed algorithm can be described in the following two steps:
Step 1: Using BIC, find the sub-optimal number of normal distributions for both f and f0 (as described above). These optimal numbers are then used as the number of clusters in K-means technique.
Step 2: Using K-means clustering technique, for both f and f0 find the best mean μi and variance Vi for all clusters.
Step 3: With the obtained values of μi, Vi and using the EM algorithm, iteratively update the values of the optimized πi for all clusters (both f and f0), i.e.
A reasonable number of clusters is expected to be obtained from the first step of the algorithm, and the estimation results of the two bellow data sets in Tables 1 and 4 show that the used K (calculated based on AIC) is satisfactory. Table 3 shows the results of the MMM and K5M methods for the run with an unequal variance and four normal distributions for both f and f0. The MMM creates the likelihood ratio (LR) statistics plotted in Figure 1, the K5M with K = 4 forms the LR statistics plotted in Figure 2, and the K5M with K = 2 results to the LR plot of Figure 3.
Table 1 Comparison of the result of the K5M with the MMM and the Mod2MMM based on the Leukaemia data.
Method Total detected genes ALL AML Total accepted genes out of 50 genes [22]
MMM 187 21 18 39
Mod2MMM 58 14 16 30
K5M, K = 3 185 25 20 45
K5M, K = 4 58 19 8 27
Table 3 Estimation of fitted and by MMM (in the optimum run) and K5M.
f0 f
MMM = (0.1859, -0.2231, 0.0322, 0.0638) = (-0.0387, 0.4381, 0.1600, -0.1933)
= (0.3215, 0.3522, 0.7692, 0.337) = (3.2288, 3.397, 2.6393, 4.6982)
= (0.1672, 0.2353, 0.4048, 0.1925) = (0.0687, 0.0509, 0.0263, 0.0725)
K5M = (1.1111, 1.1264, 0.3115, -0.3329) = (1.7867, -0.6817, -2.354, 0.3324)
= (0.4589, 0.4640, 0.1879, 0.1807) = (2.9432, 0.5583, 4.24, 0.5027)
= (0.1914, 0.1963, 0.3120, 0.3001) = (0.0583, 0.1018, 0.0294, 0.0442)
Table 4 The top ten most significant genes provided by K5M and MMM.
GenBank Accession IDs Gene/ Protein Description Rank based on MMM Rank based on K5M
D00073 Kidney/ carrier activity 1 1
AA815845 Unknown 2 2
AF085696 ion transportation/ K+ channel, inward rectifier/renal salt flow 3 3
AW047688 Brain/Hypothalamus 4 4
M12660 Kidney/ Complement protein H gene 5 5
AI847513 Brain/ Hypothalamus 7 6
AA919924 Phosphate metabolism/inositol-1(or4)-monophospha te Activity 6 7
X69966 Dilation of the proximal renal tubules and extensive vacuolization of tubule epithelium 8 8
AF103809 Elevated kidney levels of lysosomal enzymes 9 9
AA711516 Barstead mouse myotubes MPLRB5 10 10
Figure 1 Likelihood ratio statistics as a function of Z value based on the MMM method.
Figure 2 Likelihood ratio statistics as a function of z based on the K5M with K = 4.
Figure 3 Likelihood ratio statistics as a function of z based on the K5M with K = 2.
It is worth mentioning that due to the random initialization in K-means and the random initialization of the coefficients πi 's, in each run, it is expected that the number of identified differentially expressed genes fluctuate slightly. However, as indicated above, since the K- means clustering algorithm is known to a robust method, and considering the fact that in the EM estimation process, only a linear estimation is performed, it is expected that the robustness of the proposed algorithm be much more than the other version of the MMM based algorithms. This observation, as have been shown before, is supported by our experimental results. In addition, our experimental indicate that the most expressed genes are identified in all runs or the algorithm and in each run one or two new genes with less expression ratio are added to this set.
Results and discussion
In this section, first the two applications and their corresponding data sets are described and then the results produced by the proposed method (i.e. K5M) is compared with the other MMM based methods on two data sets. The detailed description of the methods is given in MMM & its recent extensions Section.
Leukaemia dataset
In this section, we apply the nonparametric MMM method with and without the proposed modifications to the Leukaemia data presented in [7]. The objective of this application is to identify the most important genes involved in development of different types of Leukaemia. The dataset used for this analysis includes 27 acute lymphoblastic leukaemia (ALL) samples and 11 acute myeloid leukaemia (AML) samples for 7129 genes. The main goal is to find genes with differential expression between ALL and AML cases. A second goal is to compare the result of MMM and Mod2MMM (as introduced in MMM & its recent extensions Section) with K5M and test the robustness of K5M. The genome-wide significance level is chosen α = 0.01 (according to Benferroni adjustment used in the MMM based methods). Each sample in the dataset is pre-processed as in [21], by subtracting its median and dividing the resulting variable by its quartile range (i.e. the difference between the first and the third quartile).
Results of Leukaemia study
Thomas et al [22] used known biological information to identify the most important genes in Leukaemia and provided biological justifications for these identified genes. They introduced 50 genes out of the identified genes as the most expressed and related genes to the disease, including 25 most expressed genes for AML and 25 for ALL. We treat Thomas et al's list as the biology knowledge base and compare the capabilities of the computational techniques to correctly identify the genes discussed in [22] by processing the dataset.
The comparison of the result obtained by the K5M with those of the MMM and the Mod2MMM is summarized in Table 1. As can be seen in Table 1, The MMM has identified 187 differentially expressed genes [21], among which the total of 39 genes are in the list of genes obtained by Thomas et al [22]. The Mod2MMM method found 30 genes of the Thomas's list. The K5M algorithm, determines 45 genes that are identified in the Thomas's list, i.e. the proposed algorithm successfully identifies 90% of biological result. This means that K5M improved the detection of expressed genes 12% compare to the MMM and 30% compare to the Mod2MMM for the Leukaemia data, i.e. our method identified more genes from the list of the 50 truly expressed genes identified by Thomas et al [22].
As the BIC suggested the optimum number of clusters K = 4 for the MMM, the K5M is applied with K = 4 also. Running K5M with different number of clusters leads to the different but reasonably similar results. As the number of the clusters increase, the number of expressed genes decreases. Table 1 shows that the K5M with K = 3 identifies the total of 185 differentially expressed genes, while with K = 4 the total of 58 genes are identified, however; the 58 genes found with K = 3 are the most expressed genes among 185 genes found by K = 4. This result shows the consistency of the K5M method.
In order to further compare the performance of the MMM and K5M on the leukaemia data, The ROC curve is plotted based on False Positive rate and True Positive rate of the data set calculated as in [5]. The area under each curve is the measure of test accuracy. As can be seen in Figure 5, the area under the K5M curve is more than the area under the MMM curve, therefore the K5M is providing a more accurate classification than the MMM.
Figure 5 ROC curves for the MMM and K5M based on the leukaemia data set. The area under the K5M curve is more than the area under the MMM which shows the K5M method is more accurate than the MMM.
Hypophosphatemia dataset
The following study is the main application for which the proposed method was specialized and therefore is described in more details. Hypophosphatemia is a common X-linked metabolic bone disorder in human. Hypophosphatemia results from phosphate wasting in the renal tubules. Phosphate that is normally reabsorbed from the urine is excreted. It appears that elevated levels of FGF-23 activate the excretion of phosphorous by the kidneys. Previous studies have demonstrated an impairment of the high- affinity, low capacity Na+ dependent phosphate co-transport system [23,24]. The main animal model used to study this disease is the Hyp mouse. Hyp mice have a mutation of the Phex gene [25,9]. The disease is characterized by low reabsorption of phosphate, bone disease, and bone abnormalities in the lower extremities. The genes active in the regulation of phosphate re-absorption in the kidney are not well understood. It is also not clear whether mutations of the Phex gene block renal adaptation to low phosphate diet. Hyp mice have a primary osteoblast defect and defects in vitamin D metabolism. Parabiosis experiments on normal and Hyp mice have revealed that there is an intrinsic osteoblast defect in Hyp mice rather than an intrinsic renal abnormality. Hyp kidneys transplanted into normal mice reabsorbed phosphorus at normal levels. Kidneys transplanted from normal mice into Hyp mice began phosphate wasting in the Hyp mice.
The mechanism that leads to the excessive excretion of phosphorous is unknown. On a low phosphate diet a normal mouse will activate systems to conserve phosphate by increasing re-absorption. The genes activated in the normal mouse on the low phosphate diet, and the genes with differential expression between normal and Hyp mice should indicate the systems involved in the phosphorus homeostasis. In an attempt to identify these genes, nutritional experiments were performed on normal and Hyp mice [[9,8-14] and [15]]. Normal and Hyp mice were placed on low phosphate diets for 3 – 5 days. Tissue samples from the kidneys of test and control mice were collected. 16 samples were analyzed using Affymetrix GeneChip mouse U74A arrays- 4 samples for each experiment state. The mRNA of 12,488 genes was analyzed. Two GeneChip microarrays were done for each diet for normal mice and three microarrays for each diet for the Hyp mice for a total of 10 arrays.
To investigate this, 5-week-old normal and Hyp were fed a control (1.0% P) or low phosphate (0.03% P) diet for five days. The four group experiments are shown in Table 2.
Table 2 Four experimental groups in the Hyp mice data sets. In this paper, The comparisons are done between group 1 and group2, and between group 3 and group 4.
Diet
Control Low Phosphate
Genotype Normal Group1 Group2
Hyp Group3 Group4
In this study, we consider the gene expression signal less than 100 as noise caused by the microarray machine, and in the pre-processing step we ignored the genes whose expression signals in both conditions are less than 100. The following two specific goals are considered in this study:
1. To identify the genes in whose mRNA expressions are altered by low phosphate diet in normal mice.
2. To determine the effect of Hyp mutation on this response, i.e. identifying the genes in Hyp condition that are differentially expressed across the normal and low phosphate diet experiments.
Results of Hypophosphatemia study
The Hyp dataset includes five samples for each group. In order to make the number of data samples even, we used four samples of each group. For this data set, since j1 = j2, the Mod2MMM cannot be applied. In MMM method, five mixture models are used to estimate f0 and f (distributions under two experimental different conditions) with number of normal basis functions ranging from 1 to 5, i.e. The MMM algorithm was run several times and the run with maximum log-likelihood was chosen as the final model. Bayesian Information Criterion (BIC) [26] was used to determine the number of components. To find the rejection region for a given model, the bisection method is used. In this paper we assume α = 0.01, and therefore the gene-specific significance level used here is calculated as:
α* = 0.01/(95.44 * 2) = 5 * 10-7
Using bisection method [17], as discussed in Section 4, the value of s is obtained as s = 3 × 10-6.. Both the MMM and K5M were run 100 times. Figure 4 presents the number of genes expressed in each run of the MMM. The difference between the number of identified differentially expressed genes in two runs with the minimum and the maximum number of genes amounts to 150 genes. This clearly indicates the high degree of inconsistency and irreproducibility of the results obtained by the MMM. The number of genes expressed in each run of the K5M indicates that all genes are the same in all runs and therefore indicates 100% repeatability and robustness of the proposed method.
Figure 4 Histogram of the number of genes expressed in each run by the MMM method which shows the strong variability (x-axis shows number of runs).
The ten most significant genes expressed by the low phosphate diet in the normal mouse identified by the MMM, and the ten most significant genes provided by K5M are represented in Table 4. As can be seen in Table 5, the most differentially expressed genes are same for the MMM and K5M. Out of these 10 genes, six are directly related to the kidney's functions. For this data set, the main advantage of the K5M is its consistency and robustness as discussed above. A similar procedure is conducted to accomplish the second goal of this study, i.e. identifying the role of Hyp condition on the most definitely expressed gene in normal and low phosphate diet microarrays. The ten most significant genes that are differentially expressed across the two experimental conditions, i.e. Normal Low Phosphate and Hyp Low Phosphate, are listed in table 6. As shown in the table 6, again eight genes are related directly to the kidney's function. These further witnesses to the capability of the proposed technique to discover the genes that are truly involved in the biological study.
Table 5 The top ten significant genes, by comparing group 3 and group 4 in table 2, provided by K5M and MMM.
Accession IDs Gene/ Protein Description Rank based on MMM Rank based on K5M
AF028071 Kidney/ apical plasma membrane, Basolateral plasma membrane 3 1
D26352 Kidney/calcium ion binding 1 2
AA815845 Unknown 2 3
D00073 Kidney/ carrier activity 5 4
AB00603 Monooxygenase activity, oxidoreductase activity 9 5
U97079 GTP binding, protein binding, phosphate binding 7 6
AI315650 Detected in Kidney 6 7
X71922 Kidney/ growth factor activity, hormone activity 11 8
D43797 Kidney/carrier activity, sodium, excitatory glutamate symporter activity Identified as a non expressed gene 9
X81059 Protein phosphate 2 Identified as a non expressed gene 10
Conclusions
In this paper, we proposed a technique to improve the repeatability, and robustness of the mixture model method by using the K-mean clustering method in estimating the distributions. Our proposed method finds the distribution of the variables partially based on a clustering procedure and an EM optimization process. The method is applied to analyze two microarray data sets, Leukaemia data set and a data set reflecting the effect of the low phosphate diet on regular and Hyp mice [8] data. The experimental results indicate 100% robustness and repeatability of the results in different runs and provide 12% improvement (compared to the mixture model method) in detecting the relevant genes in both studies.
Authors' contributions
Maryam Zaheri, and Ali A. Rad were in charge of writing the codes and programming aspects of the paper.
Siamak Najarian and Javad Dargahi's primary role was to perform a literature review on mixture model techniques, identify the aspects of the method that need to be improved, and provide suggestions to address these shortcomings.
Kayvan Najarian's primary roles were to design improvments to the algoritm (based on the literature review and overal modifications suggested by Siamak Najarian and Javad Dargahi), prepare and pre-process the data (for both datasets), partcipate in preperation of the Hyp dataset, define the Hyp problem interpret the results and finally write and edit the manuscript.
Appendix 1
The Mod2MMM makes a new z and Z based on the following formula:
Where:
And:
Acknowledgements
The authors would like to thank Belma Ford (University of North Carolina at Charlotte) for her valuable help in the interpretation of the biology data and results. The authors also thank R. Meyer and M. Meyer in Cannon Research Centre of Carolina Healthcare System for providing us with the Hyp dataset, as well for their assessment and interpretation of our results from biology standpoint.
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| 15603585 | PMC545083 | CC BY | 2021-01-04 16:36:38 | no | BMC Bioinformatics. 2004 Dec 16; 5:201 | utf-8 | BMC Bioinformatics | 2,004 | 10.1186/1471-2105-5-201 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1569620010.1371/journal.pmed.0020001Health in ActionHematologyNutritionPediatricsNutrition and MetabolismPediatricsHematology (including Blood Transfusion)Micronutrient Sprinkles to Control Childhood Anaemia Health in ActionZlotkin Stanley H *Schauer Claudia Christofides Anna Sharieff Waseem Tondeur Mélody C Hyder S. M. Ziauddin Stanley H. Zlotkin is the head of the Division of GI/Nutrition at the Hospital for Sick Children, Toronto, Ontario, Canada, a senior scientist in the Programs in Metabolism and Integrative Biology at the Research Institute of the Hospital for Sick Children, professor in the Departments of Paediatrics and Nutritional Sciences, and senior research fellow at the Centre for International Health, University of Toronto, Ontario, Canada. Claudia Schauer, Anna Christofides, Waseem Sharieff, Mélody C. Tondeur, and S. M. Ziauddin Hyder are researchers in the Programs in Metabolism and Integrative Biology at the Research Institute of the Hospital for Sick Children. Waseem Sharieff is also a graduate student in the Department of Health Policy, Management, and Evaluation at the University of Toronto.
*To whom correspondence should be addressed. E-mail: [email protected]
Competing Interests: Stanley Zlotkin is an occasional consultant to Bristol-Myers Squibb, Mead Johnson, and the Gerber Company (United States), and General Foods (Canada). He owns the intellectual property rights to Sprinkles. The H. J. Heinz Company is supporting the technical development of Sprinkles on a cost-recovery basis. Any profit from royalty fees on the technology transfer of Sprinkles is currently donated to the Hospital for Sick Children Foundation.
1 2005 25 1 2005 2 1 e1Copyright: © 2005 Zlotkin et al.2005This 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 author and source are properly credited.Over 750 million children have iron-deficiency anemia. A simple powdered sachet may be the key to addressing this global problem
A simple powdered sachet may be the key to addressing a global problem
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Recent World Health Organization (WHO)/United Nations Children's Fund estimates suggest that the number of children with iron-deficiency anaemia (IDA) is greater than 750 million [1]. Iron deficiency is the most common preventable nutritional problem despite continued global goals for its control. Historically, the problem of IDA in children largely disappeared in North America when foods fortified with iron and other micronutrients became available for children. In this group, the prevalence of IDA has fallen from 21% in 1974 to 13% in 1994 [2]. Although pockets of infants and children remain at risk, generally, the eradication of iron deficiency in developed countries is recognized as a successful public health accomplishment. This solution has not worked in developing countries where commercially purchased fortified foods are not available or are not used.
In the developing world, there are three major approaches available to address iron deficiency: dietary diversification so as to include foods rich in absorbable iron, fortification of staple food items (such as wheat flour), and the provision of iron supplements. When dietary or fortification strategies are not logistically or economically feasible, supplementation of individuals and groups at risk is an alternative strategy. For the past 150 years or more, oral ferrous sulphate syrups have been the primary strategy to control IDA in infants and young children [3]. However, adherence to the syrups is often limited owing to a combination of their unpleasant metallic aftertaste, the dark stain they leave on the child's teeth, and abdominal discomfort [4]. Thus, despite the ongoing work of the United Nations Standing SubCommittee on Nutrition and others to solve the problem of poor adherence in infants and young children, all interventions to date have been universally unsuccessful [1,5]. In this article, we describe our efforts, stage by stage, towards achieving the goal of controlling IDA.
The Strategy
Our research group at the Hospital for Sick Children in Toronto conceived the strategy of “home fortification” with “Sprinkles”—single-dose sachets containing micronutrients in a powdered form, which are easily sprinkled onto any foods prepared in the household. We hypothesized that this would be a successful method to deliver iron and other micronutrients to children at risk [6]. The idea of Sprinkles was formulated in 1996, when a group of consultants determined that the prevention of childhood IDA was a United Nations Children's Fund priority, yet available interventions (syrups and drops) were not effective [7].
In Sprinkles, the iron (ferrous fumarate) is encapsulated within a thin lipid layer to prevent the iron from interacting with food. This means that there are minimal changes to the taste, color, or texture of the food upon adding Sprinkles. Other micronutrients including zinc, iodine, vitamins C, D, and A, and folic acid may be added to Sprinkles sachets. Any homemade food can be fortified with the single-dose sachets, hence the term “home fortification”. Two formulations have been developed, a nutritional anaemia formulation (Table 1) and a complete micronutrient formulation (Table 2).
Table 1 Daily Dose and Derivation of Sprinkles Nutritional Anaemia Formulation for Home Fortification of Complementary Foods
Data for infants and young children, 6–24 months of age
a Recommended nutrient intake as determined by the WHO [10]
b Recommended dietary allowances as determined by the Institute of Medicine [9]
c Based on DRI “Adequate Intake” estimates
d Assuming medium bioavailability (10%)
e Assuming moderate bioavailability (30%)
DRI, Dietary Reference Intake
Table 2 Daily Dose and Derivation of Sprinkles Complete Micronutrient Formulation for Home Fortification of Complementary Foods
Data for infants and young children, 6–24 months of age
a Recommended nutrient intake as determined by the WHO [10]
b Recommended dietary allowances as determined by the Institute of Medicine [9]
c Recommended nutrient composition of complementary foods (per 50 g of food as daily ration) is based on a total intake of one recommended nutrient intake dose for all children six to 23 months after accounting for the amounts already present in breast milk and complementary food [11]
d Based on DRI Adequate Intake estimates
e Assuming medium bioavailability (10%)
f Assuming moderate bioavailability (30%)
DRI, Dietary Reference Intake
Clinical Trials
Efficacy
To investigate the bioavailability of the iron in Sprinkles, we used a dual stable isotope method and showed that anaemic infants absorbed iron from Sprinkles about twice as efficiently as nonanaemic infants when delivered in a maize-based diet in West Africa. The study was conducted in collaboration with the Kintampo Health Research Centre of the Ministry of Health in Accra, Ghana. The geometric mean iron absorption from two doses of iron (30 mg and 45 mg of elemental iron per sachet) was 8.3% (range, 2.9%–17.8%) in infants with anaemia and 4.5% (range, 1.1%–10.6%) in infants without anaemia [8]. Comparing these absorption values to the new American/Canadian Dietary Reference Intake standards for infants, we concluded that during infancy (i) iron absorption of Sprinkles from a maize-based porridge met and surpassed needs for absorbed iron, and (ii) iron absorption is up-regulated in infants with IDA [9,10,11]. Based on these results, we estimated through computer simulations that a 12.5-mg iron dose (as recommended by the WHO) from Sprinkles should be adequate for use in large-scale distribution programs for the prevention and treatment of mild to moderate anaemia.
It has been suggested that zinc may compete with iron for the same receptor sites on intestinal mucosal cells in the proximal duodenum, thereby compromising the absorption of both minerals [12]. To address this important issue, we recently conducted a bioavailability study in rural Ghana using the same dual stable isotope method as previously used [8]. In order to determine the effect of two doses of zinc on the absorption of iron from Sprinkles (with 30 mg of elemental iron), 63 young children, 12–24 months of age with varying haemoglobin levels were studied. We found that 10 mg of zinc (in the form of zinc gluconate) added to Sprinkles significantly reduced the absorption of iron, whereas a 5-mg dose had no effect. Thus, we concluded that adding 5 mg of zinc to the formulation of Sprinkles was appropriate (unpublished data).
Over the past five years, we have completed seven community-based trials in four different countries [13,14,15,16,17,18,19]. The goal of these studies was to test the efficacy of Sprinkles in diverse settings. When we pooled data from two of our studies that compared Sprinkles to the reference standard, ferrous sulphate drops, we had a total of 518 anaemic infants (haemoglobin < 100 g/l) who were given one of two ferrous sulphate doses (15 mg or 40 mg of elemental iron as ferrous sulphate) and 318 similar infants who received one of four doses of iron from Sprinkles (12.5 mg, 20 mg, 30 mg, or 80 mg of elemental iron as microencapsulated ferrous fumarate) [13,18]. This gave us greater than 97% power (α = 0.05) to detect whether the mean difference in the end-of-study haemoglobin concentrations between ferrous sulphate and Sprinkles regimens was within ± 5 g/l (a range of equivalence). Using a random effects model (for study and dose) that adjusted for baseline haemoglobin, we found no significant difference between Sprinkles and drops.
We further examined this through quantile-quantile plots of haemoglobin concentrations at the end of the studies for Sprinkles and ferrous sulphate drops (Figure 1). The overlaid plots of haemoglobin concentrations of the Sprinkles and drops groups show that these two distributions overlap at all quantiles. These plots clearly indicate that the haemoglobin response to the two different forms of iron was equivalent. Thus, we have concluded that Sprinkles are as efficacious as the current reference standard for the treatment of anaemia. Overall, 55%–90% of the anaemic infants who were provided with Sprinkles were cured.
Figure 1 Overlaid Quantile-Quantile Plots of Haemoglobin Concentrations at the End of Studies for Sprinkles and Ferrous Sulphate Drops
The graph shows that the two distributions overlap at all quantiles, thus proving that there is an equivalent response to the two treatments for haemoglobin concentrations. Circles represent individuals who received iron drops; crosses represent individuals who received Sprinkles.
Acceptability
During our studies we also asked about the caregivers' perception of their infants' responses to Sprinkles as compared to drops, the Sprinkles' impact on the food to which they were added (change in taste, color, or consistency), the use of sachets as a delivery vehicle, and the perceived side effects of Sprinkles [13,18,19]. Invariably, the response to Sprinkles has been positive. No appreciable change in the food with Sprinkles has been reported, no one reported stains on the infants' teeth, and Sprinkles were reported to be easy to use. The only consistently reported side effect was a darkening of the infant's stool, which is expected since most of the iron is excreted in stool. In a recently conducted study in Bangladesh, using a four-point measurement scale, 60% of the mothers “extremely liked”, 30% “liked”, and the remaining 10% “somewhat liked” the Sprinkles intervention; no one disliked Sprinkles. Major reasons cited for liking Sprinkles included ease in mixing Sprinkles with complementary (i.e., weaning) foods and that their use promoted the appropriate introduction of complementary foods, since Sprinkles could be used only if complementary foods were used [19].
Ensuring a Sustainable Supply
As the results of the first studies showing the efficacy of Sprinkles became available, the need for a reliable high-quality supply became apparent. In 2000, the H. J. Heinz Company of Pittsburgh, Pennsylvania, United States, expressed an interest in the Sprinkles program as a component of their corporate social responsibility program. Since 2001, the H. J. Heinz Company has provided support and expertise in the evaluation of consumer needs and a supply of Sprinkles for research, while the H. J. Heinz Company Foundation has provided financial support for research activities. Through a formal process of technology transfer, local overseas Sprinkles production has been encouraged. Currently, an independently licensed copacker is supporting local production for a national program in Guyana, and plans are in place for technology transfer to Bangladesh and Pakistan.
Scaling Up for Countrywide Distribution
The final stage, the scale-up process, is by far the most challenging. First, this process involves dialogue with the Ministries of Health, scientific community, civil society, and other private partners. Second, it is important to identify sustainable methods of distribution that are able to reach and provide Sprinkles to the most vulnerable populations in the developing world. From our experience in Mongolia, we have determined that it is feasible to distribute Sprinkles in partnership with a non-governmental organization called World Vision. Sprinkles sachets distributed in Mongolia over a two-year period included both iron and vitamin D. Sprinkles have been successfully distributed by World Vision field staff to over 15,000 children in seven districts. Coverage has been over 80%, at a cost of about US$0.03 per sachet. In the project area, the prevalence of anaemia (haemoglobin < 115 g/l) and rickets decreased from 42% to 24% and 48% to 33%, respectively [20].
Notwithstanding these positive results on anaemia control, without committed, long-term financial input from national governments, international agencies, or nongovernmental organizations, sustainability is not guaranteed. Clearly, sustainability over the long term can most likely be achieved if a program becomes self-financing. This may be achieved through public- and private-sector partnerships that use effective social marketing models or possibly through programs which include microcredit in order to reach poorer population groups.
When strategizing how to scale up Sprinkles from small-scale research projects to large-scale programs, we quickly realized that our research group did not have the necessary funding, experience, or personnel needed to influence health policy, develop a social marketing strategy, or maintain a distribution network at a countrywide level. We have thus partnered with organizations that specialize in each of these areas to help achieve our goal of sustainable distribution.
For example, the government of Pakistan is planning to distribute Sprinkles through their ongoing Lady Health Worker Program, which is the largest public-sector primary health-care program implemented by the Federal Ministry of Health. In Bangladesh, BRAC (formerly known as Bangladesh Rural Advancement Committee), the largest national non-governmental organization in the country, is planning to distribute Sprinkles through their ongoing Female Community Health Worker program (popularly known as Shastha Shebika). In both of these countries, Sprinkles would be produced locally through public–private partnerships via a technology transfer agreement. The cost per sachet of locally produced Sprinkles should range from US$0.010 to US$0.015, depending on the volume of production, as compared to US$0.020 to US$0.025 if imported.
Conclusion
Each stage in the evolution of the Sprinkles intervention has been evaluated in a controlled manner. We determined that the use of encapsulated iron did not appreciably change the taste or color of the food to which it was added, we showed that the haemoglobin response in anaemic infants was equivalent to the current standard of practice, and we documented the acceptability of Sprinkles among caregivers who used Sprinkles in their homes. Finally, through various partnerships, we have developed a successful model to scale up the intervention for countrywide use. Our challenge for the future is to demonstrate the cost-effectiveness of this new intervention and to advocate for the adoption of Sprinkles in the nutrition policy of developing countries.
The research describing the development of Sprinkles was generously supported by grants from the United States Agency for International Development's OMNI Research Program through the Human Nutrition Institute of the International Life Sciences Institute Research Foundation, the Canadian Institutes of Health Research, Health Canada, the International Atomic Energy Agency, and the H. J. Heinz Company Foundation.
Citation: Zlotkin SH, Schauer C, Christofides A, Sharieff W, Tondeur MC, et al. (2005) Micronutrient Sprinkles to control childhood anaemia. PLoS Med 2(1): e1.
Abbreviations
IDAiron-deficiency anaemia
WHOWorld Health Organization
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Ramakrishnan U Yip R Experiences and challenges in industrialized countries: Control of iron deficiency in industrialized countries J Nutr 2002 132 820S 824S 11925488
Andres NC Disorders of iron metabolism N Eng J Med 1999 341 1986 1995
Galloway R McGuire J Determinants of compliance with iron supplements: Supplies, side effects or psychology Soc Sci Med 1994 39 381 390 7939855
Stoltzfus R Defining iron-deficiency anemia in public health terms: A time for reflection J Nutr 2001 131 565S 567S 11160589
Schauer C Zlotkin SH “Home-fortification” with micronutrient sprinkles—A new approach for the prevention and treatment of nutritional anemias J Paediatr Child Health 2003 8 87 90
Nestel P Alnwick D Iron/multi-micronutrient supplements for young children: Summary and conclusions of a consultation held at UNICEF, Copenhagen, August 19–20, 1996 1996 Washington (D.C.) International Life Sciences Institute Available: http://inacg.ilsi.org/file/ironmicr.pdf . Accessed 24 November 2004
Tondeur MC Schauer C Christofides AL Asante KP Newton S Determination of iron absorption from intrinsically labeled microencapsulated ferrous fumarate (Sprinkles) in infants with varying iron/hematologic status using a dual stable isotope method Am J Clin Nutr 2004 80 1436 1444 15531698
Food and Nutrition Board, Institute of Medicine Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium and zinc 2000 Washington (D.C.) National Academies Press Available: http://www.nap.edu/books/0309072794/html/ . Accessed 22 November 2004
World Health Organization/Food and Agriculture Organization of the United Nations Human vitamin and mineral requirements: Report of a joint FAO/WHO expert consultation 2002 Rome Food and Agriculture Organization of the United Nations Available: http://www.fao.org/documents/show_cdr.asp?url_file=/DOCREP/004/Y2809E/y2809e00.htm . Accessed 24 November 2004
Lutter CK Dewey KG Proposed nutrient composition for fortified complementary foods J Nutr 2003 133 3011S 3020S 12949402
Rolfs A Hediger MA Metal ion transportation in mammals: Structure, function and pathological implications J Physiol 1999 519 1 12 10432334
Zlotkin SH Arthur P Antwi KY Yeung G Treatment of anemia with microencapsulated ferrous fumarate plus ascorbic acid supplied as ‘sprinkles’ to complementary (weaning) foods Am J Clin Nutr 2001 74 791 795 11722961
Zlotkin SH Antwi KY Schauer C Yeung G Use of microencapsulated ferrous fumarate sprinkles to prevent recurrence of anaemia in infants and young children at high risk Bull World Health Organ 2003 81 108 115 12756979
Zlotkin S Arthur P Antwi KY Schauer C Yeung G Home-fortification with iron and zinc Sprinkles or iron Sprinkles alone successfully treats anemia in infants and young children J Nutr 2003 133 1075 1080 (deceased) 12672922
Chan M Zlotkin S Yin SA Sharieff W Schauer C Comparison of dosing frequency and safety of micronutrient Sprinkles on iron status in preschool children in northern China [abstract] International Nutritional Anemia Consultative Group [INACG] Symposium Program Abstracts 2003 2003 February 6 Marrakech, Morocco Washington (D.C.) ILSI Research Foundation 42
Christofides A Zlotkin S Schauer C Impact of micronutrient sprinkles for the treatment and prevention of iron deficiency in Canadian first nations and Inuit infants 4–18 months old [abstract] FASEB J 2003 17 Abstract # 690.13. Available: http://select.biosis.org/faseb . Accessed 22 November 2004
Zlotkin SH Christofides A Schauer C Asante KP Owusu-Agyei S Home fortification using sprinkles containing 12.5 mg of iron successfully treats anemia in Ghanian infants and young children [abstract] FASEB J 2004 18 Abstract # 343.2. Available: http://select.biosis.org/faseb . Accessed 22 November 2004
Hyder SMZ Zlotkin SH Haseen F Zeng L Efficacy of daily vs. weekly home fortification of weaning foods with Sprinkles among infants and young children in Dhaka, Bangladesh Workshop proceedings from the National Workshop on Home Fortification of Weaning Food with Sprinkles: A new strategy to control iron deficiency anaemia among infants and young children 2004 2004 February 11 Dhaka Bangladesh Rural Advancement Committee 1 24
Schauer C Zlotkin S Nyamsuren M Hubbell CR Chan M Process evaluation of the distribution of micronutrient Sprinkles in over 10,000 Mongolian infants using a non-governmental organization (NGO) program model [abstract] International Nutritional Anemia Consultative Group [INACG] Symposium 2003 2003 February 6 Marrakech, Morocco Washington (D.C.) ILSI Research Foundation 42
| 15696200 | PMC545194 | CC BY | 2021-01-05 11:13:37 | no | PLoS Med. 2005 Jan 25; 2(1):e1 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020001 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1569620710.1371/journal.pmed.0020002Neglected DiseasesImmunologyInfectious DiseasesEpidemiology/Public HealthRespiratory MedicineInfectious DiseasesRespiratory MedicineMicrobiologyImmunology and allergyMedicine in Developing CountriesCoccidioidomycosis—A Fungal Disease of the Americas Neglected DiseasesHector Richard F *Laniado-Laborin Rafael Richard F. Hector is project director of the Valley Fever Vaccine Project at the Institute for Global Health, University of California, San Francisco, United States of America. Rafael Laniado-Laborin is a professor at the Tijuana Medical School, Universidad Autonoma de Baja California, Tijuana, Mexico.
Competing Interests: The authors both declare that they have no competing interests.
*To whom correspondence should be addressed: E-mail: [email protected] 2005 25 1 2005 2 1 e2Copyright: © 2005 Hector and Laniado-Laborin.2005This 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 author and source are properly credited.Coccidioidomycosis was first recognized as a serious disease over 100 years ago, but the disease remains an enigma and often goes undiagnosed, even in endemic areas
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It has been more than a century since coccidioidomycosis was first recognized as a serious disease, and its etiology and epidemiology have been well documented. But the disease remains an enigma to many, and it often goes undiagnosed, even in endemic areas. As management of this chronic disease remains problematic, new preventive or therapeutic options are needed.
Etiology and Epidemiology
Coccidioidomycosis is a fungal disease found only in the Western Hemisphere. It is caused by two nearly identical species, Coccidioides immitis and C. posadasii, generically referred to as the “Californian” and “non-Californian” species respectively [1]. The fungus grows in a mycelial phase (see Box 1) in the soil within a geographically delineated area of the United States known as the Lower Sonoran Life Zone [2]. This semiarid zone encompasses the southern parts of Texas, Arizona, New Mexico, and much of central and southern California (Figure 1).
Box 1. Glossary
Mycelial phase—The growth form in the soil, composed of filamentous hyphae and reproductive spores called arthroconidia.
Arthroconidia—Reproductive spores, highly resistant to dessication, which are the infectious particles inhaled by man and animals.
Spherules—The parasitic phase of this dimorphic fungus; spherules are round cells of 30–100 µM or more that reproduce the progeny endospores.
Endospores—The progeny units of the parasitic phase, derived from spherules.
Figure 1 Geographic Distribution of Coccidioidomycosis
(Illustration: Margaret Shear)
Endemic regions for coccidioidomycosis have long been identified in semiarid areas in Mexico [3], and smaller endemic foci have been described in areas of Central and South America [4,5]. More recently, Brazil has also been found to contain endemic areas in the semiarid northeastern states of the country [6]. The climatic conditions and flora of these states are similar to those in endemic regions in North, Central, and South America. In Latin America, Mexico has the largest number of reported cases, with the prevalence of infection in northern Mexico reported to be between 10%–40% [7,8]. C. posadasii is thought to be the predominant species in Mexico [3].
As the soil dries or nutrients become limiting, the fungus reproduces asexually by disarticulating the hyphae into small, environmentally-resistant arthroconidia (reproductive spores) (Figure 2). These are easily aerosolized when the soil is disturbed by wind or human activities. Consequently, it is the inhalation of the dust-borne arthroconidia that leads to infection by this pathogenic fungus in both humans and domestic or wild mammals. Upon inhalation, the fungus converts to a unique life cycle of alternating spherules and progeny endospores, which comprises the parasitic phase of this dimorphic fungus (Figure 2) [9]. Mycelial elements are only occasionally found in diseased tissue [10]. Coccidioidomycosis is not contagious; reports of human-to-human spread are extremely rare. Hence, primary exposure to contaminated dust is the sole risk factor for the acquisition of this disease.
Figure 2 Life Cycle of Coccidioides immitis
(Illustration: Michael Borjon/The Bakersfield Californian)
It is estimated that upwards of 100,000 primary coccidioidal infections occur in humans each year in the endemic areas of the United States [11]. In recent years, the incidence of the disease has increased in California and Arizona, which may be partially due to the rapid immigration of previously unexposed persons from states outside the endemic areas (in other words, the pool of susceptible people has increased) [12]. In the United States, diagnosis in patients who have symptoms is established by serodiagnosis in conjunction with patient history. In previous decades, a coccidioidal skin test antigen was a useful adjunct in the diagnosis, but it became unavailable in the 1980s [13]. The incidence of primary pulmonary disease outside the United States is not established; most reports are limited to disseminated or unusual cases [14]. Diagnosis in Latin America is usually based on microbiologic findings, as serology is not always available [14].
Clinical Features
In their pioneering epidemiologic studies, Smith and colleagues found that about 60% of exposures to the fungus result in asymptomatic infection [15]. In the 40% of patients who have symptomatic disease, there are protean manifestations. These range from a primary, or benign, pulmonary infection (commonly known as “Valley Fever”) to a progressive pulmonary or extrapulmonary disease involving the skin, bones and/or joints, the central nervous system, and other organ systems. Fortunately, most patients with primary disease recover spontaneously and retain lifelong immunity to exogenous reinfection. Chronic and disseminated disease is estimated to occur in up to 5% of infected individuals, with comparatively more cases occurring in older individuals and in males [12]. The most dangerous form of the disease is meningeal infection, which occurs in about 0.15%–0.75% of extrapulmonary coccidioidomycosis cases and requires treatment for life [16].
In regions where tuberculosis rates are high, the two diseases may occur together. Tuberculosis and coccidioidomycosis share common epidemiological, clinical, radiographic, and even histopathological features, making a correct diagnosis extremely difficult in cases where both diseases coexist. In areas where both diseases are endemic, the pertinent studies for diagnosing both conditions should be performed in every patient with compatible clinical features. The diagnosis of one of them does not exclude the possible existence of the other [17].
Treatment
Historically, patients with the primary respiratory form of the disease were not treated because the vast majority recovered on their own. Instead, such patients were given supportive care and were monitored, often with radiographs, until the disease resolved. In recent years, however, an increasing number of physicians are prescribing azole antifungals in cases of primary disease, both because drugs like fluconazole have a good safety record, and because there is a perception that treatment may prevent progression to more serious forms of the disease. This latter presumption, however, is not supported by controlled trial data.
All cases of chronic or disseminated disease call for antifungal therapy, but the choice of drugs, route, and duration of therapy is highly dependent on the form of the disease, the severity and site(s) of infection, and the immune status of the patient. Galgiani and colleagues have published clinical practice guidelines on the choice of drug and duration of therapy for a given form of the disease [18].
There are only two classes of antifungal therapy routinely used for treatment of coccidioidomycosis. The first class is the polyenes, with amphotericin B desoxycholate and the newer lipid formulations used for the more serious forms of disease. The second class is the azoles, with ketoconazole, fluconazole, itraconazole, and the newer analogue voriconazole as available options. Voriconazole, in particular, is being used more and more often in life-threatening mycoses, and was found to be better than amphotericin B in the primary therapy of invasive aspergillosis [19]. According to available reports, treatment in Latin America usually consists of one of the azoles (fluconazole or itraconazole) and/or amphotericin B desoxycholate; lipid formulations are too costly to be accessible[20].
Treatment of the more serious or aggressive forms of the disease is typically of long duration and often results in less than complete resolution of disease; relapse is common [21]. Unfortunately, information on the treatment of coccidioidomycosis is limited, due to the small numbers of controlled trials performed for what is perceived to be a niche market. Clearly, newer, more powerful drugs are needed.
In addition to drugs, surgery is sometimes indicated to remove focalized infections, such as pulmonary cavities, or to debride osseous forms of the disease [22].
Immunology and the Basis for a Vaccine
Acquired resistance to coccidioidomycosis strongly correlates with the development of a delayed-type hypersensitivity skin test response to coccidioidal antigens [23] and the production of T-helper-1 (Th1)-associated cytokines to coccidioidal antigens, such as interferon-gamma (IFN-γ) and Interleukin-2 (IL-2) [24]. Humoral immunity plays no known role in overcoming infection.
Although all humans are equally susceptible to initial infection, there is evidence of genetic predisposition to dissemination, independent of socioeconomic or environmental factors, particularly among African-Americans and Filipinos [25]. Pregnancy is also a risk factor.
In cases of marked immunosuppression, either in advanced AIDS or other forms of depressed cellular immunity, the management of coccidioidomycosis is particularly challenging and requires aggressive treatment [26].
As previously mentioned, recovery from disease confers lifelong immunity to reinfection, and is a rationale for the development and implementation of a vaccine for the prevention of symptomatic or serious forms of the disease. The combination of increasing incidence of disease, a growing population in the endemic area, and the lack of a highly effective drug treatment justifies efforts to prevent (rather than treat) this disease. To that end, a university-based consortium, the Valley Fever Vaccine Project (www.valleyfever.com), has identified and cloned immunogenic proteins that have proven effective in the prevention of deaths and fungal burdens in mouse models of coccidioidomycosis. This suggests that a vaccine for use in humans could be created [27]. A candidate vaccine comprised of a fusion protein based on two antigens has been selected and is currently in pharmaceutical development under the sponsorship of this project, with the goal of evaluating the safety and immunogenicity in humans.
Conclusion
Although the vast majority of infected individuals emerge from coccidioidomycosis without complications, an unlucky minority are faced with a debilitating disease that lacks adequate drug options for rapid and completely effective treatment. In the absence of newer therapeutics, discoveries that lead to immunologic intervention [28] or prevention by vaccines may ultimately bring a measure of relief.
Citation: Hector RF, Laniado-Laborin R (2005) Coccidioidomycosis—A fungal disease of the Americas. PLoS Med 2(1): e2.
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Rios-Olivares EO 1st human case of coccidioidomycosis in Nicaragua Rev Latinoam Microbiol 1979 21 215 218 550226
Bonardello NM de Gagliardi CG Intradermal reactions with coccidioidins in different towns of San Luis Province Sabouraudia 1979 17 371 376 545714
Wanke B Lazera M Monteiro PC Lima FC Leal MJ Investigation of an outbreak of endemic coccidioidomycosis in Brazil's northeastern state of Piaui with a review of the occurrence and distribution of Coccidioides immitis in three other Brazilian states Mycopathologia 1999 148 57 67 11220226
Laniado-Laborín R Cárdenas-Moreno RP Alvarez-Cerro M Tijuana: zona endémica de infección por Coccidioides immitis
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Padua y Gabriel A Martínez-Ordaz VA Velasco-Rodreguez VM Lazo-Sáenz JG Cicero R Prevalence of skin reactivity to coccidioidin and associated risks factors in subjects living in a northern city of Mexico Arch Med Res 1999 90 388 392
Sun SH Huppert M A cytological study of morphogenesis in Coccidioides immitis
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Weiden MA Saubolle MA Einstein HE Catanzaro C The histopathology of coccidioidomycosis 1996 Washington D.C National Foundation for Infectious Disease 12 17 Coccidioidomycosis
Galgiani JN Coccidioidomycosis: A regional disease of national importance; Rethinking our approaches to its control Ann Intern Med 1999 130 293 300 10068388
Ampel NM Mosely DG England B Vertz PD Komatsu K Coccidioidomycosis in Arizona: Increase in incidence from 1990 to 1995 Clin Infect Dis 1998 27 1528 1530 9868673
Smith CE Diagnosis of pulmonary coccidioidal infections Calif Med 1951 75 385 391 14886741
López-Márquez A Hernández-Avendaño V Durán-Padilla MA Navidad-Cervera F Chávez-Macías L Coccidioidomicosis diseminada con infección pulmonar, ganglionar y meníngea. Caso con hallazgos postmortem Rev Med Hosp Gen (Mex) 2004 67 88 93
Smith CE Beard RR Whiting EG Rosenberger HG Varieties of coccidioidal infection in relation to the epidemiology and control of the diseases Am J Public Health 1946 36 1394 1402
Cortez KJ Walsh TJ Bennett JE Successful treatment of coccidioidal meningitis with voriconazole Clin Infect Dis 2003 36 1619 1622 12802765
Castañeda-Godoy R Laniado-Laborín R Coexistencia de tuberculosis y coccidioidomicosis. Presentación de dos casos clínicos Rev Inst Nal Enf Resp Mex 2002 15 98 101
Galgiani JN Ampel NM Catanzaro A Johnson RH Stevens DA Practice guidelines for the treatment of coccidioidomycosis Clin Infect Dis 2000 30 658 661 10770727
Herbrecht R Denning DW Patterson TF Bennett JE Greene RE Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis N Engl J Med 2002 347 408 415 12167683
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| 15696207 | PMC545195 | CC BY | 2021-01-05 10:38:11 | no | PLoS Med. 2005 Jan 25; 2(1):e2 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020002 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1569621010.1371/journal.pmed.0020003Policy ForumInfectious DiseasesPharmacology/Drug DiscoveryMalariaPharmacology and toxicologyMedicine in Developing CountriesPregnancyInfantsIntermittent Presumptive Treatment for Malaria Policy ForumWhite Nicholas J Nicholas J. White is at the Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, and the Centre for Clinical Vaccinology and Tropical Medicine, Churchill Hospital, Oxford, United Kingdom. E-mail: [email protected]
Competing Interests: The author is on the editorial board of PLoS Medicine.
1 2005 25 1 2005 2 1 e3Copyright: © 2005 Nicholas J. White.2005This 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 author and source are properly credited.A better understanding of the pharmacodynamics of intermittent presumptive treatment, says White, will guide more rational policymaking
A better understanding of the pharmacodynamics will guide more rational policymaking
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Intermittent presumptive treatment (IPT) in pregnancy involves giving a curative treatment dose of an effective antimalarial drug at predefined intervals during pregnancy. IPT in pregnancy was first introduced in areas of high malaria transmission as a measure to reduce the adverse impact of Plasmodium falciparum malaria in pregnancy [1,2,3,4,5,6,7,8]. Later, based on trials showing that IPT could reduce anaemia in young children and also malaria episodes in infants, it was extended as a measure to reduce morbidity and mortality in the first year of life [9,10,11,12].
Antimalarial chemoprophylaxis for pregnant women living in endemic areas has been recommended for many years, but in practice has been limited to the use of chloroquine and pyrimethamine [13,14]. Unfortunately, there are few places left in the world where these drugs can still be relied upon to prevent P. falciparum malaria. There are insufficient safety data on the newer antimalarials to warrant their systematic use in pregnant women. IPT with sulphadoxine-pyrimethamine (SP) has been introduced as an alternative. Antimalarial chemoprophylaxis in young children has been shown to reduce the adverse impact of P. falciparum malaria [15,16,17], but this intervention never obtained the same endorsement as chemoprophylaxis in pregnancy.
Five randomised trials of IPT in pregnancy in East Africa have been reported [1,2,3,4,5], all with SP, all in high-transmission settings, and all done between 1992 and 1999 (Table S1). The alarming recent increase in resistance to SP in Africa confounds the cost-effectiveness assessments upon which subsequent policy recommendations for IPT in pregnancy were based [18,19].
There is no consensus on how IPT works, making planning difficult. This article argues that IPT provides mainly intermittent suppressive chemoprophylaxis (as opposed to treatment effect alone or some other magical effects which have never been specified). If this is correct dosing schedules should be individualised for each antimalarial depending on the drug's pharmacokinetic and pharmacodynamic properties. As increasing resistance to SP must seriously compromise IPT regimens based on this drug, the evaluation of available new effective antimalarials is needed urgently, in both high- and low-transmission areas.
Pharmacokinetics
After a treatment dose of SP (25 mg sulfadoxine/1.25 mg pyrimethamine per kilogram body weight), plasma concentrations of pyrimethamine (half-life, 3 days) and sulfadoxine (half-life, 7 days) decline log-linearly [20,21]. The antimalarial effect depends on synergy between the two components, but the effect from one treatment dose can last as long as 60 days with fully sensitive P. falciparum [20,21]. For slowly eliminated antimalarial drugs (Table S2), the terminal elimination phase crosses the in vivo dose–response curve (Figure 1). Thus, if a full treatment dose is given, concentrations at the beginning of the terminal elimination phase exceed the minimum parasiticidal concentrations (MPCs)—the lowest concentrations that give maximum effect [22]. The exceptions to this are chloroquine (and probably piperaquine), as resistance to these drugs increases, because the elimination of chloroquine is multiexponential, and the terminal elimination phase begins at concentrations that are low by comparison with the peak concentrations after treatment (Figure 2).
Figure 1 In Vivo Antimalarial Pharmacodynamics
The parasite burden in an adult (vertical axis) is shown in green. After parasite burden expands to the point where it causes illness, treatment is given (red arrow), which causes a log-linear decline in parasite numbers until concentrations of the antimalarial drug (grey shading) fall below the MPC. As the antimalarial blood levels fall further, the decline in parasite burden slows until it reaches a multiplication rate of one (the antimalarial concentration at this point is the in vivo MIC). The parasite population then expands to cause a recrudescence six weeks later. The sigmoid concentration–effect relationship is shown in brown; it is depicted in the reverse direction to that normally drawn. PMR, parasite multiplication rate.
Figure 2 Blood Concentration Profiles of Two Antimalarials with Different Elimination Profiles
The examples shown here are mefloquine (orange) and chloroquine (pink). An increase in MIC has different effects on the shortening of post-treatment suppressive prophylaxis (hatched bars). MICR, MIC for resistant parasites; MICS, MIC for sensitive parasites.
The pharmacokinetic properties of many drugs are altered in pregnancy; lower concentrations often result from an expanded volume of distribution. Strangely, despite the wide endorsement of SP IPT in pregnancy, there are no pharmacokinetic studies of sulphadoxine or pyrimethamine in pregnancy, so it is not known whether the current dosing is optimal. The absorption and disposition of many drugs are also altered in infancy, but there are very few data on antimalarial pharmacokinetics in the first year of life. For some drugs (e.g., amodiaquine) there is insufficient information for any age group.
Pharmacodynamics
Is the benefit of IPT gained only through clearing parasites from the placenta (“treatment effect”), or is the prevention of new infections (“prophylactic effect”) an important component? If only the treatment effect is important, then how long does the beneficial effect of eradicating an asymptomatic low-density infection persist for? If it lasts until the next infection becomes patent (i.e., detectable), then rapidly eliminated drugs will provide protection only for a few days longer than the average incubation period (about two weeks). Establishment of a new placental infection (i.e., pathologically significant placental sequestration) may take longer because the placenta selects and accumulates parasites that bind to the proteoglycans chondroitin sulphate and hyaluronic acid [23]. If only the treatment effect is important, then for sustained benefit we must hypothesise that the parasites that persist asymptomatically before IPT is given are a selected subpopulation that is more pathological than the parasites that cause subsequent reinfection. This seems implausible in infancy, and even in pregnancy it seems unlikely that it would take more than ten weeks in high-transmission settings to re-establish a significant placental infection. This suggests that the prophylactic effect is important for the efficacy of IPT.
The duration of prophylactic effect is compromised particularly by resistance. For most antimalarials the duration of antimalarial effect is a simple function of the in vivo concentration–effect (dose–response) relationship and the pharmacokinetic properties of the antimalarial drug [22]. But for SP this function is more complicated, as synergy between the two components needs to be considered. The duration of synergy depends on resistance levels determined by mutations in the parasites' genes encoding dihydropteroate synthase and dihydrofolate reductase, the respective targets of sulphadoxine and pyrimethamine [20,21]. Information on this temporal pattern of reinfection following IPT in pregnancy or infancy is lacking. Such information is essential if the choice of drug and the dosing is to be rationalised.
Resistance is defined by a right shift in the concentration–effect relationship and results in reduced effects for any concentration below the MPC for resistant parasites [24] (see Figure 1). As the concentration of a slowly eliminated antimalarial in the blood declines, it continues to suppress the growth of newly acquired infections as they emerge from the liver. Eventually, however, concentrations fall below the minimum inhibitory concentration (MIC) for the prevalent parasites (i.e., the concentration at which the net multiplication rate is one), and parasite expansion is possible (Figure 3). It follows, then, that the duration of “post treatment prophylaxis” (PTP) (i.e., the length of time after an antimalarial treatment dose for which newly acquired infections are suppressed) is determined by the concentrations of the drugs used (determined by dose and pharmacokinetics) and the sensitivity of the prevalent parasites. The more resistant the parasites are, the shorter is the duration of PTP; for each doubling of MIC the duration of PTP is shortened by one half-life (Protocol S1). The triple dihydrofolate reductase mutants now prevalent across much of Africa have an approximate 1,000-fold reduction in pyrimethamine susceptibility, which would translate into a reduction in PTP of one month (Figure 4). As a further confounder, folic acid, which is prescribed widely in pregnancy, is a competitive antagonist of pyrimethamine.
Figure 3 Hypothetical Parasite Burden Profiles during Pregnancy with SP IPT in a High-Transmission Setting
Entomological inoculation rate is about 50 infectious bites per person per year. Note that many infections self-cure (each infection is depicted as a green line). The hatched bars represent the duration of “suppressive prophylactic activity”, and the solid bars represent the period during which parasite multiplication is suppressed (i.e., levels exceed the in vivo MIC). The horizontal dotted line at 108 parasites represents the level at which malaria can be detected on a blood film. (A) represents a drug-sensitive area; (B) represents a moderately resistant area.
Figure 4 Relationship between MIC and PTP
The proportional increase in malaria parasite MIC with resistance is plotted against the shortening of the duration of PTP, expressed as multiples of the terminal half-life. This applies only to drugs for which suppressive antimalarial prophylaxis occurs in the terminal elimination phase (i.e., most drugs).
Preventing Placental Pathology
In a high-transmission setting infections are acquired every few days or weeks throughout life (see Figure 3). Mortality is high in childhood, but by the time of adulthood and pregnancy, infections are largely asymptomatic—although they are often still patent (which requires a total burden of greater than 100 million parasites) [22]. Thus, immunity prevents life-threatening parasite burdens, and suppresses the pro-inflammatory response (which causes illness), but it does not prevent infection. In pregnancy this immune control is impaired in the placenta, which acts as a “privileged site” for parasite multiplication. The objective of IPT in pregnancy is to reduce or eliminate the adverse effects of malaria on maternal anaemia and birth weight, and, in addition, in a low-transmission setting, to prevent severe malaria in the mother [25,26]. How malaria produces intrauterine growth retardation is still unresolved, but in P. falciparum malaria, retardation tends to be greatest in the first pregnancy, and often occurs without maternal illness. The greater the placental parasite burden, the greater is the reduction in birth weight. “Placental malaria”—histological evidence of placental accumulation of parasitized erythrocytes or malaria pigment deposition—has often been used as an endpoint in intervention studies, although the quantitative relationship between placental malaria and reduction in birth weight remains poorly characterised.
Preventing Malaria in Infancy
There are fewer data on the efficacy of IPT in infancy than in pregnancy (Table S3). The pharmacodynamics of IPT in infancy are probably similar to those in pregnancy, although there is no “privileged site” for parasite multiplication. Protection in the first months of life is mediated by a variety of factors, which include transplacentally acquired maternal antibody (IgG) and a relatively high haemoglobin F content in the infants' erythrocytes. After about six months of age, protection from these factors wanes, and the infant becomes much more vulnerable to malaria than the mother (because protective immunity has yet to be acquired). As delivery of antimalarials in the rural tropics is so difficult, for operational reasons IPT is currently being given to infants at the same time as the EPI immunisations (at 2, 3, and 9 months). This regimen leaves a six-month gap between the second and third administrations, which, even for fully SP-sensitive parasites, leaves four unprotected months. This is at a time when the infant is increasingly vulnerable to severe malaria. More information is needed on the duration of protection afforded by currently available antimalarial drugs when administered to healthy infants.
Should IPT be Used in Low-Transmission Settings?
If IPT is just a simple, albeit imperfect, way of administering chemoprophylaxis, then there are also strong arguments for evaluating this approach in low-transmission settings. The adverse impact of malaria in pregnancy is greater in low-transmission than in high-transmission settings. The reduction in the birth weight of first-borne infants is similar, but extends to the second and subsequent pregnancies; treatment failure rates are higher than in non-pregnant adults [27,28,29]; and there is a significant risk of severe malaria with attendant very high mortality. In Asia and South America, where low-transmission areas predominate, P. vivax is also an important cause of low birth weight, and so useful preventative measures must also be effective against this infection [30].
Antimalarial prophylaxis has been recommended and used in low-transmission settings, but whereas chloroquine remains generally effective against P. vivax, there are no safe and effective available drugs for P. falciparum infections. Use of IPT in these areas would provide a sterner test than in a high-transmission area because there would be little or no background immunity to assist antimalarial drug efficacy.
What Is the Correct Dose and the Correct Interval Between Doses?
The dose used in IPT is usually the full age- or weight-adjusted treatment dose derived either empirically or from dosefinding studies (usually in non-pregnant adults). The dose and dosing interval should be determined by the tolerability, absorption, distribution, and elimination kinetics of the drug used, and the in vivo MIC. Unfortunately, for the two drugs that have been used for IPT (SP and amodiaquine) there are no pharmacokinetic data in pregnant women or infants. The in vivo MIC is an important measure but it is parasite specific and difficult to assess [31,32,33,34,35]. Ideally, the interval between doses should not be more than one week longer than the time needed for plasma concentrations to fall from peak post-dose levels to the MIC value. This timing is a conservative choice as it assumes all infections are equally harmful, and it does not take into account either the delay in selecting a placenta-binding P. falciparum subpopulation in pregnancy or the delay in achieving full growth rates because of continued sub-MIC suppression. Although the MIC for drugs that are succumbing to resistance obviously varies considerably, for newer drugs such as lumefantrine or piperaquine the variance is considerably less and generalisations can be made.
Should an Artemisinin Combination Be Used for IPT?
If IPT is simply prophylaxis, then a rapidly eliminated artemisinin component provides very little direct benefit for the additional cost and risk (although the risks are thought to be very small in the second and third trimesters of pregnancy and in infancy). The addition of an artemisinin component would accelerate parasite clearance and prevent gametocyte production, but the benefits of this in an asymptomatic pregnant woman or child are uncertain. The main benefit would be in providing protection against the emergence of de novo resistance to the slowly eliminated drug, although, because parasitaemias tend to be low, the probabilities of de novo selection are much lower than in acute symptomatic infection. But there is a genuine concern that if monotherapies are made available, then they will be used and abused, and resistance may develop.
Discussion: The Policy Implications
Without a better understanding of the pharmacodynamic effects of IPT, it will be difficult to make rational improvements in this promising approach to malaria prevention. The most parsimonious explanation for its effectiveness is that IPT provides antimalarial prophylaxis that, if sufficiently lengthy and effective, is beneficial both to the pregnant woman and the infant. But how lengthy and how effective?
For IPT in pregnancy the only drug that has been evaluated is SP, at a time when the drug was more effective than it is today. A significant improvement in birth weight was found in only two of four randomised trials. In a large prospective observational study conducted in western Kenya, IPT was associated with an odds ratio of 0.65 (95% confidence interval, 0.45 to 0.95) for low birth weight [7]. A dose–response relationship was found, with an adjusted mean increase in birth weight of 61 g for each increment in the number of SP doses (up to three doses). So, two doses of SP did not provide maximal benefit. But given the alarming decline in SP efficacy in Africa (resulting from rapid spread of “quintuple” dihydrofolate reductase/dihydropteroate synthase mutants that are about 1,000 times less sensitive to pyrimethamine than wild-type parasites), there are grave doubts about whether the efficacy observed in these various studies would still be observed today, even if the dosing was increased. SP cure rates in children in Malawi—where IPT in pregnancy is widely used—have been consistently less than 40% for the past five years [36]. It has been suggested that asymptomatic pregnant women in high-transmission settings may have sufficient immunity to complement a failing drug—i.e., treatment responses would be better than in symptomatic children. However, if the duration of PTP is the main determinant of benefit, then this benefit is shortened progressively by increasing resistance (see Figure 4). Alternatives to SP are needed urgently.
Is IPT safe? There is no evidence to date that IPT is harmful. But the incidence of serious adverse effects when amodiaquine (agranulocytosis, 1:2,000) and SP (Stevens-Johnson syndrome,1:7,000) were used as antimalarial prophylaxis by Western travellers was so high that they are contraindicated [37]. Both drugs were associated with severe hepatitis. Single treatments are considered safer, but how much safer is not known. There are insufficient data on the safety of amodiaquine in pregnancy [38]. The closer IPT comes to continuous prophylaxis, presumably the higher the risks of serious adverse effects. The risk–benefit assessment is difficult to make, but with the current high levels of SP resistance, these important uncertainties also argue strongly for the evaluation of alternatives. Proguanil and quinine are regarded as safe, but both are eliminated very rapidly. Treatment doses of mefloquine are not well tolerated by healthy subjects, and there are safety concerns in pregnancy [39]. Serious contenders all require more than a single dose and will need urgent evaluation. These include artemether-lumefantrine, although more information is needed on safety and on the pharmacokinetics in pregnancy, and the duration of PTP provided by lumefantrine needs further assessment. It may be too short. Dihydroartemisinin-piperaquine may be the most promising candidate. It is very well tolerated, and piperaquine is slowly eliminated. Indirect evidence from the pattern of new infections following clinical trials suggests protracted suppressive activity. It has not yet been evaluated in pregnancy, so more information is needed on safety and pharmacokinetics in this context. For IPT in infancy, however, there seems every reason to evaluate this drug as soon as possible.
Supporting Information
Protocol S1 Calculations Showing That for Each Doubling of MIC the Duration of PTP Is Shortened by One Half-Life
(27 KB DOC).
Click here for additional data file.
Table S1 Randomised Trials of IPT in Pregnancy
(31 KB DOC).
Click here for additional data file.
Table S2 Terminal Elimination Half-Lives of Currently Available Antimalarial Drugs
(34 KB DOC).
Click here for additional data file.
Table S3 Randomised Trials of IPT in Infancy
(27 KB DOC).
Click here for additional data file.
Summary Points
Intermittent presumptive treatment (IPT) with sulphadoxine-pyrimethamine (SP) in pregnancy and with amodiaquine or SP in infancy has been proposed for use in areas with high levels of malaria transmission.
The duration of post treatment prophylaxis is likely to be an important determinant of the benefit of IPT.
Because of rapidly increasing resistance, it is very unlikely that IPT in pregnancy with SP is as effective now in east Africa as it was 5–10 years ago, when it was evaluated.
More effective antimalarial drugs such as artemether-lumefantrine and particularly dihydroartemisinin-piperaquine should be evaluated for IPT in both low- and high-transmission settings.
Choice of drug, dosing, and dose spacing for IPT should be based on a better understanding of pharmacokinetics and pharmacodynamics.
I am very grateful for advice from Francois Nosten, Feiko ter Kuile, Jane Crawley, Nick Day, and Bill Watkins. N. J. White is a Wellcome Trust Principal Fellow.
Citation: White NJ (2005) Intermittent presumptive treatment for malaria. PLoS Med 2(1): e3.
Abbreviations
IPTintermittent presumptive treatment
MICminimum inhibitory concentration
MPCminimum parasiticidal concentration
PTPpost treatment prophylaxis
SPsulphadoxine-pyrimethamine
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| 15696210 | PMC545196 | CC BY | 2021-01-05 11:13:37 | no | PLoS Med. 2005 Jan 25; 2(1):e3 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020003 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1569621410.1371/journal.pmed.0020004EssayInfectious DiseasesEpidemiology/Public HealthHealth EconomicsHealth PolicyHIV/AIDSPublic HealthMedicine in Developing CountriesInfectious DiseasesWomen's HealthHuman rightsDeadly Alliances: Death, Disease, and the Global Politics of Public Health EssayGandy Matthew Matthew Gandy teaches geography at University College London and has been a visiting scholar at Columbia University, the Humboldt University and University of California, Los Angeles. He is author of Concrete and Clay: Reworking Nature in New York City (The MIT Press, 2002) and coeditor of The Return of the White Plague: Global Poverty and the “New” Tuberculosis (Verso, 2003). E-mail: [email protected]
Competing Interests: The author declares that he has no competing interests.
1 2005 25 1 2005 2 1 e4Copyright: © 2005 Gandy.2005This 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 author and source are properly credited.Most people threatened by AIDS, tuberculosis, and unsafe drinking water are poor and have little or no influence over the global politics of public health
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The rancour surrounding the Bangkok AIDS summit of July 2004 has exposed a series of fundamental disagreements surrounding the global politics of public health. These tensions range from access to cheaper lifesaving drugs to disputes over the role of poverty and gender equality in the promotion of sexual health.
The world is now experiencing the most profound public health challenge of the last forty years: we have witnessed the appearance of new diseases such as Ebola, SARS, and in particular, AIDS, combined with the alarming resurgence of diseases previously thought to have been under control, such as malaria and tuberculosis [1,2]. The AIDS pandemic in particular threatens to devastate entire regions and has already fundamentally altered the life expectancy and demographic profile of many countries in sub-Saharan Africa. Billions of people lack access to adequate sanitation and safe drinking water (Figure 1), and the UN predicts that slums will become the dominant urban form within the next fifteen years (Figure 2) [3,4,5].
Figure 1 A Recently Completed Water Supply Project in Amukoko, Lagos, Nigeria
Unsafe drinking water is the major cause of disease in Lagos. Under 5% of households receive a piped water supply and under 1% are connected to a closed sewer system.
(Photo: Matthew Gandy)
Figure 2 Open Sewer with Drinking Water Pipes Passing Through
This photograph was taken in Amukoko, one of the largest slum areas in Lagos, Nigeria, a rapidly growing megacity with an estimated population of 15 million. Lagos is predicted to be one of the world's largest cities within the next 10 years.
(Photo: Matthew Gandy)
Yet the very idea of “public health” sits uncomfortably alongside the current emphasis of bio-medical science on the molecular realm of DNA coding and the development of lucrative Western markets for new pharmaceutical products such as sildenafil (Viagra). The needs of the majority—the global poor—scarcely feature within this tactical alliance between the biomedical sciences and corporate power. Since most people threatened by AIDS, tuberculosis, unsafe drinking water, and other health threats are poor, they have little or no influence over the global politics of public health.
Making Sense of the Crisis
If we are to make sense of the current public health crisis, we need to explore interconnections between political, economic, and social developments that are ignored by the fragmentary emphasis of the biomedical sciences. The current impetus toward economic globalization is causing widespread social and economic disruption, ranging from wild currency fluctuations to the systematic collapse of viable agricultural systems [6]. The imposition of austerity packages—widely referred to as structural adjustment programmes—in combination with the forcible extension of global markets for Western products is plunging millions of people into poverty and economic dependence. Existing primary health care services in much of the developing world and the former states of the Soviet Union have been drastically cut back, and services that were once freely available are now increasingly beyond the reach of the poor. And these harmful trends also extend even to the wealthiest global cities, such as London and New York, where a combination of poverty, homelessness, and cutbacks in primary health care since the 1980s has contributed toward the spread of tuberculosis and other diseases [7,8].
An examination of the social impact of the global public health crisis shows that it is women and children who have been most badly affected. One of the least addressed dimensions to the externally imposed structural adjustment programmes on developing countries is the harmful impact on the sexual health of women caused by their increased economic dependence on men. Sexual health programmes based on abstinence, for example, ignore the difficulties women face in negotiating safer or non-penetrative forms of sex. It is married women in southern Africa and south Asia who make up the largest and most vulnerable group of women, since they are at risk of being infected by their husbands.
A new generation of HIV-positive women activists, such as the Nigerian journalist and AIDS campaigner Rolake Odetoyinbo Nwagwu, are now engaged in a vital struggle to challenge the social attitudes and economic inequalities that have driven the devastating impact of AIDS on women and children in developing countries. The spread of AIDS in more traditional societies is closely linked with patriarchal power structures.. The sustenance of these structures is assured by the rise of poverty-fuelled ethnic and religious chauvinism, which undermines the prospects for developing more progressive approaches to social policy. And in regions where war or civil strife prevail, the vulnerability of women and children to sexual violence, economic exploitation, and disease is even greater so that we cannot consider public health questions separately from issues surrounding political stability and social justice.
Spaces of Exclusion
In order to understand better the political dynamics behind public health we need to recognize that the development of modern forms of governance has emerged in tandem with new approaches toward the administration of human populations. The rise of the 19th-century industrial city, for example, necessitated the development of much more sophisticated forms of urban governance in order to tackle the threat of epidemic disease and enable these new cities to function effectively as centres of economic activity. But these new spaces of public health control, which scholars such as Michel Foucault have so vividly described, had a shadowy “other”. This “other” was represented by the increasingly squalid conditions endured by the mass of the population in European colonies, exemplified by the devastating modern outbreaks of bubonic plague in cities such as Baghdad, Lagos, and Bombay [9].
The Italian political philosopher Giorgio Agamben has elaborated on this distinction between “inside” and “outside” under modern systems of governance to reveal the persistence of what he terms conditions of “bare life” within even the most sophisticated legal and political systems [10]. The contemporary exclusion of the world's poor from adequate medical care is thus a form of state-sponsored violence, in which millions are denied even the most basic human rights. These “wasted lives”, to use the sociologist Zygmunt Bauman's phrase, represent a literal as well as metaphorical process of permanent and deadly exclusion for the poor, the marginalized, and others who have no value within the global economy [11].
Excluding the Poor from Medical Care
A striking manifestation of this systematic exclusion of the poor from medical care is provided by the Bush administration's efforts to stymie access to affordable antiretroviral drugs. A dramatic standoff in 2001 between the global pharmaceutical industry and public health activists in South Africa, following the import of cheap generic antiretroviral drugs manufactured in Brazil, led to the historic Doha Declaration on intellectual property rights and public health [12]. Generic drug production in countries such as India, Brazil, and Thailand has succeeded in bringing down treatment costs per patient from $10,000 to $300 per year, yet the World Health Organization has revealed that less than one in 20 people who need antiretroviral treatment in the developing world are currently receiving it [13].
In order to widen access to affordable drugs, the current production of generic medicines will have to be massively expanded, but the Bush administration and its corporate allies in the pharmaceutical industry has worked assiduously to undermine the potential impact of the Doha agreement [14]. The United States government, for example, has negotiated bilateral trade deals with countries such as Chile and Thailand in an effort to dissuade them from the production of cheaper drugs [15]. Tensions exploded into the open at the Bangkok AIDS summit of 2004, where lobbyists on behalf of the US government put forward a series of specious assertions about the distribution of much-needed AIDS drugs in sub-Saharan Africa. The lobbyists asserted that the costs of drugs have been lowered—yet discounted brands remain at least twice as expensive as generic drugs. And the lobbyists for the Bush administration claimed that generic drugs undermine profitability and hence the incentive for new research (which is, in any case, overwhelmingly focused on the bloated market for prescription drugs in the US).
Of the $15 billion pledged by the Bush administration in the fight against AIDS most of this money will be focused on 15 selected countries. The countries were chosen by their willingness to abide by bilateral trade deals to prevent the production of cheaper generic drugs and their willingness to stress the centrality of abstinence as a strategy for AIDS prevention (a strategy that the religious Right has pushed for) [16,17]. It should also be noted that some of the most vocal critics of US policy, among them France, currently make a derisory financial contribution to global efforts to tackle HIV/AIDS so that the inadequacies of US policy must be viewed in a wider context of Western negligence toward the public health needs of the world's poorest countries [18].
The US government has sought to highlight the inadequacies of health care infrastructure in developing countries as a further justification for the diversion of attention from the cost of drugs. But primary health care services have themselves been undermined by the structural adjustment and trade policies promoted by Western financial institutions and their corporate backers. The global politics of AIDS is therefore caught in a neo-liberal vicious spiral from which it is impossible to disentangle the needs for social and institutional reform in the worst affected regions from the challenge of widening access to available treatments.
The Contemporary Politics of Public Health
Public health pioneers of the past, such as the German bacteriologist Robert Koch, were not only scientists but also political advocates for social change. Improvements in health care were perceived as part of a nexus of reforms that ranged from better housing and nutrition to an extension of voting rights to ensure that the poor had adequate political representation.
The contemporary politics of public health needs to be considered, however, in relation to wider discourses on security and human welfare that are quite different from those of the 19th century. The current Western preoccupation with the threat of international terrorism, for example, has distracted attention from the much more real threats to human well-being created by poverty and ill health. The vast resources—both financial and logistical—now being poured into “security” contrast starkly with the inadequate funding provided for the Global Fund for HIV/AIDS, Tuberculosis, and Malaria. The increasingly unilateral and self-interested stance of the US illustrates the tensions between attempts to build international forms of governance for health care and the continuing centrality of national interests or global institutions that explicitly represent the economic power of a relatively small group of nations [19].
The paradox is that widening global disparities in wealth and poverty play a role in fostering the social and political conditions in which religious fanaticism and hatred for the West can flourish. So any “war on terror” that fails to address the causes of poverty, despair, and insecurity in the lives of the world's poor will ultimately create a more dangerous world for everyone.
Citation: Gandy M (2005) Deadly alliances: Death, disease, and the global politics of public health. PLoS Med 2(1): e4.
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Gandy M Zumla A The return of the White Plague: Global poverty and the “new” tuberculosis 2003 London Verso 320
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Agamben G Heller-Roazen D Homo sacer: Sovereign power and bare life 1998 Stanford (California) Stanford University Press 199
Bauman Z Wasted lives: Modernity and its outcasts 2004 Cambridge Polity 152
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Angell M The truth about drug companies: How they deceive us and what to do about it 2004 New York Random House 336
Alagiri P Summers T Kates J Henry J. Kaiser Family Foundation/Ford Foundation Spending on HIV/AIDS in Resource-Poor Settings 2002 Available: http://www.kff.org/hivaids/loader.cfm?url=/commonspot/security/getfile.cfm&PageID=28514 . Accessed 14 November 2004
Kohlmorgen L Global health governance und UN AIDS: Elemente eines globalen integrationsmodus Peripherie: Zeitschrift für Politik und Ökonomie in der Dritten Welt 2004 93/94 139 165
| 15696214 | PMC545197 | CC BY | 2021-01-05 10:38:13 | no | PLoS Med. 2005 Jan 25; 2(1):e4 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020004 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1569621510.1371/journal.pmed.0020005PerspectivesInfectious DiseasesInfectious DiseasesMedicine in Developing CountriesPublic HealthEnvironmental healthSeasonal Patterns of Infectious Diseases PerspectivesPascual Mercedes *Dobson Andy Mercedes Pascual is an associate professor in the Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan, United States of America. Andy Dobson is a professor in the Department of Ecology and Evolutionary Biology, Princeton University, Princeton, New Jersey, United States of America.
Competing Interests: The authors declare that no competing interests exist.
*To whom correspondence should be addressed. E-mail: [email protected] 2005 25 1 2005 2 1 e5Copyright: © 2005 Pascual and Dobson.2005This 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 author and source are properly credited.
Climate Drives the Meningitis Epidemics Onset in West Africa
Why is that many infectious diseases, like cholera, malaria, and meningococcal meningitis, show seasonal patterns? And how can we accurately determine these patterns?
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Meningococcal meningitis in western Africa shows recurrent seasonal patterns every year. Epidemics typically start at the beginning of February and last until May. We can try to explain the observed patterns on the basis of some seasonally varying environmental factor that favors disease transmission. Air dryness produced by strong dust winds is the most likely candidate.
But while there are qualitative “stories” of this kind in the literature for many seasonal phenomena, convincing quantitative evidence to support them remains largely elusive. Instead, we tend to see weak associations between environmental and transmission variables when measured by simple, linear correlations. The study of meningococcal meningitis in Mali by Sultan and colleagues in this issue of PLoS Medicine is a remarkable exception [1]. The study reports a strong association between the yearly onset of epidemics and a large-scale regional index for atmospheric circulation related to the Harmattan winds in Sahelo-Sudanian Africa.
The Importance of Seasonality
Why is a focus on the seasonality of infectious diseases and its variation from year to year so important? Isn't it more important for us to instead understand the effects of long-term climate change on human health?
At first sight, understanding seasonal patterns seems disconnected from understanding the impact of long-term climate change. However, seasonal patterns are one major pathway for the subtle but potentially drastic effects of climate change on disease dynamics. Long-term climate change affects seasonal patterns through the lengthening of the transmission season and the crossing of environmental and demographic thresholds that underlie seasonal outbreaks [2]. Thus, identifying the specific environmental factors underlying seasonal transmission is a critical step towards predicting and understanding how long-term environmental trends in mean climate and their variability will impact human health.
The Problem of Scale
One important difficulty in uncovering seasonal drivers of infectious diseases is to identify the appropriate scale of analysis. The relationship between disease and climate described by Sultan and colleagues only becomes apparent at large spatial scales. The authors argue that these large scales are necessary to eliminate “idiosyncratic” variability in the relationship between cases and climate at the local level. In other words, there are only weak correlations between seasonal variations and climate variables at small scales because of the multiple other factors that play a local role and act as noise.
But we should be cautious about the suggestion that appropriate larger scales will always resolve the problem of local variability and present strong linear associations between climate and disease. Public health measures might require predictions not only at national and regional scales, but also at a variety of smaller scales.
Moreover, one important source of variation in how infectious diseases respond to climate is the fraction of susceptible individuals in the population. This fraction varies over time as the result of immunity acquired by previous infection, and by the input of births and migrants into the pool of susceptible people. The constant waxing and waning of this pool of hosts underlies the intrinsic potential of the population dynamics of infectious diseases to oscillate and create epidemic outbreaks. The tendency of these intrinsic cycles to go up and down in synchrony at different locations in space will determine whether susceptibility levels act as noise at small scales or, alternatively, whether their effect must be considered in conjunction with climate at larger scales. Because the number of susceptible individuals is a hidden variable in most epidemiological analyses, recently proposed methods for its reconstruction from data on cases must be combined with studies on climate variation if we are to understand the interaction between susceptibility levels and climate variation [3,4,5].
The problem of scale also arises when we need to identify the appropriate timing (the temporal window) to detect strong associations between disease outbreaks and environmental covariates. This is particularly important when strong couplings between environment and transmission occur only transiently. This seems to be the case for cholera in Bangladesh, where couplings are strong during El Niños, but considerably weaker the rest of the time [6]. Intermittent couplings provide insight into how the system might behave if pushed into specific dynamic regions by a change in climate. Intermittent couplings also suggest the existence of thresholds in the response to climate, an area of research that remains in need of quantitative approaches.
Seasonal Drivers May Be Elusive
Besides scale, specific seasonal drivers are often elusive because of the simpler reason that in nature seasonality is ubiquitous. Multiple and covarying drivers have been proposed for the seasonal nature of cholera, including temperature, rainfall, and plankton blooms [7]. Yet the specific roles of these drivers in the bimodal seasonal cycle of cholera, and particularly in the second peak in endemic regions in south Asia, have not been convincingly shown (Figure 1) [8]. We still don't have predictive explanations of the geographic variation in seasonal patterns. We won't find such explanations by considering the average seasonal pattern; instead, we must consider the anomalies in amplitude and onset of the peaks that occur in different years.
Figure 1 The Role of Rainfall in Driving the Seasonal Nature of Cholera Is Unclear
This photograph was taken during a cholera and nutrition survey during flooding in Bangladesh in 1974. In Bangladesh, monsoon rains appear to have a seasonal “dilution” effect on transmission, producing a decrease in cholera cases during that season. We don't know whether extreme rains also produce a lagged increase in cases later on in the cycle. In other parts of the world, cases typically peak during the rainy season. (Photo: Jack Weissman, Centers for Disease Control and Prevention)
Ecologists have considered seasonality in mathematical models of the population dynamics of infectious diseases. Models of populations with seasonally forced, dynamic interactions (births, deaths, aggregation, or disease transmission) reveal an array of possible responses, from simple yearly cycles, through cycles that repeat with longer periods, to irregular chaotic fluctuations. Some models also predict intermittent switching between different dynamic infectious disease behaviors. But typical models consider only simple seasonal forcing functions (mathematical functions that are periodic in time and therefore describe in a generic way the seasonal variation in the transmission rate or some other seasonal parameter—a sine wave is an example). There are some important exceptions to this—some models do incorporate more complicated seasonal forcing functions that describe the actual processes underlying the seasonal drivers of transmission. Examples are models of childhood diseases that describe the regular stopping and starting of school terms [9,10,11], and recent malaria models that include the seasonal dynamics of mosquito births and pathogen incubation as functions of temperature and rainfall [12].
The explicit way in which models treat seasonal environmental drivers may be critical in addressing the links between within-year seasonal cycles and those of longer period that are observed in many infectious diseases. For meningococcal meningitis, we still need to examine the connection between the seasonal association described by Sultan and colleagues and the previously proposed role of humidity in inter-annual cycles [13].
The Complexity of Infectious Disease Dynamics
Sultan and colleagues' study is exceptional in that it illustrates a clear relationship between an external environmental variable and the initiation of disease outbreaks. In contrast, many studies seeking environmental drivers are plagued by the many confounding factors, particularly the impact that other components of global change have on the transmission dynamics of infectious diseases. Thus, when we examine datasets for malaria, we must also consider the evolution of drug resistance and a growing human population that is increasingly forced to live in areas that are marginal for agricultural production but optimal for malaria transmission.
Given this complexity, a serious limiting factor to quantitative analyses and predictive models of ecological and disease patterns is the lack of long-term disease records with similar data collected over a network of spatial locations. The handful of extremely valuable records that have allowed progress in understanding long-term patterns in disease dynamics pale in comparison to the spatiotemporal coverage available for climate studies and modeling. The need to resolve these issues of scale and confounding variability only underscores the urgency and importance of maintaining and developing systematic surveillance programs for infectious diseases around the world.
We thank the participants of the working group on “Seasonality and the population dynamics of infectious diseases” at the National Center of Ecological Analysis and Synthesis (NCEAS, Santa Barbara, California).
Citation: Pascual M, Dobson A (2004) Seasonal patterns of infectious diseases. PLoS Med 2(1): e5.
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Rodó X Pascual M Fuchs G Faruque ASG ENSO and cholera: A nonstationary link related to climate change Proc Natl Acad Sci U S A 2002 99 12901 12907 12228724
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Grenfell BT Bolker BM Kleczkowski A Mollison D Seasonality, demography, and the dynamics of measles in developed countries Epidemic models: Their structure and relation to data 1993 Cambridge Cambridge University Press 248 270
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Besancenot JP Boko M Oke PC Weather conditions and cerebrospinal meningitis in Benin (Gulf of Guinea, west Africa) Eur J Epidemiol 1997 13 807 815 9384271
| 15696215 | PMC545198 | CC BY | 2021-01-05 11:13:37 | no | PLoS Med. 2005 Jan 25; 2(1):e5 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020005 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1569621610.1371/journal.pmed.0020006Research ArticleInfectious DiseasesInfectious DiseasesClimate Drives the Meningitis Epidemics Onset in West Africa Climate and Meningitis EpidemicsSultan Benjamin
1
*Labadi Karima
2
Guégan Jean-François
3
Janicot Serge
1
1IRD–Laboratoire d'Océanographie Dynamique et du Climat (LODYC)–UMR 7617 (CNRS/IRD/P6/MNHN)–Université Pierre et Marie CurieParisFrance2Université de Paris 7 Denis Diderot UFR GHSS (c.c. 7001) Dynamique des Milieux et Risques–2ParisFrance3IRD–GEMI-UMR 2724 IRD-CNRS, Evolution des Systèmes SymbiotiquesMontpellierFranceHales Simon Academic EditorAustralian National UniversityAustralia
Competing Interests: The authors have declared that no competing interests exist.
Author Contributions: BS and SJ designed the study. BS, KL, and SJ analyzed the data. BS, KL, JFG, and SJ contributed to writing the paper.
*To whom correspondence should be addressed. E-mail: [email protected] 2005 25 1 2005 2 1 e620 7 2004 13 10 2004 Copyright: © 2005 Sultan et al.2005This 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 author and source are properly credited.
Seasonal Patterns of Infectious Diseases
Meningitis and Climate in West Africa
Background
Every year West African countries within the Sahelo-Sudanian band are afflicted with major meningococcal meningitis (MCM) disease outbreaks, which affect up to 200,000 people, mainly young children, in one of the world's poorest regions. The timing of the epidemic year, which starts in February and ends in late May, and the spatial distribution of disease cases throughout the “Meningitis Belt” strongly indicate a close linkage between the life cycle of the causative agent of MCM and climate variability. However, mechanisms responsible for the observed patterns are still not clearly identified.
Methods and Findings
By comparing the information on cases and deaths of MCM from World Health Organization weekly reports with atmospheric datasets, we quantified the relationship between the seasonal occurrence of MCM in Mali, a West African country, and large-scale atmospheric circulation. Regional atmospheric indexes based on surface wind speed show a clear link between population dynamics of the disease and climate: the onset of epidemics and the winter maximum defined by the atmospheric index share the same mean week (sixth week of the year; standard deviation, 2 wk) and are highly correlated.
Conclusions
This study is the first that provides a clear, quantitative demonstration of the connections that exist between MCM epidemics and regional climate variability in Africa. Moreover, this statistically robust explanation of the MCM dynamics enables the development of an Early Warning Index for meningitis epidemic onset in West Africa. The development of such an index will undoubtedly help nationwide and international public health institutions and policy makers to better control MCM disease within the so-called westward–eastward pan-African Meningitis Belt.
An analysis of winds and yearly meningitis epidemics in West Africa shows a surprisingly close correlation that could help to predict disease outbreaks
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Introduction
Meningococcal meningitis (MCM) has affected Sahelian Africa for centuries and became endemic over the past 25 y. During the 1980s, the World Health Organization (WHO) registered between 25,000 and 200,000 disease cases per year, with about 10% of them resulting in death, and with the highest infection rates observed in younger children [1]. MCM became, therefore, a public health concern in the poorest regions in the world following the severe drought at the end of the 1970s.
MCM is an infection of the meninges, caused by the bacteria Neisseria meningitidis, that causes high death rates in African communities. The agent is highly contagious, and person-to-person aerial transmission occurs through respiratory and throat secretions [2]. Interaction between different environmental parameters (e.g., immune receptivity of individuals, a poor socioeconomic level, the transmission of a more virulent serotype [such as the recent emergence of the serogroup W135 in West Africa], social interactions [such as pilgrimages, tribe migrations, and meetings], and some specific climatic conditions) may play a part in MCM disease outbreaks and spread within local populations [2].
Among favorable conditions for the resurgence and then dispersion of the disease, climatic conditions may be important as environmental forces inducing periodic fluctuations of disease incidence. Recent findings concerning the population dynamics of some infectious diseases have clearly identified the importance of climate as a major driver [3,4]. MCM outbreaks in West Africa usually start at the beginning of February, and then disappear in late May. The geographical distribution of disease cases is called the “Meningitis Belt” and is roughly circumscribed to the biogeographical Sahelo-Sudanian band [5,6]. This Sahelo-Sudanian region has a dry winter, dominated by northern winds, called the Harmattan, followed by a wet season starting in spring with the monsoon. The co-occurrence in both space and time of MCM disease cases and climate variability within the Sahelo-Sudanian area suggests that the occurrence of MCM might be directly related to climate. So far, very few studies have tried to quantify the potential linkages that could exist between climate and MCM outbreaks (Figure 1).
Figure 1 The “Meningitis Belt” in West Africa
Modified from the WHO [9].
The winter climate causes damage to the mucous membranes of the oral cavity through dry air and strong dust winds, and creates propitious conditions for the transmission of the bacteria responsible for MCM; low absolute humidity and dust may enhance meningococcal invasion by damaging the mucosal barrier directly or by inhibiting mucosal immune defenses. In contrast, higher humidity during both the spring and summer seasons strongly reduces disease risk by decreasing the transmission capacity of the bacteria [7,8], and MCM epidemics generally stop with the onset of rainfall [9]. In addition to the seasonal cycle, the link between climate and meningitis has also been documented at the interannual scale in northern Benin, where Besancenot et al. [10] have suggested a positive relationship between low absolute humidity and interannual variability in meningitis. Meanwhile, although the global influence of climate is quite clear, the effects of climatic variability on MCM population dynamics are still only partially known because of the mixing of different processes acting at different spatial hierarchical scales, and the interactions between disease outbreaks and medical, demographical, and socioeconomic conditions.
Most studies thus far have focused on very small spatial scales, and the need remains to discriminate between local properties and potential large-scale effects in disease patterns, to go beyond data heterogeneities and idiosyncratic details in order to identify important disease patterns influenced by large-scale forces such as climate variability (see Methods). The aim of the present work is thus to document the climatic context of MCM disease outbreaks and population dynamics in a highly affected Sahelian country, that is, Mali, and to show, if it exists, the presence of a correlation between climate and seasonal resurgence of disease. The idea behind the present study was to explain MCM disease dynamics in Mali in a statistically robust way, which will permit us to propose some tools for predicting disease risks for the benefit of public health.
Methods
The Scaling-Up Approach: From Local to Global Scales
Recent findings concerning the population dynamics of some infectious diseases have clearly identified the importance of climate as a major driver [3,4]. With evidence of the impact of large-scale meterological phenomena such as El Niño on infectious disease patterns, modern epidemiology is now confronted with a scale problem in the identification of the spatiotemporal scales that might be relevant to explain patterns and processes [11]. Since most previous studies have focused on very small spatial scales, there is a need for “bottom-up” approaches to discriminate between local properties and potential large-scale effects on disease patterns. One of the simplest assumptions of these “bottom-up” approaches is the assumption that local scales are random processes overlaying a driving large-scale phenomenon such as climate variability. As such, this scaling-up approach seeks to point out the emerging patterns conditioned by the large-scale processes, with a random or deterministic function f such that:
Local data = f(large-scale forces, idiosyncratic details).
The aggregation of local data in the scaling-up approach is a simple way to go beyond data heterogeneities and idiosyncratic details so that only the important disease effects of large-scale forces remain. That is the rationale for our study: to show that large-scale phenomena such as the seasonal Harmattan winds over the whole of Mali can contribute to the periodic resurgence of MCM and its variation in time on a national scale, even if this aggregate analysis for the entire country is not able to capture variations at smaller space scales.
Epidemiological Data: The WHO Weekly Reports
The diagnosis of MCM is based on both physical examination and on evaluation of the cerebrospinal fluid (CSF) from a lumbar puncture. As the disease is characterized by a sudden onset of intense headache, fever, nausea, vomiting, photophobia, and stiff neck, in association with neurological symptoms (lethargy, delirium, coma, and convulsions), the WHO [9] recommends that the clinical diagnosis include an examination for meningeal rigidity, neurological signs, purpura, blood pressure, and focal infection. A lumbar puncture and CSF examination are then used to confirm the diagnosis based on physical examination and to identify the meningococcus [9]. This diagnosis is the basis for disease surveillance and case reporting using a standard case definition for MCM (Box 1) that can be implemented in any health-care setting. Meningitis reports are included in the weekly reports of notifiable diseases and are aggregated at different spatial scales from the health site to the country level. The present study is based on these weekly reports by the WHO's Department of Communicable Disease Surveillance and Response of cases and deaths due to MCM over the whole of Mali from 1994 to 2002.
Box 1. Standard Case Definition of MCM Modified from the WHO [9]
This case definition allows the detection of cases of meningococcal septicemia.
Suspected case of acute meningitisa. Sudden onset of fever (>38.5 °C rectal or 38.0 °C axillary) with stiff neck. In patients under 1 y of age, a suspected case of meningitis occurs when fever is accompanied by a bulging fontanelle.
Probable case of bacterial meningitisb. Suspected case of acute meningitis as defined above with turbid CSF.
Probable case of MCMb. Suspected case of either acute or bacterial meningitis as defined above with Gram stain showing Gram-negative diplococcus or ongoing epidemic or petechial or purpural rash.
Confirmed casec. Suspected or probable case as defined above with either positive CSF antigen detection for N. meningitides or positive culture of CSF or blood with identification of N. meningitides.
aOften the only diagnosis that can be made in dispensaries (peripheral level of health care).
bDiagnosed in health centers where lumbar punctures and CSF examination are feasible (intermediate level).
cDiagnosed in well-equipped hospitals (provincial or central level).
The Typical Seasonal Pattern of the Disease
In this study, an epidemic is defined in terms of population dynamics following Anderson and May [12] and Grenfell and Dobson [13]. This definition considers disease resurgence and its variation in time and allows us to focus on the cyclic character of the disease resurgence each year, even if the number of annual cases is low, and it makes it easier to find temporal correlations between climate and disease. To represent a typical seasonal cycle of a meningitis epidemic, we computed the weekly mean of standardized anomalies M(w) of the number of cases as
where X̄y
and σy represent, respectively, the mean and the standard deviation of the 54 weekly values of cases Xy(w) for the year y, and N = 9 represents the number of years for the 1994–2002 period.
The Onset of the Epidemic
We determined the week of the onset of the epidemic for each year by characterizing a breaking slope in the annual cycle of the number of cases. The dates of the breaking slope have been determined objectively by using the Mann–Whitney–Pettitt test [14], which is a nonparametric test used here to detect a “change point” in a time series. A change point is defined as a point on either side of which values are on average higher or lower than the whole of the other data points. Considering the studied time series Xy(w), we computed Uy(w) as
where 2 ≤ w ≤ 54 and Uy(1) = Vy(1), where Vy(w) is defined by
Then the the most significant change point of the year y is the point for which the value|Uy(w)| is maximized. The probability Py(w) of a given week being a change point is defined by
where T = 54 (the length of the time series in weeks).
Atmospheric Data: The NCEP/NCAR Reanalysis
The National Centers for Environmental Prediction (NCEP) and the National Center for Atmospheric Research (NCAR) have completed a reanalysis project with a current version of the Medium Range Forecast model [15]. This dataset consists of a reanalysis of the global observational network of meteorological variables (wind, temperature, geopotential height [i.e., the height of a pressure surface above mean sea level], humidity on pressure levels, surface variables, and flux variables such as precipitation rate), with a “frozen” state-of-the-art analysis and forecast system at a triangular spectral truncation of T62, performing data assimilation throughout the period from 1948 to the present. This analysis enables circumvention of problems involving previous numerical weather prediction analyses due to changes in techniques, models, and data assimilation. Data are reported on a 2.5° × 2.5° grid every 6 h (00.00, 06.00, 12.00, and 18.00 UTC) on 17 pressure levels from 1,000 hPa to 10 hPa, a good resolution for studying synoptic weather systems. For this study we used the wind speed fields at 1,000 hPa (near the surface) for the 9 y of the 1994–2002 period: first we averaged the four outputs of each day, and then we averaged these daily means for each week to obtain a weekly value.
Computation of the Harmattan Wind Index
The principal component analysis (PCA) [16] is a multivariate procedure that extracts the common variance that exists in a set of variables. The main use of PCA is to reduce the size of a dataset while retaining as much information as possible in principal components (PCs), which are linear combinations of the initial variables. In this study, this technique has been used in order to summarize the spatiotemporal variability of wind fields at low levels. As the input data are spatial objects (grid points), the PCA gives for each mode a spatial pattern associated with a time series (the PC). We performed the PCA on the weekly values from 1994 to 2002 of wind speed at 1,000 hPa over the Mali window (in red in Figure 1) by taking into account all the grid points from 12.5° N to 25° N and from 12.5° W to 2.5° E. The input matrix was thus composed of 6 × 7 = 42 loadings (the number of grid points over the spatial window) and 486 scores (the number of weeks in the 1994–2002 period). Data was first standardized in order to extract the correlation matrix C = X′X, where X represents the input matrix and X′ its transpose. The αth PC time series ψα can thus be obtained by a linear combination of the initial variables through
where u
α is the α
th eigenvector of the correlation matrix C associated with the eigenvalue λ
α.
The α
th spatial pattern is the correlation map between the initial wind fields and the α
th PC time series. The examination of the different spatial patterns and PC time series (not shown here but previously discussed in [17]) reveals a close relationship between Harmattan wind dynamics and the third PC with negative values, which represents a strong wind in southern Mali. The Harmattan wind index of the study thus represents the third PC, with a temporal pattern very similar to the seasonal cycle of wind speed associated with the Harmattan winds over Mali.
Results
The “Epidemic Seasonality” in Mali
The weekly records of the WHO's Department of Communicable Disease Surveillance and Response of cases and deaths due to MCM for the 1994–2002 period allowed us to describe the seasonal evolution of MCM epidemics in Mali. Two important parameters were used: the date of the onset of the epidemic and the date of the seasonal maximum number of cases. We determined for each year the week of the onset of the epidemic (here called “W
o”) as determined by a breaking slope in the annual cycle of the number of cases. The dates of breaking slope have been determined objectively by using the Mann–Whitney–Pettitt test [14], which is a nonparametric test used here to detect a change point in a time series. This test has the advantage of being adapted to small samples, giving the point of the most significant change and the probability of it being a significant change point (see Materials and Methods). The mean date of epidemic onset fell between the fifth and sixth week of the year (7–15 February), with a standard deviation of about 2 wk (5.2 ± 1.7 wk). For the 1994–2002 period, the maximum number of cases occurred between week 13 and week 14, that is, between 1 April and 15 April, with a standard deviation of about 2 wk (13.7 ± 1.6 wk) (Figure 2).
Figure 2 The Seasonal Periodicity of Meningitis Cases
Mean seasonal pattern of the number of cases of MCM over the 1994–2002 period in standardized anomalies (bars). The red curve represents the same evolution, but in composite mean, using the week of epidemic onset as the reference date, W
o, each year. Time series in red is shown from “W
o − 3 wk” to “W
o + 30 wk.”
In order to mitigate the effect of strong variability from one year to another during the 1994–2002 period, we computed the average of standardized anomalies of the number of cases (bars in Figure 2) to represent a typical seasonal pattern of a meningitis epidemic. The first 5 wk are characterized by negative anomalies. The average length of the “epidemic year,” as defined by the number of consecutive weeks with positive anomalies, is 4 mo (16 wk).
To improve the description of the seasonal pattern of the epidemic and to reduce noise due to the variability of the onset date year to year, we determined the composite mean of the number of cases over the 1994–2002 period by using the week of epidemic onset for each year as the respective reference date, W
o. The red curve of Figure 2 shows the mean seasonal course before and after the onset of the epidemic, showing an abrupt increase of the number of cases—the “upward phase”—until the sixth week after the onset, a highly active period of the disease—the “active phase”—from “W
o + 6” to “W
o + 10,” followed by a decrease of the number of cases—the “downward phase”—until the end of the epidemic around 16 wk after the onset. Both upward and downward phases lasted on average 1.5 mo.
The Atmospheric Circulation during an Epidemic in Mali
Rainfall distribution over West Africa is controlled by the meridional migration of the intertropical convergence zone following the seasonal excursion of the sun [18]. The latitudinal shift of this zone of high humidity and instability leads to an opposition of two main annual regimes: the bimodal regime of the Guinean latitudes (from the equator to 7° N) with two rainy seasons during spring and autumn, and the unimodal regime of monsoon over Sudano-Sahelian Africa and succession of a dry winter and a wet summer [19]. North of the intertropical convergence zone, the intertropical front is defined as the confluence line between moist southwesterly monsoon winds and dry northeasterly Harmattan [20,21]. The seasonal progression of this system, involving a migration toward the summer pole of moisture and winds converging in the low layers, can be documented by using weekly fields surface wind speed from NCEP and NCAR data, which provide gridded atmospheric parameters with a 2.5° resolution [15]. The relationship between atmospheric circulation and the seasonal course of the MCM epidemic in Mali was studied using a regional index summarizing the spatiotemporal evolution of the low-layer circulation. This index was obtained from a dominant mode of a PCA (see Materials and Methods) applied to weekly fields of surface wind speed over the 1994–2002 period in Mali. The seasonal pattern shows the Harmattan wind dynamics (Figure 3) with negative values representing strong winds, and positive values representing weak winds, in the southern part of Mali, the area under study in the present work.
Figure 3 Temporal Patterns of Epidemics and Climate
Weekly means of the Harmattan wind index over the 1994–2002 period and mean seasonal pattern of the number of cases of MCM (in standardized anomalies).
Using this atmospheric index, we defined the date of “winter maximum” as the first minimum of the wind index for each year of the period 1994–2002. The winter maximum thus corresponds to the week where wind speed is the strongest. The mean date of winter maximum is around the sixth week, with a standard deviation of 2 wk, corresponding to the week when the Intertropical Front is located at its southern latitude. The Harmattan wind index shows a temporal pattern very similar to the that of the number of cases of MCM, with a clear breaking slope at the sixth week, on 15 February, corresponding to the onset of the epidemic and to the winter maximum, and with a recession of the disease at the 16th week concomitant with the onset of the wet season in the south part of Mali in early May. It is interesting to note that although they are determined from two different datasets, the mean weeks of winter maximum and of the onset of the epidemic are identical, 7–15 February. This coherence is reinforced by a very strong correlation between the two dates (0.92) for the years 1994 to 2002. Figure 4 shows the linear regression analysis between week of winter maximum and week of epidemic onset. Although the number of years under consideration is low, the scatter plot points out the close statistical linkage between these two events, suggesting that the winter maximum explains more than 85% of the variance in the week of epidemic onset in Mali: An earlier winter is associated with an earlier onset of the epidemic, and a later winter with a later onset. However, even though the results of the correlation analysis are strongly significant, the high R
2 is partially due to the low number of considered years (only nine); this low number is the main limitation of the present analysis.
Figure 4 The Onset of Epidemics and the Winter Maximum
Scatter plot of the week of epidemic onset and the week of winter maximum over the 1994–2002 period.
Discussion
In this paper and a previous publication of ours [17], by using the weekly number of cases of MCM disease in Mali and large-scale fields of surface wind speed, we clearly identify a strong relation between climate and the seasonal pattern of MCM cases in Mali. It is shown that the onset of disease outbreak is characterized by a clear breaking slope in the seasonal cycle of the number of cases at the sixth week of the year, that is, 15 February. The computation of an atmospheric index based on surface wind speed over Mali points out that this abrupt shift is also present in the atmospheric signal, corresponding to the winter wind maximum, when Harmattan winds are the strongest in Sahelo-Sudanian Africa. The similarity in the seasonal patterns of both Harmattan winds and MCM disease cases is obvious, with a strong correlation between the week of winter maximum and that of the onset of epidemic. Similar results, not illustrated here, have been obtained by using surface temperatures and specific humidity for the computation of atmospheric indexes, attesting to the robustness of the analysis.
However, whatever the climatic index is used, this analysis does not allow us to link the intensity of the “epidemic” (the annual number of cases) to the intensity of winter in terms of absolute humidity and surface wind speed. This lack of a relation may be due to the time series length, with an insufficient number of years to study interannual variations, or it may imply that the climatic influence is limited to explaining the occurrence of the seasonal cycle of the epidemic and its geographical range distribution, but not its intensity. Although they fail to forecast epidemic intensity, such climatic indexes, with their correlation with the onset and the seasonal course of the epidemic in Sahel, provide an important means of disease monitoring and prediction in Africa. Indeed, the seasonal pattern of humidity and Harmattan winds can be easily tracked, thus promoting the emergence of an Early Warning Index (EWI) for the onset of MCM epidemics. The seasonal forecast of this EWI based on Harmattan winds could thus be implemented routinely by using comprehensive coupled models of the atmosphere, oceans, and land surface that provide a degree of predictability of climate fluctuations with a seasonal lead time in many parts of the world [22]. The ability of the climate models to predict the winter maximum could be tested by using the outputs of the Development of a European Multi-Model Ensemble System for Seasonal to Interannual Prediction (DEMETER) project, which was conceived and funded under the European Union Fifth Framework Environment Programme. The principal aim of DEMETER was to advance the concept of multimodel ensemble prediction by using a number of state-of-the-art global-coupled ocean–atmosphere models and to produce a series of 6-mo multimodel ensemble hindcasts. The DEMETER project already has application partners in agronomy and in tropical disease prediction [22].
This EWI parameter, in association with other environmental parameters implicated in disease resurgence [23], could help to more precisely characterize disease risk maps at regional scales. The natural extension of this work is to relate this information on the timing of disease outbreaks with specific spatial environmental characteristics at finer scales, in an Early Warning System based on the monitoring of the impact of climate variability and environmental change on epidemic occurrence in West Africa. Recent findings by Molesworth et al. [23] have already quantified the relationship between the environment and the location of the epidemics to propose a model based on environmental variables and to identify regions at risk for meningitis epidemics. The combination of the EWI for MCM epidemic onset and risk maps at regional scales could be a starting point to more optimally direct national and international health policy strategies and to optimize mass vaccination campaigns. In addition, more general measures can be taken by national authorities to improve the control of MCM disease, such as closing markets and schools and discouraging social gatherings when an outbreak is likely to occur [9].
Patient Summary
Background
Climate is known to be one of the factors that can affect when and how epidemics occur; for example, floods often increase the risk of waterborne disease. However, there are many more subtle climatic changes that might also be important in affecting when and how diseases occur.
What Did the Researchers Do?
They looked at the relationship between a recurring epidemic of a disease called meningococcal meningitis in Mali in West Africa and the local climatic conditions, especially the winds. Meningococcal meningitis is a serious infection of the lining of the brain and spinal cord by a bacterium (called Neisseria meningitides). These researchers had previously published some detailed work on the local climate in a French journal. In this paper they have focussed more on the aspects that deal with disease. They found out that over several years the onset of the epidemic coincided with the peak of the winds.
Who Will Use These Results?
People who would find these results useful are those who plan for epidemics. Such information will allow them to plan in advance, and even predict whether an epidemic will occur at all. However, these results were based on only the years between 1994 and 2002, and so will need to be confirmed in more years.
We are grateful to the National Oceanic and Atmospheric Administration–Cooperative Institute for Research in Environmental Sciences Climate Diagnostics Center (Boulder, Colorado, United States) for providing the NCEP/NCAR reanalysis dataset and J.-L. Monge (Laboratoire de Meteorologie Dynamique) for the access to data on CLIMSERV. We also thank J.-P. Chippaux and G. Beltrando, who helped to clarify this paper. BS, JFG, and SJ thank the Institut de Recherche pour le Développement for financial support.
Citation: Sultan B, Labadi K, Guégan JF, Janicot S (2005) Climate drives the meningitis epidemics onset in West Africa. PLoS Med 2(1): e57.
Abbreviations
CSFcerebrospinal fluid
DEMETERDevelopment of a European Multi-Model Ensemble System for Seasonal to Interannual Prediction
EWIEarly Warning Index
MCMmeningococcal meningitis
NCARNational Center of Atmospheric Research
NCEPNational Centers for Environmental Prediction
PCprincipal component
PCAprincipal component analysis
WHOWorld Health Organization
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World Health Organization Meningococcal meningitis: Fact sheet 2003, number 141. Available: http://www.who.int/mediacentre/factsheets/2003/fs141/en/index.html
2003 Accessed 23 December 2003
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Pascual M Rodo X Ellner S Colwell R Bouma M Cholera dynamics and El Nino–Southern Oscillation Science 2000 289 1766 1769 10976073
Lapeyssonnie L [The meningococcal meningitis in Africa] (in French) Bull World Health Organ 1963 28 3 114p
Cheesbrough JS Morse AP Green SDR Meningococcal meningitis and carriage in western Zaire: A hypoendemic zone related to climate? Epidemiol Infect 1995 114 75 92 7867746
Molesworth A Cuevas LE Morse AP Herman JR Thomson MC Dust clouds and spread of infection Lancet 2002 359 81 82
Molesworth AM Thomson MC Connor SJ Cresswell MP Morse AP Where is the Meningitis Belt? Defining an area at risk of epidemic meningitis in Africa Trans R Soc Med Hyg 2002 96 242 249
World Health Organization Guidelines on the control of epidemic meningococcal disease. 2nd ed. Geneva: WHO. Report Number WHO/EMC/BAC/98.3 1998 84
Besancenot JP Boko M Oke PC Weather conditions and cerebrospinal meningitis in Benin (Gulf of Guinea, West Africa) Eur J Epidemiol 1997 13 807 815 9384271
Guégan JF Morand S Poulin R Thomas F Renaud F Guégan JF Are there general laws in parasite community ecology? The emergence of spatial parasitology and epidemiology Parasitism and ecosystems 2005 Oxford Oxford University Press In press
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Kalnay E Kanamitsu M Kistler R Collins W Deaven D The NCEP/NCAR 40-year reanalysis project Bull Amer Meteorol Soc 1996 77 437 471
Richman MB Rotation on principal components J Climatol 1986 6 293 335
Sultan B Labadi K Beltrando G Janicot S [Meningococcal meningitis and the atmospheric dynamics in West Africa] Environnement, Risques et Santé 2004 3 23 24
Hastenrath S Climate dynamics of the tropics 1995 Dordrecht Kluwer Academic 488
Nicholson SE Kim J Hoopingarner J Atlas of African rainfall and its interannual variability 1988 Tallahassee Florida State University, Department of Meteorology 237
Moron V Guinean and Sahelian rainfall anomaly indices at annual and monthly scales (1993–1990) Int J Climatol 1994 14 325 341
Sultan B Janicot S The West African monsoon dynamics, Part II: The “pre-onset” and the “onset” of the summer monsoon J Clim 2003 16 3407 3427
Palmer TN Alessandri A Andersen U Cantelaube P Davey M Development of a European multi-model ensemble system for seasonal to inter-annual prediction (DEMETER) Bull Am Meteorol Soc 2004 85 853 872
Molesworth AM Cuevas LE Connor SJ Morse AP Thomson MC Environmental risk and meningitis epidemics in Africa. Emerg Infect Dis. Available: http://www.cdc.gov/ncidod/EID/vol9no10/03–0182.htm
2003 Accessed 23 December 2003
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1569621710.1371/journal.pmed.0020007Best PracticeGeriatricsHealth PolicyNeurology/NeurosurgeryDementiaGeriatric MedicineEducating the Brain to Avoid Dementia: Can Mental Exercise Prevent Alzheimer Disease? Best PracticeGatz Margaret Margaret Gatz is in the Department of Psychology, University of Southern California, Los Angeles, California, United States of America, and the Department of Medical Epidemiology and Biostatistics, The Karolinska Institute, Stockholm, Sweden. E-mail: [email protected]
Competing Interests:The author declares that she has no competing interests.
1 2005 25 1 2005 2 1 e7Copyright: © 2005 Margaret Gatz.2005This 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 author and source are properly credited.
Test Your Knowledge: Ten Questions about Dementia
Physicians often recommend to older adults that they should engage in mentally stimulating activity to reduce the risk of dementia. But is this recommendation based on sound evidence?
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Physicians now commonly advise older adults to engage in mentally stimulating activity as a way of reducing their risk of dementia. Indeed, the recommendation is often followed by the acknowledgment that evidence of benefit is still lacking, but “it can't hurt.” What could possibly be the problem with older adults spending their time doing crossword puzzles and anagrams, completing figural logic puzzles, or testing their reaction time on a computer? In certain respects, there is no problem. Patients will probably improve at the targeted skills, and may feel good—particularly if the activity is both challenging and successfully completed.
But can it hurt? Possibly. There are two ways that encouraging mental activity programs might do more harm than good. First, they may offer false hope. Second, individuals who do develop dementia might be blamed for their condition. When heavy smokers get lung cancer, they are sometimes seen as having contributed to their own fates. People with Alzheimer disease might similarly be viewed as having brought it on themselves through failure to exercise their brains.
What Does the Evidence Show?
Three types of evidence are cited to support the idea that mental exercise can improve one's chances of escaping Alzheimer disease.
Epidemiological studies
Having more years of education has been shown to be related to a lower prevalence of Alzheimer disease in cross-sectional, population-based studies [1] and to a lower incidence of Alzheimer disease in cohorts followed longitudinally [2]. Typically, the risk of Alzheimer disease is two to four times higher in those who have fewer years of education, as compared to those who have more years of education. Other epidemiological studies, albeit with less consistency, have suggested that those who engage in more leisure activities, especially activities that are mentally stimulating, have a lower prevalence and incidence of Alzheimer disease [3,4]. Additionally, longitudinal studies have found that older adults without dementia who participate in more intellectually challenging daily activities show less decline over time on various tests of cognitive performance [5].
How effective is mental exercise in holding back dementia?
(Illustration: Sapna Khandwala, Public Library of Science)
In epidemiological studies, people cannot be randomly assigned to different levels of education, or to different kinds and levels of participation in leisure activities. Consequently, researchers must try to identify confounders and take them into account analytically. However, uncertainties remain. Both education and leisure activities are imperfect measures of mental exercise. For instance, leisure activities represent a combination of influences. Not only is there mental activation, but there may also be broader health effects, including stress reduction and improved vascular health—both of which may contribute to reducing dementia risk [6]. It could also be that a third factor, such as intelligence, leads to greater levels of education (and more engagement in cognitively stimulating activities), and independently, to lower risk of dementia. Research in Scotland, for example, showed that IQ test scores at age 11 were predictive of future dementia risk [7].
Another problem with these epidemiological studies is that reverse causation could be involved—in other words, that incipient dementia could be causing reduced engagement in leisure activities, although some prospective studies have been particularly attentive to controlling for this possibility [8]. Clinical trials are needed to test the hypotheses that emerge from the best epidemiological research. Moreover, because the onset of Alzheimer disease can be hard to pinpoint, and early changes may occur years before the disease is diagnosed, conclusions must be based on large samples, followed over a long period of time.
Randomized clinical trials
Many studies support the possibility of enhancing memory and other cognitive performance, or of slowing cognitive decline in older adults without dementia [9]. The most effective programs teach mnemonic strategies, provide practice, and give supportive feedback. Mnemonic strategies include the organization of items into meaningful groups, the use of imagery, and the method of loci (visualizing items to be remembered in a sequence of specific, well-learned locations). Comprehensive programs can also include: encouraging memory aids (such as appointment books), teaching relaxation techniques, and providing instruction about memory changes in normal aging. However, improvements are not found in all studies. When improvements are found, they are often modest, may not be maintained over time, and do not generalize beyond the skill being trained. Often, the subjective gains rival the objective ones; for example, participants do tend to report fewer complaints about their memory.
These limitations are evident in one of the largest randomized controlled trials of cognitive training with older adults, a large, multisite study named ACTIVE (Advanced Cognitive Training for Independent and Vital Elderly) [10]. Participants were assigned to receive training in one of three cognitive skills: memory, reasoning, or speed of processing. Tests of cognitive abilities given immediately after training showed large improvements on the particular cognitive skill on which the individual had been trained, but no transfer to the other two cognitive domains. Additionally, for the control group that received no training, simply taking the test battery at pre-test led to improvement on the post-test. The effects of training were maintained over a two-year follow-up. However, the cognitive training program had no significant effect on measures of everyday functioning. Finally, for participants in ACTIVE or in other memory training programs, it remains unknown whether eventual rates of Alzheimer disease will be reduced.
Neurobiology studies
The third type of evidence suggesting that mental exercise may help to prevent Alzheimer disease comes from neurobiology studies that show greater brain complexity in those with higher levels of mental activity. Many such studies, done with animals, show greater neural complexity after having been exposed to an enriched environment that provides lots of stimulation, for example by including wheels, tunnels, toys, and gnawing sticks [11]. One human study with magnetic resonance spectroscopy showed changes in the hippocampus in elderly memory training participants compared to controls [12]. Another report found changes on positron emission tomography scanning following two weeks of a comprehensive memory program that included memory training, special diet, physical exercise, and stress reduction [13].
Mental Exercise and Cognitive Reserve
The concept of cognitive reserve is often used to explain why education and mental stimulation are beneficial. The term cognitive reserve is sometimes taken to refer directly to brain size or to synaptic density in the cortex. At other times, cognitive reserve is defined as the ability to compensate for acquired brain pathology. This definition encompasses coping skills as well as recruitment of other brain areas, with cognitive reserve thus accounting for individual differences in severity of cognitive dysfunction when there are pathological neural changes. People with a higher level of education have greater cognitive reserve. In some studies, education or occupation are even used as proxy measures of cognitive reserve, while others are beginning to measure neural substrates that correspond to reserve [14].
Taken together, the evidence is very suggestive that having greater cognitive reserve is related to a reduced risk of Alzheimer disease. But the evidence that mental exercise per se can increase cognitive reserve and stave off dementia is weaker. Epidemiological studies suggest that individual differences in cognitive reserve may actually be lifelong. In addition, people with greater cognitive reserve may choose mentally stimulating leisure activities and jobs, leading to a chicken-and-egg dilemma for the interpretation of the relationship between mentally stimulating activities in adulthood and dementia risk. Cognitive training has demonstrable effects on performance, on views of self, and on brain function—but the results are very specific to the skills that are trained, and it is as yet entirely unknown whether there is any effect on when or whether an individual develops Alzheimer disease. Further, the types of skills taught by practicing mental puzzles may be less helpful in everyday life than more prosaic “tricks,” such as concentrating, or taking notes, or putting objects in the same place each time so that they won't be lost.
Conclusion
So far, we have little evidence that mental practice will help prevent the development of dementia. We have better evidence that good brain health is multiply determined, that brain development early in life matters, and that genetic influences are of great importance in accounting for individual differences in cognitive reserve and in explaining who develops Alzheimer disease and who does not. At least half of the explanation for individual differences in susceptibility to Alzheimer disease is genetic, although the genes involved have not yet been completely discovered [15]. The balance of the explanation lies in environmental influences and behavioral health practices, alone or in interaction with genetic factors.
For older adults, health practices that could influence the brain include sound nutrition, sufficient sleep, stress management, treatment of mood or anxiety disorders, good vascular health, physical exercise, and avoidance of head trauma. But there is no convincing evidence that memory practice and other cognitively stimulating activities are sufficient to prevent Alzheimer disease; it is not just a case of “use it or lose it.”
The author is grateful to Hans-Olov Adami for helpful discussions, and for grant support from the National Institutes of Health (R01 AG08724, P30 AG17265, P50 AG05142) and from the Alzheimer's Association (ZEN-02-3895).
Citation: Gatz M (2005) Educating the brain to avoid dementia: Can mental exercise prevent Alzheimer disease? PLoS Med 2(1): e7.
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Ott A Breteler MM van Harskamp F Claus JJ van der Cammen TJ Prevalence of Alzheimer's disease and vascular dementia: Association with education. The Rotterdam study BMJ 1995 310 970 973 7728032
Ott A van Rossum CT van Harskamp F van de Mheen H Hofman A Education and the incidence of dementia in a large population-based study: The Rotterdam Study Neurology 1999 52 663 666 10025813
Scarmeas N Levy G Tang MX Manly J Stern Y Influence of leisure activity on the incidence of Alzheimer's disease Neurology 2001 57 2236 2242 11756603
Wilson RS Bennett DA Bienias JL Aggarwal NT Mendes de Leon CF Cognitive activity and incident AD in a population-based sample of older persons Neurology 2002 59 1910 1914 12499482
Hultsch DF Hertzog C Small BJ Dixon RA Use it or lose it: Engaged lifestyle as a buffer of cognitive decline in aging? Psychol Aging 1999 14 245 263 10403712
Fratiglioni L Paillard-Borg S Winblad B An active and socially integrated lifestyle in late life might protect against dementia Lancet Neurol 2004 2004 343 353
Whalley LJ Starr JM Athawes R Hunter D Pattie A Childhood mental ability and dementia Neurology 2000 55 1455 1459 11094097
Verghese J Lipton RB Katz MJ Hall CB Derby CA Leisure activities and the risk of dementia in the elderly N Engl J Med 2003 348 2508 2516 12815136
Mohs RC Ashman TA Jantzen K Albert M Brandt J A study of the efficacy of a comprehensive memory enhancement program in healthy elderly persons Psychiatry Res 1998 77 183 195 9707301
Ball K Berch DB Helmers KF Jobe JB Leveck MD Effects of cognitive training interventions with older adults: A randomized controlled trial JAMA 2002 288 2271 2281 12425704
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Valenzuela MJ Jones M Wen W Rae C Graham S Memory training alters hippocampal neurochemistry in health elderly Neuroreport 2003 14 1333 1337 12876468
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Ashford JW Mortimer JA Non-familial Alzheimer's disease is mainly due to genetic factors J Alzheimers Dis 2002 4 169 177 12226536
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1569621810.1371/journal.pmed.0020008EssayEpidemiology/Public HealthHealth PolicyPublic HealthInternational healthHealth PolicyMedicine in Developing CountriesTrade and Health: Is the Health Community Ready for Action? EssayLee Kelley *Koivusalo Meri Kelley Lee is co-director of the Centre on Global Change and Health, London School of Hygiene and Tropical Medicine, United Kingdom. Meri Koivusalo is a senior researcher in the Globalism and Social Policy Programme at the Finnish National Research and Development Centre for Welfare and Health, Helsinki, Finland, and is co-editor of Global Social Policy.
Competing Interests: KL is on the editorial board of PLoS Medicine. MK declares that she has no competing interests.
*To whom correspondence should be addressed. E-mail: [email protected] 2005 25 1 2005 2 1 e8Copyright: © 2005 Lee and Koivusalo.2005This 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 author and source are properly credited.There are greater tensions than ever before between promoting trade and protecting health. Human health could lose out to trade liberalization unless the health community fights its case
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Trade is the lifeblood of all commerce. The exchange of goods and services has played a defining role in human history, creating vast empires, encouraging mass migration, and sometimes tipping the balance between peace and conflict. It is thus unsurprising that protecting and encouraging international trade has remained a top priority for governments, businesses and international organisations.
Historically, the protection of health has been a permitted reason for restricting trade. Trade brought plague to Athens in 430 BC, killing as much as one third of the population, as well as to fourteenth century Europe after which quarantine practices were introduced. During the nineteenth century, flourishing trade also facilitated the spread of diseases such as cholera. This prompted a series of International Sanitary Conferences among leading trading nations, and the adoption of International Sanitary Conventions (forerunners of the present day International Health Regulations). While protecting health was a clear aim, in reality the primary task was to minimise interference by health matters on trade.
Today, there are greater tensions than ever before between promoting trade and protecting health because of globalisation. Successive rounds of trade negotiations held since the Second World War, under the General Agreement on Trade and Tariffs and, since 1995, the World Trade Organization (WTO), have substantially reduced tariff levels and standardised trading practices across countries. This process of trade liberalisation has significantly increased trade volumes, bringing more and more countries into the world trading system. For the public health community, trade has raced ahead of corresponding measures to protect health. Efforts to ensure that there is an appropriate balance between the two policy areas has become a difficult challenge.
Tensions between Trade and Health
The right to restrict trade to protect the health of humans, animals, and plants is recognised by the General Agreement on Trade and Tariffs (Article XX) under two conditions: (1) the restriction is applied in a nondiscriminatory way; and (2) the restriction is based on recognised scientific evidence. Countries are allowed to restrict trade, for example, of certain goods such as radioactive waste or infected food products.
Disputes can arise if the restriction is believed to be discriminatory or there is disagreement about the scientific evidence supporting it (see sidebar). The ban introduced by the European Community in 1989 on hormone-treated beef imported from the US. led to two rulings by the Dispute Settlement Body of the WTO in favour of the American government. The assessment of the evidence primarily by trade experts, rather than public health experts, is a clear problem of the existing dispute settlement process. The process also makes it difficult to regulate inappropriate production methods which do not lead to problems in the end product but may be of public health concern. For example, the practice of using hormones to boost meat production may prove problematic in future research, even if residues in the meat are not judged high enough at present to warrant sufficient proof of health concerns.
Moreover, tight regulations on trade that are intended to protect health can come under fire from the trade lobby. Two World Bank studies argue, for instance, that European Union (EU) regulations on pesticides in bananas, as well as aflatoxins, could be interpreted as barriers to trade and market access [1,2].
The short shrift given to precautionary measures to protect health, where existing scientific evidence is deemed insufficient, reflects a further inbuilt priority given to trade. Growing concerns over the development and use of genetically modified organisms (GMOs), for example, have been dismissed by major companies such as Monsanto and Cargill on the basis of a lack of existing scientific evidence of harm to health. Consumer groups and public health advocates, however, argue that the subject is still in its scientific infancy. Where new causal pathways or systemic impacts of environmental exposures are of concern, such as with GMOs, at best one can say that the jury is still “out”. Hence, allowing GMOs to be spread widely, rather than taking precautionary measures, could prove to have irreversible consequences.
There is a fine balance between protecting patents and ensuring access to essential drugs in the developing world
(Illustration: Margaret Shear)
The classification of certain goods as a risk to health, and thus subject to trade restrictions, is also a source of dispute. The best example is tobacco products which manufacturers argue should be treated like other traded goods. Public health advocates, however, argue that because tobacco is harmful to health, it should be subject to special restrictions. A battle over tobacco is currently being played out in regional trade negotiations and will be raised at forthcoming multilateral negotiations over agricultural trade. Whether the public health community will be able to argue successfully to protect health, when pitted against the vast resources of a multi-billion dollar industry, remains to be seen.
The TRIPs Agreement
Two new sources of tension have arisen in recent years—intellectual property rights (IPRs) and trade in services. The protection of IPRs is a new feature of international trade law, coming into force under the Agreement on Trade-Related Aspects of Intellectual Property Rights (TRIPS) adopted in 1994. The agreement sets out minimum standards for protecting and enforcing patents, trademarks, and copyrights.
Since the mid-1990s there has been increasing concern over access to drugs in the developing world, and especially drugs for treating HIV/AIDS. This issue gained international attention when the South African government sought to access cheaper versions of patent protected drugs, but was faced with strong opposition by the pharmaceutical industry. The importance of public health priorities, and the existence of flexibility within TRIPS, was eventually confirmed in the Doha Declaration on the TRIPS Agreement and Public Health signed in 2001 [3]. The agreement allows for “compulsory licensing”, which means that local manufacturers in poor countries are allowed to make cheap versions of patented drugs during public health emergencies, provided that they give a royalty payment to the patent holder. Nonetheless, IPRs protection has remained a problem given continued disagreement by the US over which diseases and countries are covered under the declaration. The capacity to protect IPRs has also been enhanced by the shift towards bilateral trade negotiations as a result of the breakdown in multilateral negotiations under the WTO.
While international debates over TRIPS have so far focused on the developing world, it is clear that there are issues relevant to the public health community as a whole concerning open access to a wide range of health-related knowledge and information. The possible benefits and costs of the TRIPS agreement had not been openly discussed beforehand. Rather, its measures were heavily influenced by industries seeking to exert ownership over intellectual goods such as research, information, and other data sources. Within an increasingly competitive world market, companies are driven to recoup their investment in such resources through IPRs. For the public health community, however, there is a vital need for affordable and open access by all to such resources. Leaving the commercial market to drive research and development (R&D) can lead not only to problems of access in developing countries but it can also lead to the neglect of research and public health priorities in all countries, such as research on antibiotics, which has been deemed insufficiently profitable [4]. Concern over the impact of intellectual property rights and marketing monopolies on overall pharmaceutical policies, pricing and R&D of pharmaceuticals has also led to proposals for a new trade framework for global health care R&D efforts [5].
Trade in Health Services
The General Agreement on Trade in Services (GATS) is another expanded area of trade law. Services are the fastest-growing segment of the world economy, providing more than sixty percent of global output and employment. In the past, most services were not considered to be tradable across borders. Advances in communications technology, including the rise of e-commerce, and regulatory changes have made it easier to deliver services across borders. For trade in health services, the implications are not yet clear.
It is generally expected that GATS would not apply to public services due to a general exemption on the matter. However, this exclusion is very narrowly worded, and based on a model of public services which may no longer hold. Health sector reforms have changed the ways in which publicly financed services are provided in many countries, including the extensive use of contracting out and managed competition. In most countries, this includes both medical care and related services such as laboratory or ambulance services. Health services, which are contracted out to the non-profit or for-profit sector, are likely to be no longer considered purely “public” and thus protected by the exclusion. Recent legal reviews of the GATS [6,7] confirm this concern, describing how the agreement would apply to any health-related service supplied on a “commercial” basis. Whether paid for directly by the patient or through a social security fund, the expectation that GATS negotiations will not apply to public services may no longer hold.
Given this, the two reviews warn that there are valid grounds to suggest that negotiations on trade in services, and full commitments in health services in GATS, will have important implications for the ways in which national health systems and policies are implemented. While GATS may not directly limit the aims of national health policies, commitments under GATS can influence the ability of governments to implement health policies and regulate commercial service providers. This could apply especially to efforts to introduce new regulations that restrict market forces. The current GATS negotiations on domestic regulation include requirements of least trade restrictiveness and necessity tests for introducing regulatory measures in committed sectors. These requirements could pose difficulties if a government sought, for example, to oblige hospitals to operate on a non-profit basis. This might be interpreted under the GATS as restricting market forces. In this way, GATS could effectively influence the scope of national health policy, even challenging the capacity of governments to pursue health policies that prioritise universal access, cost containment, and quality control.
Challenges to the Health Community
As world trade continues to expand in scale and scope, the health community faces a number of key challenges. We must be armed with a better understanding of the world trading system, notably the legal framework for international trade. Comprehending the potential health implications of various bilateral, regional, and multilateral agreements regulating trade today is a daunting task. Although the public health community has fewer resources at its disposal than the proponents of trade liberalisation, it is clear that health priorities do have strong support by citizens all over the world.
It is critically important for the health community to challenge the value-based assumption that trade liberalisation, rather than human welfare, should be given automatic priority. Indeed, ignoring health can lead to problems in the trade sphere. The outbreaks of bovine spongiform encephalopathy in Europe and North America and severe acute respiratory syndrome in Asia emphasised that trade can be severely disrupted by insufficient measures to protect health. The health community has to press for a much louder voice in the setting of trade policy at the national and international levels. A balance between trade and health policies can only be achieved if the health community is prepared to be far more vocal, and the trade policy community is prepared to listen.
Recent Disputes between Trade and Health
Growth-Promoting Hormones
In 1998 WTO ruled that a European Community ban of the use of certain growth-promoting hormones was not based on an appropriate risk assessment. Rather than lift the ban, the EU sought new evidence of the risk to human health of hormone residues in meat products. In 1999 Canada and the US imposed trade sanctions worth US$116.8 million and Cdn$11.3 million. In 2003 the EU announced that, based on new scientific evidence, the ban will remain in place and asked the US and Canada to lift sanctions.
Chrysotile Asbestos
In 2001 WTO ruled that the European Community was permitted to ban the import of chrysotile asbestos on the grounds of protecting public health, and rejected the Canadian government's claim that the ban was discriminatory and an unnecessary barrier to trade. While the decision shows that WTO panel decisions can prioritise health, the process and grounds of the decision indicates that, even with such a well-established carcinogen, the dispute settlement process required extensive argument by the public health community.
Citation: Lee K, Koivusalo M (2005) Trade and health: Is the health community ready for action? PLoS Med 2(1): e8.
Abbreviations
EUEuropean Union
GATSGeneral Agreement on Trade in Services
GMOsgenetically modified organisms
IPRsintellectual property rights
R&Dresearch and development
TRIPSAgreement on Trade-Related Aspects of Intellectual Property Rights
WTOWorld Trade Organization
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References
Otsuki T Wilson J Global trade and food safety: Winners and losers in a fragmented system Working paper 2689 2001 October Washington (DC) World Bank 34
Otsuki T Wilson J To spray or not to spray ? Pesticides, banana exports and food safety Working paper 2805 2002 March Washington, DC World Bank 28
World Trade Organization Declaration on the TRIPS agreement and public health 2001 Adopted on 14 November 2001. Available: http://www.wto.org/english/thewto_e/minist_e/min01_e/mindecl_trips_e.htm . Accessed 11 November 2004
Nelson R Antibiotic pipeline runs dry Lancet 2003 362 1726 1727 14655659
Hubbard T Love J A new trade framework for global healthcare R&D PLoS Biology 2004 2 e52 14966544
Luff D Mattoo A Sauve P Regulation of health services and international trade law Domestic regulation and service trade liberalization 2003 Oxford Oxford University Press and World Bank 244
Fidler D Legal review of the General Agreement on Trade in Services (GATS) from a public health perspective 2003 Geneva World Health Organization
| 15696218 | PMC545201 | CC BY | 2021-01-05 10:39:38 | no | PLoS Med. 2005 Jan 25; 2(1):e8 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020008 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1569621910.1371/journal.pmed.0020009The PLoS Medicine DebateOtherMedical EthicsEthicsInformed ConsentClinical TrialsShould an Institution That Has Commercial Rights in a New Drug or Device Be Allowed to Evaluate the Technology? The PLoS Medicine DebateMcKinney Ross Korn David Ross McKinney is Vice Dean for Research, Duke University School of Medicine, Durham, North Carolina, United States of America. E-mail: [email protected]
David Korn is Senior Vice President for Biomedical and Health Sciences Research at the Association of American Medical Colleges, Washington, D.C., United States of America. He is also Vice President, Dean of Medicine, and Professor of Pathology, Emeritus, at Stanford University, Stanford, California, United States of America. E-mail: [email protected]
Competing Interests: RM reports that he is employed by an academic medical center that has both intellectual property and performs clinical trials. DK declares that he has no competing interests.
1 2005 25 1 2005 2 1 e9Copyright: © 2005 McKinney and Korn.2005This 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 author and source are properly credited.Background to the debate: In the United States, the passage of the Bayh–Dole Act in 1980 encouraged universities to license inventions for commercial development. Although this financial incentive can stimulate academic researchers to discover new drugs and devices, there is concern that the possibility of monetary reward could distort investigators' objectivity.
In the US, universities are encouraged to license inventions for commercial development. Although this financial incentive can stimulate academics to discover new treatments, it might also distort the investigators' objectivity
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Ross McKinney's Viewpoint: Universities Should Be Allowed, Provided the Trial Is Approved by an External Review Board
One of the principal missions of an academic health center is to advance the understanding and treatment of disease through clinical research. In this pursuit, there is a need for checks and balances. When Jesse Gelsinger, a relatively healthy young adult, died in Philadelphia during a clinical trial of a novel adenovirus-based genetic therapy for ornithine transcarbamylase deficiency, it was a tragedy [1]. In retrospect, there were many clues that there were problems with the adenovirus vector, clues that neither the investigator nor the institution pursued.
Attorney Alan Milstein made the case that the investigator and institution were both blinded to these problems by their heavy financial investment in the technology, an investment worth millions of dollars [2]. Though the legal case was settled out of court, it created a de facto standard that institutions with commercial rights in a new drug or technology should not be allowed to pursue clinical trials involving that new technology. I do not believe that such a blanket prohibition is necessary.
At its core, the issue revolves around conflicts of interest. In clinical research, the investigator should be primarily an advocate for the patient or volunteer. The core reason to perform clinical research is to create generalizable knowledge about a therapy, patient population, or a disease process with the long-term intent of improving human health. The interests of the patient and investigator should be fully aligned. However, most physicians in clinical research have other, more personal motivations, intermixed with the desire for progress. Successful research projects can lead to publications, promotions, grant renewals, and per case clinical trial enrollment fees. Some investigators have intellectual property rights that may have very substantial financial value if the drug or device reaches the level of approval by the United States Food and Drug Administration. These investigators stand to gain personally if the clinical trial is successful, a situation that has the potential to distort the investigator's objectivity, and may lead to a less honest relationship with study volunteers.
An external IRB could provide independent oversight of the trial
(Illustration: Giovanni Maki)
In order to ensure that investigators are honest with potential research volunteers, the system of institutional review boards (IRBs) evolved. The IRB approves the informed consent document, which should describe the clinical experiment in a clear and dispassionate way to patients and their families. IRBs are largely made up of faculty and staff from the institution, although there are also public members and nonscientists on most IRB panels. The IRB must remain autonomous and be able to hold up or stop an investigation. There is an obligation that the IRB first and foremost think about patient rights and safety.
The passage of the Bayh–Dole Act in 1980 enabled universities to license inventions for commercial development [3]. The closer to Food and Drug Administration approval the drug or technology is at the time of licensure, the more valuable it becomes. Therefore, universities have an incentive to advance the clinical development of inventions by their faculty. In this regard, they are very much like corporate sponsors of research, subject to the same Food and Drug Administration oversight as corporations.
In terms of performing clinical trials using new technologies in which it has a financial interest, how is a university different from a corporate sponsor? In regard to patient safety, one primary distinction rests with the IRB. The corporate sponsor will present the research protocol to an independent commercial IRB, the university to its own IRB. Yet in both cases, there are potential conflicts of interest. The university IRB members will have a conflict of interest between the investments of their employer and the rights of the research volunteers. Independent commercial IRBs depend on pleasing corporate customers for their continued existence, and there is an unstated expectation that they will both be fast and produce rulings consistent with corporate expectations (which in most cases include a desire to do the research ethically).
A university's financial conflicts could influence the conduct of the trial
(Illustration: Giovanni Maki)
At the university level a logical and conservative solution to the problem of institutional conflicts is to require that an IRB from outside the institution become the IRB of record when such a conflict arises. This external IRB could be either an independent commercial IRB or one of another university. The key is to grant the IRB independence and the authority to provide real oversight.
There are other elements of conflicts of interest that need to be considered when the institution has a commercial interest, but most have more to do with the management of personal conflicts than institutional conflicts. The institution needs to assure the presence of an independent data safety monitoring board, thorough audits of good clinical practice, and a publications committee that will ensure submission of all meaningful study results, whether positive, negative, or neutral. Anyone subjectively evaluating patient data should be as free of conflicts as possible. These steps can be formulaically required, which should allow for performance of clinical research despite the presence of institutional commercial interests.
David Korn's Viewpoint: Academic Biomedical Research Must Be Free from the Taint of Financial Compromise
United States research universities, and especially their academic medical centers, have greatly benefited from their uniquely privileged status in our society. That status is rooted in public confidence and trust that these institutions and their faculties will be independent and impartial in fulfilling both their academic mission to create, transmit, and preserve knowledge, and their duty to the general society to serve as credible, trustworthy arbiters of knowledge. One important mark of this status has been the remarkably consistent generosity of public support for biomedical research. Another has been the noteworthy deference of the federal government to university autonomy, and the light hand with which the sponsoring agencies historically have overseen the conduct of university research.
When federal interposition occurred, it typically responded to widely publicized episodes of research misconduct, sometimes intertwined with egregious financial self interests of investigators; these episodes legitimately questioned the effectiveness of institutional oversight. Nevertheless, regulations consistently focused more on defining the metes and bounds of the permissible than on prescriptive mandates, and their implementation was effected largely through the mechanism of “assurances”—commitments that institutions would faithfully safeguard the specified perimeters of acceptable conduct.
Awardee institutions thus bear primary responsibility for assuring the credibility and integrity of federally sponsored research. Public confidence in the trustworthiness of these institutions is critical, and yet nowhere is it more fragile than in biomedical research involving human participants. That confidence eroded in the 1980s and 1990s because of reports of scientific misconduct and of individual and institutional financial self interests in clinical trials. Scathing reports from federal oversight agencies and angry congressional hearings questioned whether financially self-interested institutions could any longer be trusted to guard the welfare of research participants or the integrity of clinical research.
In 2001, the Association of American Medical Colleges convened a task force to examine and make recommendations on individual and institutional financial self interests in clinical research. The task force began by recognizing four important trends over the past three decades. First, the nature and culture of academic biomedical research have changed, bringing the potential of commercial relevance even to the most fundamental of scientific discoveries. Second, there has been enormous growth in the extent and depth of interactions between research universities and industry, especially in biomedicine. Third, the public has become increasingly impatient that its extraordinary investments in research yield more effective disease preventions and therapies. Fourth, the involvement of academic researchers in the translation of their discoveries has been essential in bringing those discoveries to market and to the benefit of public health.
But the task force, in its two reports, asserted that both individual and institutional financial conflicts of interest in clinical research could be problematic [4,5]. It recommended urgent and substantial refinement and strengthening of institutional policies and practices for monitoring, managing, and—when necessary—extinguishing such conflicts.
Both reports rest on a common set of core principles. The most important is that institutions should regard all significant financial interests in research involving human participants as potentially problematic. Where such interests exist, there should be a rebuttable presumption that the concerned individual or institution should not conduct the research, absent compelling circumstances. Importantly, the task force, after intense debate, rejected categorical prohibitions lest they unintentionally impede the translation of research discoveries into tangible public benefits.
The task force acknowledged that the issue of institutional financial self interests is extraordinarily complex and sensitive, since it touches the very core of institutional autonomy. But the fact that an institution has a financial interest per se should raise a strong presumption against its participation in the clinical testing of that product. Public accountability and scientific integrity require that all research results emanating from academic medicine be as free as possible from the taint of financial compromise. Adding human participants to the research mix should raise the barrier to the highest level and require compelling justification for any participation by a financially self-interested institution.
The task force did not define “compelling,” believing that each institution should make that determination based on disinterested scrutiny of the facts and circumstances of each case. For example, there may exist in a given institution a unique capability, without which the proposed research involving human participants could not be conducted as effectively or safely, or at all. In these instances, the public and science deserve access to that capability, provided the necessary safeguards are put in place to mediate the conflicting interests. In all such instances, protection of scientific integrity and the welfare of research participants must remain the foremost priority of both investigator and institution.
This narrow window avoids absolute prohibition while striving to prevent institutional participation where credible alternatives exist. Only by such stringent self-policing can we sustain the trustworthiness and credibility of biomedical research, researchers, and their institutions, while continuing vigorously to promote the translation of biomedical discovery for the public's benefit.
Ross McKinney's Response to David Korn's Viewpoint
The public has every right to expect that academic institutions are working first for the public's interest. This value is even codified in the laws granting these institutions tax-exempt status. The public also expects that new, more effective therapies will be developed swiftly as a consequence of its support for academic research.
The inventor of a new technology is always more motivated to see it through to widespread use than anyone else. This motivation, which may be as simple and benign as curiosity or as easy to understand as a financial incentive, is a powerful force driving human research. This force can be disciplined and controlled by the IRB and policies on conflicts of interest. Personal investment in research is, nevertheless, an important driver of scientific progress.
When society makes an unnecessarily broad assumption that nearly all research with financial implications for investigators or their institution is potentially corrupted, a brake is placed on progress. Society will be better served by establishing clear guidelines and formalizing oversight of the research process than by rigidly limiting clinical research affected by conflict of interest. As examples, clinical trials should have independent data safety monitoring boards charged to review the study design, execution, data analysis, and publication of results. The IRB system should be strengthened in its independence through the use of community members. And, to be certain that institutional conflict of interest is avoided, an IRB from outside the institution in most cases will be preferable
Society wants better treatments. The fact that an inventor has mixed motives for developing a new treatment has always been acknowledged. The need to carefully manage the experimental process in human studies has always been understood. However, when rules minimize the role of inventors at academic centers, by forcing trials of their new ideas to go to outside institutions, society loses more than it gains. The incremental gain in safety is likely to be small (particularly if oversight is well established), while the decrease in speed of development will be significant.
David Korn's Response to Ross McKinney's Viewpoint
There are many similarities between the position espoused by Ross McKinney and my position. Most saliently, we both recognize the critically important role played by academic biomedical scientists in making discoveries and in facilitating their efficient translation into beneficial products. Neither of us proposes that academic investigators, or their institutions, should be flatly prohibited from trying to foster that translation in the presence of financial self interests.
But there is an important difference. McKinney's approach for dealing with institutional conflicts of interest depends critically on the engagement of external agents to monitor closely both scientific integrity and the welfare of human participants. That, in my view, would require such deep interposition of those agents into the conduct of academic research as to be not only unprecedented but unfeasible. Beyond that, the approach falls short with respect to the maintenance of institutional trustworthiness and protection of public trust.
Routine clinical assessment of technologies by financially interested institutions fosters public cynicism and distrust of the motives of academic biomedical researchers. The protective mechanisms recommended by McKinney are opaque to the public and reflect a “business as usual” image that fails fully to account for the markedly changed circumstances and perceptions of academic biomedical research. Most important, the mechanisms appear to be aimed primarily at protecting institutions' financial interests.
By contrast, the American Association of Medical Colleges formulation urges that any institutional involvement in clinical research involving human participants in the presence of financial conflicts must be predicated on the presence or absence within the institution of demonstrably unique capability. This approach offers a much higher and more credible standard that aims to protect not only participant well-being and scientific integrity, but also institutional trustworthiness and public trust.
Citation: McKinney R, Korn D (2005) Should an institution that has commercial rights in a new drug or device be allowed to evaluate the technology? PLoS Med 2(1): e9.
Abbreviation
IRBinstitutional review board
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References
Ciment J Gene therapy experiments put on “clinical hold” BMJ 2000 320 336
Sibbald B Death but one unintended consequence of gene-therapy trial CMAJ 2001 164 1612 11402803
Moses H Martin JB Academic relationships with industry: A new model for biomedical research JAMA 2001 285 933 935 11180737
Association of American Medical Colleges Task Force on Financial Conflicts of Interest in Clinical Research Protecting subjects, preserving trust, promoting progress—Policy and guidelines for the oversight of individual financial interests in human subjects research 2001 Washington (D.C.) Association of American Medical Colleges Available: www.aamc.org/members/coitf/firstreport.pdf . Accessed 11 October 2004
Association of American Medical Colleges Task Force on Financial Conflicts of Interest in Clinical Research Protecting subjects, preserving trust, promoting progress II: Policy and guidelines for the oversight of an institution's financial interests in human subjects research 2002 Available: www.aamc.org/members/coitf/2002coireport.pdf . Accessed 11 October 2004
| 15696219 | PMC545202 | CC BY | 2021-01-05 11:13:37 | no | PLoS Med. 2005 Jan 25; 2(1):e9 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020009 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1569620110.1371/journal.pmed.0020010PerspectivesGenetics/Genomics/Gene TherapyGene TherapyGeneticsMicrobiologyIntegration of Retroviruses: A Fine Balance between Efficiency and Danger PerspectivesFischer Alain *Cavazzana-Calvo Marina Alain Fischer is Professor of Pediatric Immunology and Head of the Pediatric Immunology Department and the Inserm Research Unit 429 at Necker University Hospital, Paris, France. Marina Cavazzana-Calvo is Professor of Hematology and Head of the Biotherapy Department at Necker University Hospital, Paris, France.
*To whom correspondence should be addressed. E-mail: [email protected]
Competing Interests: The authors declare that they have no competing interests.
1 2005 25 1 2005 2 1 e10Copyright: © 2005 Fischer and Cavazzana-Calvo.2005This 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 author and source are properly credited.Since retroviruses can integrate a copy of their DNA into the host cell DNA, they are good vectors for gene therapy. But such vectors also have oncogenic potential
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One of the key characteristics of retroviruses is their ability to integrate a copy of the DNA reverse-transcribed from their viral RNA genome into host cell DNA. This integration is mediated by a preintegration complex (PIC) comprising viral DNA, reverse transcriptase, and integrase, as well as poorly characterized host proteins [1]. It is this property that makes retroviruses good vectors for the transfer of therapeutic genes, and many such vectors have been made.
The Perspectives section is for experts to discuss the clinical practice or public health implications of a published study that is freely available online.
Stages in Vector Development
Early vectors included those derived from murine oncoretroviruses (RVs) such as murine leukemia virus (MLV). A limitation of these viruses was that their PIC requires that the nuclear membrane is dissolved so that the PIC can come in direct contact with host cell DNA; hence, there is efficient integration only in dividing cells. Later vectors were based on lentiviruses (LVs) such as human immunodeficiency virus and simian immunodeficiency virus (SIV), whose PIC can penetrate into the nucleus so that integration can occur in nondividing cells that are in the G1 phase of the cell cycle.
Insertional Mutagenesis
MLV-derived vectors have been used with success to achieve sustained correction of two forms of severe combined immunodeficiency (SCID)—SCID-X1 (Gamma-c deficiency) [2] and adenosine deaminase deficiency [3]. In both cases, hematopoietic progenitors were infected out of the patient's body with nonreplicative MLV-derived vectors. However, integration carries the risk of insertional mutagenesis. This mutagenesis has been demonstrated in the chicken by using replicative RVs. RV integration close to protooncogenes has been shown to induce their activation, leading to tumorigenesis. Nonreplicative MLV vectors have also been reported to induce insertional mutagenesis in a murine model [4] and, more worryingly, in two patients from the SCID-X1 trial [5]. In both instances, it is suggested that cooperation between vector-associated transgene expression (dLNGFR in one case and common Gamma chain in the other) and long terminal repeat–driven enhancement of protooncogene expression (evl-1 and LMO-2, respectively) was responsible for aberrant clonal proliferation. No such events have been reported yet in the use of LV vectors in experimental settings.
Sites of Retroviral Integration
It was initially believed that integration of retroviruses occurred randomly, but the advent of technology allowing the assessment of RV or LV integration into host cell genomes has led to a reassessment of this assumption. Using a combination of ligation-mediated polymerase chain reactions and sequencing of amplified integration sites (unique sequences made from the viral long terminal repeat and the host-genome-associated sequence), it is possible to determine all the integration sites that can be found in a given transduced cell population (or its progeny). Exact mapping can be done back to the human (or relevant animal) genome database. However, even this methodology may not detect all of the integration sites present in a given transduced cell population.
Key papers have determined the “rules of the game” for RV and LV integration into a variety of cell lines [6,7,8]; these rules seem to differ between the virus types. In both cases, however, the integration pattern is not random. RV integration tends to be close to transcription start sites of active genes—close enough to regulatory sequences to potentially exert a long terminal repeat–mediated enhancer effect [7,8]. By contrast, LVs integrate mostly in transcription units, with a preference for actively transcribed genes, but do not target the region downstream of transcription start sites [6,8]. These data indicate that there are virus-specific PIC-associated determinants that cause specific targeting with the host cell genome. However, much remains to be done to identify viral factors and host ligands involved in these interactions.
Data from these pioneering papers were obtained by in vitro infection of mature cells or cell lines with the relevant RV or LV [6,7,8]. However, it is possible that the pattern of integration might differ in other cell subsets, particularly immature cells such as hematopoietic progenitors. In fact, as shown by Mitchell et al. [8], infection of mature cells of different tissue types leads to a partially distinct pattern of integration sites related to the set of genes transcribed in these different cell types [8].
In Vivo Models
In a paper published in last month's PLoS Biology, Hematti et al. [9] took the in vivo analysis further by analyzing the pattern of integration sites in cells derived from simian hematopoietic progenitor cells transduced either with a RV (MLV) or a LV (SIV) vector and transplanted into monkeys. This experimental setting is the closest possible to human trials. The results essentially confirm the nonrandom insertion pattern of both types of vectors as shown by the analysis of cell lines transduced in vitro. The analysis showed the Gaussian distribution of insertions centered on the transcription start site—thus very close to regulatory elements—of RV (Figure 1), and the preference of LV for the transcription units, with a concentration of integrations into some gene-dense regions [9]. A study such as this is therefore a useful piece of preclinical work that will help the interpretation of the analysis of clinical samples.
Figure 1 Distribution of MLV and SIV Integration Sites within a 60-kb Window Centered on Transcription Start Sites
The vertical arrow points to 0 kb. Each gray bar corresponds to the percentage of SIV integration sites within a 5-kb interval, and black bars correspond to the percentages of MLV integration sites in a 5-kb interval. The distribution of a set of 65,000 in silico–generated random integration sites is represented by the dashed line. (Source: [9].)
Similar data were also obtained by Laufs et al. in the analysis of a set of RV integration sites into human hematopoietic progenitors xenotransplanted into immunodeficient mice [10]. It thus appears that the overall distinct pattern of RV and LV integration could be independent of transduced cell types (immature versus differentiated, and tissue type). However, the targeted genes could differ considerably depending on the set of genes expressed in the target cell. Several parameters could potentially influence the transcription profile in a clinical setting, including stage of cell maturity, tissue type, ex vivo transduction culture conditions, patient age [5], underlying genetic disease, and any modification of the vector.
Designing Future Vectors
It will thus be essential to build a free, accessible database incorporating all relevant information gathered from both experimental and clinical settings. In this respect, information gathered from the SCID-X1 and adenosine deaminase deficiency trials is awaited with great interest. Combining all available information will be the only way to determine the frequency of insertions that have a potential to induce activation of a protooncogene (a risk primarily associated with the use of RV) or to induce disruption of a regulatory gene (a risk primarily associated with the use of LV). Studies of relevant gene expression activation or suppression should therefore be carried out in parallel. It is only from these multiple analyses, including a careful comparison of the oncogenic potential of vectors in relevant animal models, that a precise assessment of the risk associated with use of retroviruses for gene therapy will come. These data will thus be the basis for objective comparison between technologies and for the design of safer vectors.
Citation: Fischer A, Cavazzana-Calvo M (2005) Integration of retroviruses: A fine balance between efficiency and danger. PLoS Med 2(1): e10.
Abbreviations
LVlentivirus
MLVmurine leukemia virus
PICpreintegration complex
RVmurine oncoretrovirus
SCIDsevere combined immunodeficiency
SIVsimian immunodeficiency virus
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Aiuti A Slavin S Aker M Ficara F Deola S Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning Science 2002 296 2410 2413 12089448
Li Z Dullmann J Schiedlmeier B Schmidt M von Kalle C Murine leukemia induced by retroviral gene marking Science 2002 296 497 11964471
Hacein-Bey-Abina S Von Kalle C Schmidt M McCormack MP Wulffraat N LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1 Science 2003 302 415 419 14564000
Schroder AR Shinn P Chen H Berry C Ecker JR HIV-1 integration in the human genome favors active genes and local hotspots Cell 2002 110 521 529 12202041
Wu X Li Y Crise B Burgess SM Transcription start regions in the human genome are favored targets for MLV integration Science 2003 300 1749 1751 12805549
Mitchell RS Beitzel BF Schroder AR Shinn P Chen H Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences PLoS Biol 2004 2 e234 15314653
Hematti P Hong BK Ferguson C Adler R Hanawa H Distinct genomic integration of MLV and SIV vectors in primate hematopoietic stem and progenitor cells PLoS Biol 2004 2 e423 15550989
Laufs S Gentner B Nagy KZ Jauch A Benner A Retroviral vector integration occurs in preferred genomic targets of human bone marrow-repopulating cells Blood 2003 101 2191 2198 12424203
| 15696201 | PMC545203 | CC BY | 2021-01-05 10:38:13 | no | PLoS Med. 2005 Jan 25; 2(1):e10 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020010 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1569620210.1371/journal.pmed.0020012Research in TranslationCancer BiologyHematologyOncologyOncologyChemotherapyHematology (including Blood Transfusion)Retinoic Acid and Arsenic for Treating Acute Promyelocytic Leukemia Research in TranslationZhou Guang-Biao Zhao Wei-Li Wang Zhen-Yi Chen Sai-Juan Chen Zhu *The authors are at the Shanghai Institute of Hematology and State Key Laboratory for Medical Genomics, Rui Jin Hospital, Shanghai Second Medical University, Shanghai, China.
Competing Interests: The authors declare that they have no competing interests.
*To whom correspondence should be addressed: E-mail: [email protected] 2005 25 1 2005 2 1 e12Copyright: © 2005 Zhou et al.2005This 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 author and source are properly credited.What were the critical steps in the development of ATRA and arsenic as treatments for APL? Researchers in Shanghai tell the story and look to the future
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Acute promyelocytic leukemia (APL) was first identified as a distinct subtype of acute myeloid leukemia in 1957 by Leif Hillestad. It is called M3 in the French–American–British classification, with a variant type referred to as microgranular (M3v in the French–American–British nomenclature) [1]. APL is characterized by three features: (1) the presence of an accumulation of abnormal promyelocytes (see Glossary) that do not differentiate into mature granulocytes, (2) the occurrence of fibrinogenopenia and disseminated intravascular coagulation that is often worsened by chemotherapy, and (3) the presence of the specific chromosomal translocation t(15;17)(q22;q21) (Figure 1).
Glossary
Apoptosis: A genetically determined process of cell death in which the cell uses specialized cellular machinery to kill itself and is then eliminated by phagocytosis or by shedding.
Caspase: A family of cysteine proteases with aspartate specificity that are essential intracellular death effectors.
Disseminated intravascular coagulation: A hemorrhagic disorder that occurs following the uncontrolled activation of clotting factors and fibrinolytic enzymes throughout small blood vessels, resulting in depletion of clotting factors and tissue necrosis and bleeding.
Fibrinogenopenia: A decrease in concentration of fibrinogen in the blood.
Granulocyte: Terminally differentiated myelocyte or polymorphonuclear white blood cell (as a basophil, eosinophil, or neutrophil) with granule-containing cytoplasm.
Ligand-inducible transcription factors: Transcription factors that structurally have domains associated with DNA binding and ligand (hormone) recognition. When binding to its specific ligand, the transcription factor initiates a series of conformational changes and interacts efficiently with its specific DNA response element to recruit components of the transcriptional machinery.
Nuclear receptor superfamily: One of the most abundant classes of transcriptional regulators including receptors for steroid hormones (e.g., estrogens, glucocorticoids, and vitamin D3), RAs, thyroid hormones, and so on. These transcription factors regulate diverse functions such as homeostasis, reproduction, development, and metabolism in animals.
Promyelocyte: Granule-containing cell in bone marrow that is in an intermediate stage of development between myeloblasts and myelocytes and that gives rise to a granulocyte.
Proteasome: Proteolytic complex that degrades cytosolic and nuclear proteins.
Sumoylation: Post-translational modification of proteins by the small ubiquitin-like modifier SUMO.
Ubiquitin: A chiefly eukaryotic protein that when covalently bound to other cellular proteins marks them for proteolytic degradation.
Figure 1 The Three Features of APL
The three features of APL are (A) accumulation of abnormal promyelocytes, (B) fibrinogenopenia and disseminated intravascular coagulation, and (C) the chromosomal translocation t(15;17)(q22;q21), the resultant fusion transcripts, and variants.
APL accounts for 10%–15% of all cases of acute myeloid leukemia, with several thousand new cases diagnosed worldwide each year. Before the advent of differentiation therapy, APL was treated with anthracycline-based chemotherapy with a complete remission rate of 60%–76% and a 5-year event-free survival rate of 23%–35% [1,2].
Differentiation Therapy: From Hypothesis to Practice
Failure to differentiate terminally characterizes most, if not all, cancer cells of every origin. Whether the induction of differentiation could be a treatment strategy for cancers was hotly debated for decades before the advent of differentiation therapy.
An important discovery of the early 1970s was that myeloid leukemic cells could be reprogrammed to resume normal differentiation and to become non-dividing mature granulocytes or macrophages as a result of stimulation by various cytokines [3,4]. Based on this discovery, Leo Sachs at the Weizmann Institute of Science, Rehovot, Israel, hypothesized in 1978 that treatment with agents that induce cancer cells to complete differentiation could be a potential therapeutic option for patients with cancer [5]. In the early 1980s, Breitman and colleagues showed that retinoic acid (RA; Figure 2), a derivative of vitamin A, could induce terminal differentiation of human promyelocytic leukemic cells in vitro [6,7]. But the first clinical reports of using RA showed conflicting results. Some case reports showed beneficial effects of 13-cis RA in people with refractory or relapsed APL [8,9,10], but other reports showed that 13-cis RA was ineffective in treating APL [11].
Figure 2 Isomers of RA
Beginning in the early 1980s, the Shanghai Institute of Hematology conducted a series of experiments on differentiation therapy for APL. These experiments showed that all-trans RA (ATRA) could induce terminal differentiation of HL-60, a cell line with promyelocytic features, and fresh leukemic cells from patients with APL. These intriguing results were the impetus for a clinical trial. Twenty-four patients with APL were treated with ATRA (45 to 100 mg/m2/day). The result was dramatic: 23 patients (95.8%) went into complete remission (CR) without developing bone marrow hypoplasia or abnormalities of clotting. The remaining one patient achieved CR when chemotherapy was added [12]. Morphological maturation of bone marrow cells was found in all patients studied.
These results were later confirmed by many randomized studies in centers around the world. Further trials showed improved rates of CR, a decrease in severe adverse effects, and lengthening of the duration of remission. Table 1 summarizes the CR rates obtained in most large series of patients. Currently, ATRA combined with anthracycline-based chemotherapy can achieve CR in 90%–95% of patients with APL and overall 5-year disease-free survival in up to 75% of patients [13].
Table 1 CR Rate in Patients with APL Treated with ATRA (in Series Including More Than 50 Cases)
Chemo, chemotherapy
Arsenic: “Ancient Remedy Performs New Tricks”
Arsenic is a common, naturally occurring substance that exists in organic and inorganic forms in nature. The organic arsenicals consist of an arsenic atom in its trivalent or pentavalent state linked covalently to a carbon atom. There are three inorganic forms of arsenic: red arsenic (As4S4, also known as “realgar”), yellow arsenic (As2S3, also known as “orpiment”), and white arsenic, or arsenic trioxide (As2O3) [14].
Arsenic was used to treat chronic myelogenous leukemia in the 18th and 19th centuries, but was discarded as a treatment in the early 20th century because of its toxicity and the advent of radiation and cytotoxic chemotherapy. In the early 1970s, a group from Harbin Medical University in China found that intravenous infusions of Ailing-1, a crude solution composed of 1% arsenic trioxide with a trace amount of mercury chloride, induced CR in two-thirds of patients with APL. There was an impressive 30% survival rate after 10 years [15,16]. Pure arsenic trioxide at 0.16 mg/kg/day for 28–54 days was shown to induce CR in 14 out of 15 (93.3%) patients with relapsed APL [17]. Tetra-arsenic tetra-sulfide was also reported to be effective in APL treatment [18].
Since 1996, a large number of reports have shown that arsenic compounds induce a CR in 85% to 90% of patients with both newly diagnosed and relapsed APL [13]. Furthermore, after CR is achieved by arsenic compounds, a molecular remission (i.e., negative for promyelocytic leukemia RA receptor a [PML-RARa] transcript detected by reverse transcriptase polymerase chain reaction) is obtainable either with arsenic compounds or with ATRA and chemotherapy as consolidation treatment. It seems likely that arsenic compounds appropriately used in post-remission therapy could prevent recurrence and achieve a longer survival time [13,18].
Studies have shown that arsenic trioxide exerts dose-dependent dual effects on APL cells—it induces apoptosis (programmed cell death) preferentially at relatively high concentrations (0.5 × 10−6 to 2 × 10−6 M) and induces partial differentiation at low concentrations (0.1 × 10−6 to 0.5 × 10−6 M). The rapid modulation and degradation of the PML-RARa oncoprotein by arsenic trioxide could contribute to these two effects [19].
How Do ATRA and Arsenic Work at the Molecular Level?
To understand how ATRA and arsenic compounds act at the molecular level in treating APL, it is first important to understand the role of the PML-RARa fusion protein in the pathogenesis of APL.
Retinoids that are crucial for normal myeloid differentiation act via RA receptors (RARs) and retinoid X receptors (RXRs). These belong to the steroid/thyroid/retinoid nuclear receptor superfamily of ligand-inducible transcription factors. Both RAR and RXR families consist of three subtypes: α, β, and γ [20]. RARα forms a heterodimer with RXR and binds to RA response element to control the expression of target genes in the presence of physiological concentrations (10−9–10−8 M) of retinoids (Figure 3A).
Figure 3 Leukemogenic Effects of PML-RARá and Mechanisms of ATRA/Arsenic Trioxide in the Treatment of APL
(A) In the absence of RA, RARα/RXR heterodimers recruit the transcription corepressor (CoR), which mediates transcriptional silencing by mechanisms that include direct inhibition of the basal transcription machinery and recruitment of chromatin-modifying enzymes. Chromatin modification includes histone deacetylation, which leads to a compact chromatin structure that impairs the access of transcriptional activators. In the presence of physiological concentrations (10−9–10−8 M) of RA, the transcription corepressor is released and the coactivator is recruited to the RARα/RXR heterodimer, resulting in histone acetylation (AC) and overcoming of the transcription blockage.
(B) PML-RARα fusion protein binds to RARα target genes either on its own or with RXR and then recruits corepressors, leading to transcriptional repression and myeloid differentiation inhibition. PML-RARα oncoprotein sequesters the normal RXR and PML, inhibits the PML/P53 apoptotic pathway, and delocalizes PML and other proteins from the nuclear body. PML-RARα also may affect interferon (IFN) and other signal pathways. Abnormalities in protein tyrosine kinases (e.g., FLT3, c-fms) may collaborate with PML-RARα to cause APL.
(C) In the presence of pharmacological doses of ATRA or arsenic trioxide, the PML-RARα fusion is degraded in ways that are dependent on caspases and proteasomes. The degradation of PML-RARα may lead to derepression of transcription suppression and restoration of PML nuclear body structure. The blockade of other signaling pathways is also released, and the anti-apoptotic effect of PML-RARα is lost. ATRA also induces cyclic AMP (cAMP), which reverses the silencing of RXR, induces the expression of RA-induced genes and cyclooxygenase 1 (Cox 1), inhibits angiogenesis, and downregulates tissue factor. Subsequently, ATRA induces terminal cell differentiation, while arsenic trioxide induces partial differentiation and/or apoptosis of APL cells. The effects of ATRA and arsenic trioxide are indicated with red and blue arrows, respectively. AF2, the ligand-dependent transcriptional activation domain contained within the C-terminal E domain of RARα; D522, aspartate at residue 522; K160, lysine at residue 160; K490, lysine at residue 490; RARE, retinoic acid response element; SUG-1, a component of proteasome 19S complex that can bind with the activated AF2 domain of RARα.
More than 95% of patients with APL have the t(15;17)(q22;q21) translocation. This results in the fusion of the RARα gene on 17q21 and the promyelocytic leukemia (PML) gene on 15q22, which generates a PML-RARaα fusion transcript [21,22]. Variant translocations can also be detected in APL. The PML-RARα chimeric protein acts as a dominant negative mutant over wild-type RARα. The chimeric protein prevents activation of key target genes required for normal myeloid differentiation by sequestering RXR and other RARa cofactors and inhibiting normal RARα functions. The PML-RARα oncoprotein binds to RAR target genes either on its own or with RXR and then recruits histone deacetlyase complexes, which act as repressors of transcription.
PML-RARa may affect transcription in other pathways including those in which the transcription factor AP1 and interferon-responsive factors are involved. PML-RARα also binds to promyelocytic leukemia zinc finger (PLZF) protein and potentially affects its functions (e.g., growth suppression and transcription repression; control of developmental programs and differentiation) [20]. In addition, PML-RARα prevents apoptosis through delocalization of PML and other proteins from the nuclear body. Finally, PML-RARα may cooperate with activated mutations in protein tyrosine kinases, such as FLT3 [23], which confer proliferative and/or survival advantage to hematopoietic stem/progenitor cells. Whether PML-RARα affects the protein tyrosine kinase activity directly or indirectly is unclear. All these interactions of PML-RARα could be involved in the leukemogenesis of APL (Figure 3B).
ATRA and arsenic trioxide degrade and cleave the PML-RARα oncoprotein. Although we now have a good understanding of the molecular mechanisms underlying ATRA in differentiation therapy for APL, these mechanisms were shown long after the identification of the efficacy of this drug in treating the disease. Now it is well established that pharmacological concentrations of ATRA (10−7–10−6 M) exert their effects through targeting the PML-RARα oncoprotein, triggering both a change in configuration and degradation of the oncoprotein and the activation of transcription, leading to differentiation. Cleavage of the PML-RARα fusion protein by caspases at residue D522 has been shown in APL cells induced to differentiate by ATRA [24].
Further dissecting of the pathways involved in PML-RARα catabolism led to the discovery of ubiquitin/proteasome-mediated degradation of PML-RARα and RARα, which was dependent on the binding of SUG-1 in the AF2 transactivation domain of RARα with 19S proteasome [25,26]. In contrast to ATRA, which targets the RARα moiety of the fusion, arsenic targets the PML moiety of PML-RARα, through a still unclear mechanism, and causes PML to localize to the nuclear matrix and become sumoylated. Sumoylation at K160 is necessary for 11S proteasome recruitment and arsenic-trioxide-induced degradation, whereas sumoylation at K490 is needed for nuclear localization [27,28]. These results provide a striking similarity in the effect of these two otherwise unrelated agents (Figure 3C).
The final result of treatment with ATRA and arsenic trioxide is the degradation of the PML-RARa oncoprotein, which results in restoration of normal retinoid signaling. RXR and PML sequestration is abrogated, and PML nuclear body is restored. The corepressor is released and the coactivator is recruited and bound with RARα; thus, the transcription of target genes is derepressed. ATRA also induces cyclic AMP, a differentiation enhancer that boosts transcriptional activation, reverses the silencing of the transactivating function of RXR, and restores ATRA-triggered differentiation in mutant ATRA-resistant APL cells [29]. Additionally, ATRA induces the expression of RA-induced genes [30] and cyclooxygenase 1 [31], inhibits angiogenesis [32], downregulates the expression of tissue factor [33], and restores other signal pathways (e.g., the interferon pathway). Consequently, the abnormal promyelocytes differentiate and die through programmed cell death (Figure 3C).
Combining ATRA and Arsenic: A Cure for APL?
Since ATRA and arsenic trioxide degrade the PML-RARa oncoprotein via different pathways, and since studies in animal models have shown synergic effects of both drugs in prolonging survival or even eliminating the disease [34,35], the group at the Shanghai Institute of Hematology hypothesized that the combination of the two drugs might synergize in treating APL. To test this, 61 patients newly diagnosed with APL were randomized into three treatment groups: ATRA, arsenic trioxide, or a combination of the two drugs [36]. Although CR rates in all three groups were high (>90%), the time to achieve CR differed significantly—the time was shortest in patients treated with the combination. The disease burden (as reflected by fold change of PML-RARα transcripts at CR) decreased significantly more with combined therapy than with ATRA or arsenic trioxide monotherapy (p < 0.01), and this difference persisted after consolidation therapy (p < 0.05). Notably, all 20 patients in the combination group remained in CR whereas seven of 37 cases treated with monotherapy relapsed (p < 0.05) after a follow-up of 8–30 months (median, 18 months).
It seems that a combination of ATRA and arsenic trioxide for remission and maintenance treatment of APL produces better results than either of the two drugs used alone, in terms of the time required to achieve CR and the length of disease-free survival. We hope that the use of three treatments—ATRA, arsenic trioxide, and chemotherapy—will ultimately make APL a curable human acute myeloid leukemia [36].
Conclusion
The story of ATRA in the treatment of APL shows that by targeting the molecules critical to the pathogenesis of certain diseases, cells can be induced to return to normal. Differentiation therapy is therefore a practical method of treating human cancer that has shown consistent effectiveness in trials. The clarification of the underlying molecular abnormalities of APL is an example of the benefits of a close collaboration between bench and bedside, and is necessary for our understanding of the mechanisms of ATRA in differentiation therapy. It is clearly important to elucidate the molecular and cellular basis of a particular cancer if we are to further develop mechanism-based target therapies.
The sequencing of the human genome and ongoing functional genomic research are now accelerating the dissection of disease mechanisms and identification of therapeutic targets. This in turn may facilitate the screening of promising treatments. What we learn from developing curative treatment approaches to APL may help to conquer other types of leukemias and cancers.
This work was supported by the Chinese National Key Basic Research Project (973), the Chinese National High Tech Program (863), the National Natural Science Foundation of China, the Shanghai Municipal Commission for Science and Technology, the Shanghai Municipal Commission for Education, the Shanghai Municipal Commission for Health, the Shanghai Leading Academic Discipline Program, and the Samuel Waxman Cancer Research Foundation of the Shanghai Institute of Hematology. We appreciate long-term fruitful collaboration with Professors Hugues de Thé, Laurent Degos, Roland Berger, Michel Lanotte, and Christine Chomienne from Saint Louis Hospital, Paris; Professor Samuel Waxman from Mount Sinai Medical Center, New York; Dr. Arthur Zelent from Leukemia Research Fund Center, London; Professor Zhi-Xiang Shen from Rui-Jin Hospital, Shanghai; and Professor Ting-Dong Zhang from Harbin Medical University, Harbin, China.
Citation: Zhou GB, Zhao WL, Wang ZY, Chen SJ, Chen Z (2005) Retinoic acid and arsenic for treating acute promyelocytic leukemia. PLoS Med 2(1): e12.
Abbreviations
APLacute promyelocytic leukemia
ATRAall-trans retinoic acid
CRcomplete remission
PMLpromyelocytic leukemia
PML-RARápromyelocytic leukemia retinoic acid receptor á
RAretinoic acid
RARretinoic acid receptor
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| 15696202 | PMC545204 | CC BY | 2021-01-05 10:38:13 | no | PLoS Med. 2005 Jan 25; 2(1):e12 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020012 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1569620310.1371/journal.pmed.0020013PerspectivesCancer BiologyOncologyRespiratory MedicineCancer: LungChemotherapyGene Mutations in Lung Cancer: Promising Predictive Factors for the Success of Molecular Therapy PerspectivesInoue Akira *Nukiwa Toshihiro Akira Inoue is a research assistant and Toshihiro Nukiwa is a professor in the Department of Respiratory Oncology and Molecular Medicine, Institute of Development, Aging, and Cancer, Tohoku University, Sendai, Japan.
Competing Interests: The authors declare that they have no competing interests.
*To whom correspondence should be addressed. E-mail: [email protected] 2005 25 1 2005 2 1 e13Copyright: © 2005 Inoue and Nukiwa.2005This 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 author and source are properly credited.Gefitinib and erlotinib are two new treatments for advanced lung cancer. Gene mutations in the cancer may help predict which patients will respond to these treatments
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Lung cancer is the leading cause of death in many countries. To date, cause of cancer death in chemotherapy with cytotoxic agents has been the mainstay of treatment for advanced lung cancer. However, the activity of these agents is quite limited, and they have severe adverse effects. Recent, rapid advances in molecular biology have led to the development of many new agents that inhibit the activities of specific molecules related to tumor growth, invasion, or metastasis [1], and these agents have the potential to improve the outcome of lung cancer treatment. But we have not yet managed to successfully deliver these agents from the bench to the bedside.
Molecular Therapy for Lung Cancer
Gefitinib is the first “molecular-target agent” for lung cancer that inhibits the tyrosine kinase of the epidermal growth factor receptor (EGFR; also known as ERBB1). EGFR is frequently overexpressed in non-small-cell lung cancer (NSCLC), especially in squamous cell carcinoma, and its expression is related to the cancer's proliferation. Initial clinical trials of gefitinib showed its modest clinical activity in patients who had failed previous standard chemotherapy. But subsequent randomized trials in patients with previously untreated, advanced NSCLC have not shown a clinical advantage of gefitinib combined with standard chemotherapy over chemotherapy alone [2,3,4,5].
Erlotinib, another EGFR tyrosine kinase inhibitor for treating NSCLC, which was approved by the United States Food and Drug Administration in November 2004, has a therapeutic profile similar to that of gefitinib. Erlotinib has shown a survival benefit in a phase III trial for chemo-refractory NSCLC that compared erlotinib to best supportive care [6]. (To date, there have been no trials showing a survival benefit of EGFR inhibitors over standard chemotherapy.) Interestingly, in these trials of EGFR inhibitors, EGFR expression levels in the tumors were not correlated with the response; high response rates were seen in women, patients with adenocarcinoma, nonsmokers, and Japanese patients. Recent studies have now shed light on why certain patients are more likely to respond than others—the key lies in the presence of gene mutations.
EGFR Gene Mutations in NSCLC
In April and May 2004, two new studies published in Science and the New England Journal of Medicine helped to explain at the molecular level the low clinical activity of EGFR inhibitors [7,8]. These studies identified somatic mutations in the EGFR gene, especially around the region encoding the ATP-binding pocket of the receptor's tyrosine kinase domain. These mutations increased the sensitivity of tumor cells to gefitinib. A high incidence of mutations was detected in patients with NSCLC that had a durable clinical response to gefitinib, and subsequent studies revealed that these mutations were also related to the response to erlotinib. Moreover, such mutations were more frequently detected in patients with adenocarcinoma, in women, in Japanese patients, and in nonsmokers [9]—results that were compatible with previous clinical data.
These findings should hopefully lead to the identification of subgroups of patients who are likely to benefit substantially from such EGFR inhibitors. But another question was raised by these studies: is it only EGFR gene mutations that determine the response to EGFR inhibitors?
RAS Gene Mutations in NSCLC
The RAS proteins are low-molecular-weight GTPases that are bound to the inner side of the cell membrane. They are involved in signal transduction pathways: they regulate downstream effector proteins such as Raf/MAP kinase and PI3 kinase, under the influence of various cell surface receptors including EGFR (Figure 1). Mutations of the K-ras gene have been found in up to 30% of lung adenocarcinomas and have been considered a poor prognostic factor [10]. A study by Pao and colleagues published in this issue of PLoS Medicine suggests that the K-ras mutation is an important predictive factor in defining which patients will benefit from receiving EGFR inhibitors [11]. In Pao and colleagues' study, the K-ras mutations were completely associated with a lack of response to EGFR inhibitors (0/14 tumors with K-ras mutations were sensitive). The EGFR mutations were significantly related to response (17/17 tumors with EGFR mutations were sensitive), as observed in previous studies.
Figure 1 EGFR Signal Transduction in Cancer Cells
Arrows indicate stimulation, and T-bars, inhibition. EGFR-I, EGFR inhibitor; MEK, MAPK kinase.
Although the number of tumors examined in this study may be too small to lead to a definite conclusion—and, furthermore, about half of tumors were retrospectively collected—this study is the first to show that mutations of EGFR and K-ras are not related and that K-ras mutations are associated with a lack of sensitivity to EGFR inhibitors. However, since the sensitivity of the method for finding each mutation influences how often it is detected, standardization of detection methods is important. Hence, the true incidence of K-ras mutations in NSCLC (including non-adenocarcinoma) and their refractoriness to EGFR inhibitors need to be established in further studies. In fact, the reported incidence of EGFR mutations differs between institutions. In addition, although Pao and colleagues' study examined such mutations only by DNA sequence, mRNA or protein expression would show mutation status more accurately.
Therapeutic Implications
In the clinical setting, prolonged disease stabilization with no measurable reduction in tumor size is seen in about half of patients treated with EGFR inhibitors, and a significant survival benefit in this group was shown in a phase III trial [6]. The mutation status of the EGFR gene and K-ras gene among those stabilized tumors should be evaluated; it may reveal two different groups differentiated by mutation status.
It will also be important to establish whether, in tumors with an EGFR mutation that eventually acquire resistance to EGFR inhibitors, the resistance is associated with a change of EGFR status (mutation or change in expression) or is associated with a change of K-ras status. Such changes can be revealed by re-examination of tumors at the time of relapse. If resistance is K-ras dependent, the new “K-ras inhibitors” (unfortunately not yet available) may be of help for patients who have developed a K-ras mutation, as well as for patients whose tumors harbor K-ras mutations from the beginning.
Although many issues still need to be resolved step by step through prospective trials to show the benefit of this new strategy, these mutations are promising predictive factors for the success of EGFR inhibitors. Even if the population who may benefit from EGFR inhibitors (such as patients who are positive for an EGFR mutation and negative for a K-ras mutation) is very small, the response rate of over 80% is encouraging, and has never before been achieved in advanced NSCLC. Finally, it is important to detect which patients derive no benefit from EGFR inhibitors because severe adverse effects such as acute lung injury can occur in any patient treated with these drugs [12]. By combining all the factors that relate to response or resistance, patients who will benefit from treatment can hopefully be identified. Undoubtedly we have taken a great step forward in molecular therapy for lung cancer treatment.
Citation: Inoue A, Nukiwa T (2005) Gene mutations in lung cancer: Promising predictive factors for the success of molecular therapy. PLoS Med 2(1): e13.
Abbreviations
EGFRepidermal growth factor receptor
NSCLCnon-small-cell lung cancer
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Shepherd FA Pereira J Ciuleanu TE Tan EH Hirsh V A randomized placebo-controlled trial of erlotinib in patients with advanced non-small cell lung cancer (NSCLC) following failure of 1st line or 2nd line chemotherapy. A National Cancer Institute of Canada Clinical Trials Group (NCIC CTG) trial [abstract] J Clin Oncol 2004 22 July 15 Suppl 7022 Available: http://www.asco.org/ac/1,1003,_12-002643-00_18-0026-00_19-00678,00.asp . Accessed 24 November 2004
Paez JG Janne PA Lee JC Tracy S Greulich H EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy Science 2004 304 1497 1500 15118125
Lynch TJ Bell DW Sordella R Gurubhagavatula S Okimoto RA Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib N Engl J Med 2004 350 2129 2139 15118073
Pao W Miller V Zakowski M Doherty J Politi K EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib Proc Natl Acad Sci U S A 2004 101 13306 13311 15329413
Huncharek M Muscat J Geschwind JF K-ras oncogene mutation as a prognostic marker in non-small cell lung cancer: A combined analysis of 881 cases Carcinogenesis 1999 20 1507 1510 10426799
Pao W Wang TY Riely GJ Miller VA QiuluPan KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib PLoS Med 2005 2 e17 15696205
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| 15696203 | PMC545205 | CC BY | 2021-01-05 10:38:12 | no | PLoS Med. 2005 Jan 25; 2(1):e13 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020013 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1569620410.1371/journal.pmed.0020015Research ArticleImmunologyInfectious DiseasesEpidemiology/Public HealthInfectious DiseasesImmunology and allergyEpidemiologyAre Anticapsular Antibodies the Primary Mechanism of Protection against Invasive Pneumococcal Disease? Immunity to Pneumococcal DiseaseLipsitch Marc
1
*Whitney Cynthia G
2
Zell Elizabeth
2
Kaijalainen Tarja
3
Dagan Ron
4
Malley Richard
5
1Departments of Epidemiology and Immunology and Infectious Diseases, Harvard School of Public HealthBoston, MassachusettsUnited States of America2Active Bacterial Core Surveillance and Division of Bacterial and Mycotic Diseases, National Center for Infectious DiseasesCenters for Disease Control and Prevention, Atlanta, GeorgiaUnited States of America3National Reference Laboratory for Pneumococcus, National Public Health InstituteOuluFinland4Pediatric Infectious Disease Unit, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the NegevBeer-ShevaIsrael5Children's Hospital and Harvard Medical SchoolBoston, MassachusettsUnited States of AmericaGreenwood Brian Academic EditorUniversity of LondonUnited Kingdom
Competing Interests: The authors have declared that no competing interests exist.
Author Contributions: ML and RM designed the study. TK and RD conducted the studies. ML and EZ analyzed the data. CGW, EZ, and the Active Bacterial Core Surveillance Team contributed reagents/materials/analysis tools. ML, RM, CGW, and RD contributed to writing the paper. The members of the Active Bacterial Core Surveillance Team are listed in the Acknowledgments.
*To whom correspondence should be addressed. E-mail: [email protected] 2005 25 1 2005 2 1 e152 8 2004 28 11 2004 Copyright: © 2005 Lipsitch et al.2005This 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 author and source are properly credited.
Rethinking Immunity against Pneumococcal Disease
Background
Antibody to capsular polysaccharide has been the basis of several vaccines that offer protection against invasive disease from Streptococcus pneumoniae. The success of such vaccines has led to the inference that natural protection against invasive pneumococcal disease is largely conferred by anticapsular antibody. If this is so, one would expect that the decline in disease from different serotypes would vary significantly, and that the appearance of substantial concentrations of anticapsular antibodies would coincide temporally with the decline in age-specific incidence.
Methods and Findings
Using incidence data from the United States, we show that, on the contrary, the decline in incidence with age is quite similar for the seven most important serogroups, despite large differences in exposure in the population. Moreover, only modest increases in antibody concentration occur over the second and third years of life, a period in which serotype-specific incidence declines to less than 25% of its peak. We also present detailed data on the distribution of antibody concentrations in Israeli toddlers, which are consistent with the United States findings. The same conclusion is supported by new data on age-specific incidence in Finland, which is compared with published data on antibody acquisition in Finnish toddlers.
Conclusion
We suggest some additional studies of the mechanisms of protection that could distinguish among potential alternative mechanisms, including acquired immunity to noncapsular antigens, maturation of nonspecific immune responses, or changes in anatomy or exposure.
Anticapsular antibodies induced by vaccination can protect against invasive streptococcal disease, but natural immunity seems to involve other mechanism as well
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Introduction
The protective effects of antibody to pneumococcal capsular polysaccharides have been appreciated since the development of serum therapy, in which passively transferred, serotype-specific antipneumococcal serum reduced mortality from pneumococcal pneumonia by half [1]. The development of pneumococcal polysaccharide vaccines for adults [2] and the efficacy of pneumococcal polysaccharide–protein conjugate vaccines in infants and children [3,4] have confirmed that active immunity to the polysaccharide can provide excellent protection against invasive disease from pneumococci of the same serotype, and in some cases protection against cross-reacting serotypes within the same serogroup.
While the ability of passive or vaccine-induced anticapsular antibodies to protect against pneumococcal disease is clear, less is known about the natural development of immunity to pneumococcal disease in unimmunized persons. In unimmunized populations, the incidence of invasive disease follows a well-known age distribution, peaking in the first 2 y of life, declining by more than an order of magnitude by the second and third decades of life, and then rising at an accelerating pace, with incidence in persons over 70 y approaching that in infants [5]. The reason for the decline in incidence has not been conclusively determined, yet it is often suggested that the acquisition of anticapsular antibodies plays a critical role in this decline [6,7]. Indeed, it has been proposed that the human immune system sees each serotype of Streptococcus pneumoniae as a distinct, independent pathogen [8].
The hypothesis that protection from invasive pneumococcal disease is caused by the acquisition of anticapsular antibodies directed against each of the pneumococcal serotypes yields two simple predictions about the age-specific epidemiology of pneumococcal disease. First, it predicts that the age-specific timing of the decline in invasive disease should be different for different serotypes: those that are rare, poorly immunogenic, or both should decline later in life than those that are common and immunogenic. Second, it predicts that protection against invasive disease from a given serotype should coincide temporally with the acquisition of anticapsular antibody to that serotype, both at an individual level and at a population level. We tested these predictions using data from the United States, Finland, and Israel.
Methods
United States Dataset
Incidence of invasive pneumococcal disease was measured in eight sites around the United States participating in the Centers for Disease Control and Prevention's Active Bacterial Core Surveillance between 1994 and 1999. The data used here are restricted to those periods during which serotyping was routinely performed: 1994–1999 for the Georgia site, 1995–1999 for the Minnesota site, and 1998–1999 for all other sites [5]. Data were not available on the timing of anticapsular antibody acquisition in these same populations, but we compared the timing of the decline in pneumococcal disease against previously published data on age-specific prevalence of anticapsular antibody levels greater than 0.2 mcg/ml [9].
Israel Dataset
Antibody concentrations were measured by enzyme-linked immunosorbent assay (ELISA) (with absorption by cell wall polysaccharide but not by 22F polysaccharide) in blood samples that were obtained from 130 toddlers at enrollment and at approximately 12 and 24 mo after enrollment in a double-blind, controlled trial of a nine-valent pneumococcal conjugate vaccine. The toddlers analyzed for this study were those in the control group, which received meningococcal C conjugate vaccine; the details of the trial [10] were previously described. Preliminary analyses of these data confirmed previous findings [11] that ELISA measurements were highly correlated (and therefore likely revealed cross-reactions) for all pairs of serotypes, except for type 14, for which correlations were minimal, consistent with previous findings of little cross-reaction. For this reason, we chose to analyze age trends only in serotype 14 antibodies.
Finland Dataset
Mandatory reporting from all microbiological laboratories in Finland to the National Register of Infectious Disease (http://www3.ktl.fi/stat/) identified all blood and cerebrospinal fluid isolates of S. pneumoniae obtained in the years 1995–2001. Incidence within 6-mo age groups was calculated using population denominators obtained from Statistics Finland (Helsinki, Finland). Since the primary purpose of examining incidence in Finland was to compare age-specific rates against published distributions of antibody concentrations for the same age groups [12], we restricted our attention to serotype 14 and serogroup 6, for which subsequent investigations suggested antibody measurements in Finland were relatively unaffected by cross-reactions [13] (ELISA measurements for published data from Finland used a special type 6B polysaccharide that was found to minimize cross-reactions [13]).
Results
United States Findings
Figure 1 shows the age-specific incidence of invasive pneumococcal disease, by capsular serogroup, obtained from population-based active surveillance in the United States prior to the introduction of the conjugate vaccine. Figure 2 shows age-specific incidence by type of infection, for the same age range.
Figure 1 Age-Specific Incidence of Invasive Pneumococcal Disease in the United States by Serogroup, Based on Data from Active Bacterial Core Surveillance
Serogroups 4 and 23 are shown only up to 48 mo, after which incidence is less than 1/100,000 person-years. All serogroups besides those in the heptavalent vaccine are shown combined as non-vaccine serogroups (NVG).
Figure 2 Age-Specific Incidence of Invasive Pneumococcal Disease in the United States by Disease Type, Based on Data from Active Bacterial Core Surveillance
Meningitis incidence is plotted only up to 30 mo, after which it remains at or below 1/100,000 person-years. “Pneumonia” indicates bacteremic pneumonia, while “bacteremia” indicates nonfocal bacteremia. “Total” includes other invasive diagnoses.
Incidence peaks between the ages of 9 and 15 mo, and falls in an approximately parallel fashion thereafter, for each of the seven most important serogroups (which are those included in the seven-valent conjugate vaccine) and for the remaining serogroups put together. The same pattern is observed for both pneumonia and bacteremia. For each serogroup, incidence by age 24 mo is approximately half that in the peak age group, and by 36 mo, incidence for each serogroup has fallen to 10%–25% of its peak.
The consistent timing of the pattern across multiple serogroups argues for a common mechanism, rather than for independent acquisition of immunity to each serogroup as a separate event. Since most individuals do not suffer from invasive pneumococcal disease in this age range, carriage or mucosal disease (otitis media) from pneumococci may be the immunizing event for anticapsular antibodies in the general population [12] (although in principle immunity to some serogroups could be generated in response to cross-reacting antigens from other bacterial species or other sources [14]). Different serogroups have vastly different frequencies among pneumococci isolated from carriage [12,15,16,17] and otitis media [12,18]; for example, serogroups 4 and 18 and the non-vaccine serogroups are isolated far less commonly than several of the other pneumococcal types identified in Figure 1. One could postulate that these differences in frequency of carriage are offset by differences in immunogenicity; however, there is little evidence that serotypes 4 or 18C are more immunogenic than other, far more common serotypes [4,15]. One could also postulate that the frequency of isolation of serotypes from carriage depends on duration as well as incidence, so that the serotypes for which carriage appears rare are simply carried for a shorter duration. While the data to address this speculation are limited, the duration of carriage of types 4 and 18C seems to be comparable to that of other, more frequently carried serotypes [15,16]. Thus, the most parsimonious interpretation of the data on the timing of the decline in age-specific susceptibility is that one or more common mechanisms are responsible for the decline in disease from all serotypes.
Testing the second prediction against data is hampered by the fact that, to our knowledge, no study has characterized the age-specific distribution of antibody concentration in a large population using the currently accepted methodology, which includes absorption with both cell wall polysaccharide and serotype 22F polysaccharide [13,19]. Analyses by Soininen and colleagues have found that antibodies measured by standard ELISA in unimmunized children are highly cross-reactive between different serotypes, and that cross-reactive antibodies lack opsonophagocytic function and often appear in the absence of any documented exposure to a given capsular serotype. As a result, age-specific antibody concentration data for any given serotype are “contaminated,” to a greater or lesser degree, by cross-reactive antibodies with other specificties.
The most important exception to this problem occurs for antibodies to serotype 14, for which cross-reaction is minimal [11]. A recent publication describes the age-specific proportion of children in the United States with anti-type-14 polysaccharide antibody concentration exceeding the putative protective concentration of 0.2 μg/ml (Figure 3 of [9]). At 12 mo, 90%–95% of the population falls below this level, and at 24 mo, 80%–85% remains below it—despite a 40%–50% drop in disease incidence from 12 mo to 24 mo. At 36 mo, 75% of children remain below the putative protective level, although by this age incidence has fallen more than 80% from its 12-mo peak. In summary, if the 0.2-μg/ml concentration were truly the threshold for “protection,” the 20%–30% reduction in the unprotected population between ages 12 and 36 mo would be inadequate to explain the 90% decline in disease incidence. Clearly, 0.2 μg/ml is not a precise dividing line between being “protected” and “unprotected,” a threshold that (if it exists) may vary by serotype, but given the available data, there is reason to doubt that anti-type-14 antibody alone is responsible for the decline in disease in this age range.
Figure 3 Box-Whisker Plot of Anti-Type-14 Polysaccharide Antibodies in Israeli Toddlers, by 6-Mo Age Groups
Central boxes indicate median and 25th and 75th percentiles; whiskers indicate upper and lower adjacent values.
Israel Findings
To assess whether the limitations of the United States antibody described above—i.e., the availability of only one cutoff point for antibody concentrations—might be providing an incomplete picture of the distribution of antibody levels by age, we examined an additional dataset from Israeli toddlers. For the reasons described above, we examined only antibodies to serotype 14, for which the distribution of concentrations by age is shown in Figure 3. These data indicate that between the ages of 12–17 and 36–41 mo, the median antibody concentration increases by about 2-fold. These data are broadly consistent with those published for the United States; antibody levels rise very gradually, though detectably, during the second and third years of life. It is difficult to believe—albeit not impossible—that the dramatic declines in disease incidence over these years are explained simply by this small rise in antibody concentrations.
Finland Findings
Incidence of serogroup 6 and serotype 14 invasive pneumococcal disease by 6-mo age groups in Finland, shown in Figure 4, is broadly similar to that found in the United States, albeit with lower absolute incidence for both serogroups. Peak incidence occurs in the 12–17-mo age group, and incidence declines to 25%–30% of its peak rate by 24–29 mo of age. This decline in incidence may be compared against the cumulative distributions of antibody concentrations in Finnish toddlers shown in Figure 2 of [12]. Between ages 12 and 24 mo, there is a discernible increase in the concentration of antibodies in the population, but the median concentration increases by only about 2-fold in this period. Moreover, the proportion of the population with antibody concentration below any particular threshold that may indicate protection changes little in this period. For example, the proportion of the population with anti-type-14 antibody concentrations less than 0.2 μg/ml declines from approximately 55% to approximately 40%, and the proportion with less than 0.5 μg/ml is reduced from about 95% to about 80%. Similar patterns are seen in the Finnish antibody data for type 6B [12]. Thus, as in the Israeli data, only a very small shift in the distribution of type-specific anti-polysaccharide antibody concentration is observed during the second year of life, yet incidence of invasive disease from the serotypes in question declines substantially.
Figure 4 Age-Specific Incidence of Invasive Pneumococcal Disease Caused by Serogroups 6 and 14 in Finland, Based on Active Laboratory-Based Surveillance
Discussion
We have assessed two lines of epidemiological evidence, analyzed ecologically, that bear on the role of anticapsular polysaccharide antibody as the determinant of protection against invasive disease that develops during the second and third year of life. The simultaneous and approximately parallel nature of the decline in disease incidence for the seven most important serogroups in the United States suggests that one mechanism, rather than seven independent mechanisms, account for the declines in invasive disease from these serogroups. Moreover, only a slight increase in anticapsular antibody concentration is measurable in Finnish, United States, and Israeli toddlers during the same age range. As we discuss below, each of these lines of evidence is subject to caveats, but we believe that, taken together, these observations make a strong case for the importance of one or more factors other than acquisition of anticapsular antibodies in the development of protection against pneumococcal disease.
There are several possible candidates for mechanisms that could explain this age-related decline in pneumococcal disease. These include the following: acquisition of antibodies or cellular immune responses to noncapsular pneumococcal “species” antigens; age-related changes in host biology that are not related to acquired immunity, such as maturation of the innate immune system or changes in anatomy or receptors for pneumococcal attachment; changes in other risk factors, such as exposure; or changes related to other microorganisms, including changes in the resident flora or changes in the incidence of viral infections.
Systemic antibodies to several pneumococcal protein antigens, which are conserved across pneumococcal strains and serotypes, develop following pneumococcal carriage and otitis media and are present by the beginning of the second year of life [20,21]. In both Finland [20] and Kenya [21], there is an increase in the concentration of antibodies to the pneumococcal proteins pneumolysin and pneumococcal surface protein A over the first two or more years of life. In Kenya, antibodies to another conserved protein, pneumococcal surface adhesin A, showed similar distributions in the first, second, and subsequent years of life, while in Finland, levels of these antibodies were already high (equivalent to adult levels) in the first year of life, and increased above these levels in the second year. In mice, either passively transferred human serum IgG against pneumococcal surface protein A or vaccine-induced antibodies to pneumococcal surface protein A and/or pneumolysin are protective against invasive disease. Such data are consistent with the hypothesis that antibodies to these, or perhaps other, conserved pneumococcal proteins are in part responsible for the decline in invasive disease in the second and subsequent years of life.
A number of investigators have tested the hypothesis that antibodies to the pneumococcal teichoic acid, known as cell wall polysaccharide (CWPS), are capable of protecting individuals against pneumococcal invasive disease. While studies in animals [22] and humans [23] have failed to find a protective effect of antibodies to CWPS or its components, a recent study showed that passive transfer of human IgG against phosphorylcholine, a component of CWPS, could protect mice against invasive pneumococcal infections [24]. Notably, such antibodies might be elicited by a number of bacteria in addition to pneumococci, such as Haemophilus influenzae, which also produce phosphorylcholine. We are unaware of studies on the timing of acquisition of anti-CWPS antibodies.
We have recently shown that mice that are exposed thrice at weekly intervals to intranasal colonization with encapsulated pneumococci are protected against subsequent carriage, that this protection is effective for heterologous as well as homologous capsular types, and that it is effective even in MuMT mice, which lack the ability to produce antibodies (Malley R, Trzcinski K, Srivastava A, Thompson CM, Anderson PW, et al., unpublished data). We have also shown that intranasal immunization with unencapsulated, killed pneumococci protects against nasopharyngeal colonization, in a fashion that is independent of antibody but requires CD4+ T cells at the time of challenge. The relevance of cellular immune mechanisms in protecting humans against pneumococcal colonization or disease is not known.
Another candidate for a factor that may be changing with age is susceptibility to viral infections, especially influenza, which may predispose to pneumococcal colonization [25] or disease [26,27]. Recent evidence from clinical trials of pneumococcal conjugate vaccines shows that the vaccines can reduce the incidence of infections such as bronchiolitis that are usually associated with viruses [28] and of documented, virus-associated pneumonia [27]. These findings raise the possibility that the decline in pneumococcal disease with age reflects, in part, a decline in the incidence or severity of viral infections, so that fewer such infections lead to secondary pneumococcal disease.
Exposure to pneumococci probably changes in some fashion over the first 5 y of life. However, for changes in exposure to account for the sharp drop in disease incidence following the first birthday, it would be necessary for exposure also to drop severalfold per year over this age range. Studies of pneumococcal carriage do show gradual changes in the prevalence and serotype composition of the nasopharyngeal flora in these years, but the prevalence of carriage changes much more gradually than the incidence of invasive disease [29].
We are not aware of data that bear strongly on the plausibility of other possible mechanisms for the age-related decline in pneumococcal disease, such as changes in anatomy, physiology, receptor expression, or resident bacterial flora. However, factors other than antibody—such as innate or acquired cellular immune responses, age-related anatomical changes, or changes in exposure to pneumococci—cannot be ruled out, and more than one factor may be involved. Indeed, the peak of pneumococcal meningitis incidence in the 3–6-mo age group (see Figure 2) suggests that the mechanism of protection against meningitis may differ from those against pneumonia and bacteremia.
Although we suggest that anticapsular antibody is not primarily responsible for the age-specific decline in invasive pneumococcal disease, there is no question that the capsule is an important virulence factor that interacts with the innate and acquired immune system in a number of ways. It is clear that the pneumococcal capsule interferes with various host clearance mechanisms [30]. It would be unsurprising if different capsular types were differentially effective in permitting pneumococci to evade phagocytosis and other host defenses [31] (M. Melin, H. Jarva, S. Meri, and H. Käyhty, unpublished data). If this were the case, then one could envision that certain capsular types might in fact follow a different age-specific incidence. In particular, recent analyses suggest that serotypes 1 and 5 have relatively stable incidence over a range of age groups (W. P. Hausdorff, D. R. Feikin, and K. P. Klugman, unpublished data).
The evidence adduced here is subject to several limitations. With respect to the relative timing of acquisition of protection against different serotypes, one could postulate that because some of the most common pneumococcal serotypes, such as 6B, 19F, and 23F, are also among the least immunogenic [12], the effective exposure of the immune system is more consistent across serogroups than it appears from serogroup frequency alone. However, this pattern is not general; for example, serotype 14 is both very common and highly immunogenic [12]. With respect to the absolute timing of protection relative to the acquisition of antibody, one could argue that low levels of anticapsular antibody, perhaps of low affinity, may be present and even active at levels below those that can be reliably detected by current assays, or that B cell memory may be present and protective at an earlier age than that at which high levels of antibody are measurable. Inferences about protective antibody concentrations from animal studies and from concentrations achieved by vaccines suffer from several uncertainties. Making allowances for all of these limitations, we nonetheless believe the data suggest that mechanisms other than anticapsular antibody are primarily responsible for the age-specific decline in pneumococcal invasive disease that starts at the age of 1 y.
The likelihood that mechanisms other than anticapsular antibody confer immunity to pneumococcal disease has important implications with respect to vaccine design. As experience with conjugate pneumococcal vaccines in children unfolds, it is becoming increasingly clear that such a strategy suffers from several limitations, including the possibility of serotype replacement (already confirmed in several clinical trials), a modest effect on nasopharyngeal colonization, limited serotype coverage, cost, and difficulties in production that have led to shortages since licensure. A better understanding of the mechanisms that underlie natural immunity to pneumococcus could pave the way for the development of more effective, species-specific pneumococcal vaccines.
Patient Summary
Background
Streptococcus pneumoniae is a common bacterium that lives in the upper respiratory tract of many children, and some adults. The bacterium generally causes no harm in healthy individuals, but in some circumstances it can cause mild infections, such as ear infections, or more severe ones, such as lung infection (pneumonia), bloodstream infection (bacteremia), or infection of the lining of the brain (meningitis). These more severe forms, called invasive pneumococcal disease, occur especially in children, elderly people, and others with weakened immune systems. The bacterium exists in different versions, or serotypes. The different versions of the bacterium each have a different outer shell (the so-called bacterial capsule). Scientists have developed vaccines against Streptococcus pneumoniae that protect against the most common serotypes. These vaccines consist of a cocktail made up of material from the capsules of the most common serotypes. This material causes the body's immune system to produce antibodies that can fight Streptococcus pnemoniae and protect vaccinated individuals against disease caused by the common serotypes. In many developed countries vaccination is recommended for all children and elderly people.
Why Was This Study Done?
Most people get exposed to many different versions of the bacterium over the course of their lives. These encounters cause little or no disease in most people, and the risk of disease declines sharply and remains low through middle age, before climbing again in the elderly. Based on experience with vaccines, scientists have thought that this “natural” protection that develops with age was also based on antibodies against the bacterial capsule. The authors of this study wanted to test whether this was actually true.
What Did the Researchers Do?
If in the healthy population protection against invasive disease is in fact due to anticapsular antibodies, one can make certain predictions about the frequency of invasive disease among certain age groups. The researchers tested those predictions against actual disease records from the United States, Israel, and Finland.
What Did They Find?
The actual records did not match the predictions very well, suggesting that natural protection against invasive pneumococcal disease is not based on anticapsular antibodies alone.
What Does This Mean?
These results suggest that there are elements of natural protection against invasive pneumococcal disease that we do not understand yet. Moreover, these elements seem to involve more general protection against various forms of the bacterium rather than individual protection against particular serotypes.
What Next?
Given the importance of the disease, we should try to understand all elements of natural protection. Such understanding might help researchers develop better vaccines to prevent invasive pneumococcal disease, and maybe even improve treatment of patients who have become ill.
More Information Online
World Health Organization information page on pneumococcal vaccines: http://www.who.int/vaccines/en/pneumococcus.shtml
United States Centers for Disease Control and Prevention factsheet on pneumococcal vaccine: http://www.cdc.gov/nip/publications/VIS/vis-PneumoConjugate.pdf
Health Canada information on pneumococcal vaccine: http://www.hc-sc.gc.ca/english/iyh/medical/pneumococcal.html
Information for health-care providers from the United Kingdom Nation-al Health Service: http://www.prodigy.nhs.uk/guidance.asp?gt=Immunizations%20-%20pneumococcal
PneumoADIP Web page on childhood pneumococcal disease: http://www.pneumoadip.com/
Tamara Pilishvili is thanked for assistance with assembling the database used for Figures 1 and 2. Porter Anderson and Michael Wessels are thanked for helpful criticism of the manuscript. ML is funded by National Institutes of Health grant 1R01 AI48935. RM acknowledges funding from the Meningitis Research Foundation and from National Institutes of Health grant 1K08 AI51526–01. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The Active Bacterial Core Surveillance Team includes Anne Schuchat (Centers for Disease Control and Prevention), Monica M. Farley (Emory University), Ruth Lynfield (Minnesota Emerging Infectious Program), Lee H. Harrison (Johns Hopkins University), Nancy N. Bennett (Monroe County Health Department, New York), William Schaffner (Vanderbilt University), Arthur Reingold (University of California at Berkeley), James Hadler (Connecticut Emerging Infections Program), and Paul Cieslak (Oregon Emerging Infections Program).
Citation: Lipsitch M, Whitney CG, Zell E, Kaijalainen T, Dagan R, et al. (2005) Are anticapsular antibodies the primary mechanism of protection against invasive pneumococcal disease? PLoS Med 2(1): e15.
Note Added in Proof
A recent report from McCool and Weiser showed that antibody-deficient mice clear pneumococcal colonization at rates similar to those of wild-type mice [32].
Abbreviations
CWPScell wall polysaccharide
ELISAenzyme-linked immunosorbent assay
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1569620510.1371/journal.pmed.0020017Research ArticleGenetics/Genomics/Gene TherapyOncologyOncologyCancer: LungGenetics
KRAS Mutations and Primary Resistance of Lung Adenocarcinomas to Gefitinib or Erlotinib KRAS Mutations Predict Drug ResistancePao William
1
2
*Wang Theresa Y
1
Riely Gregory J
2
Miller Vincent A
2
Pan Qiulu
3
Ladanyi Marc
3
Zakowski Maureen F
3
Heelan Robert T
4
Kris Mark G
2
Varmus Harold E
1
1Program in Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer CenterNew York, New YorkUnited States of America2Department of Medicine, Memorial Sloan-Kettering Cancer CenterNew York, New YorkUnited States of America3Department of Pathology, Memorial Sloan-Kettering Cancer CenterNew York, New YorkUnited States of America4Department of Radiology, Memorial Sloan-Kettering Cancer CenterNew York, New YorkUnited States of AmericaHerbst Roy Academic EditorMD Anderson Cancer CenterUnited States of America
Competing Interests: VAM has received research funding from Genentech (co-developer of erlotinib). He has received honoraria from AstraZeneca (maker of gefitinib) for consultancy. MGK has received research funding from AstraZeneca and research funding and consulting fees from Genentech and has represented AstraZeneca before the US Food and Drug Administration. WP, VAM, MFZ, and HEV, represented by the Sloan-Kettering Institute for Cancer Research, filed on June 1, 2004, a provisional patent application entitled “Use of mutations in EGFR kinase as an indicator of therapeutic efficacy of erlotinib in the treatment of NSCLC,” serial number 60/576,275. HEV is Co-founder and Chair of the Board of Directors of the Public Library of Science.
Author Contributions: WP and HEV designed the study. QP and ML designed and performed more sensitive methods to detect EGFR mutations. WP, TYW, GJR, VAM, MFZ, MGK, and RTH acquired and analyzed the data. WP, TYW, and HEV contributed to writing the paper.
*To whom correspondence should be addressed. E-mail: [email protected] 2005 25 1 2005 2 1 e1714 10 2004 28 11 2004 Copyright: © 2005 Pao et al.2005This 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 author and source are properly credited.
Predicting Tumor Responses to Gefitinib and Erlotinib
Background
Somatic mutations in the gene for the epidermal growth factor receptor (EGFR) are found in adenocarcinomas of the lung and are associated with sensitivity to the kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva). Lung adenocarcinomas also harbor activating mutations in the downstream GTPase, KRAS, and mutations in EGFR and KRAS appear to be mutually exclusive.
Methods and Findings
We sought to determine whether mutations in KRAS could be used to further enhance prediction of response to gefitinib or erlotinib. We screened 60 lung adenocarcinomas defined as sensitive or refractory to gefitinib or erlotinib for mutations in EGFR and KRAS. We show that mutations in KRAS are associated with a lack of sensitivity to either drug.
Conclusion
Our results suggest that treatment decisions regarding use of these kinase inhibitors might be improved by determining the mutational status of both EGFR and KRAS.
Mutational analysis of the KRAS gene in lung cancer patients treated with two different kinase inhibitors suggests that tumors with KRAS mutations do not respond to these drugs
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Introduction
Genes of the ERBB family encode receptor tyrosine kinases that mediate cellular responses to growth signals. Somatic mutations in the tyrosine kinase domains of two ERBB genes, epidermal growth factor receptor (EGFR) and HER2, have been found in a proportion of lung adenocarcinomas [1,2,3,4]. For EGFR, mutations are associated with sensitivity to the small-molecule kinase inhibitors gefitinib (Iressa) [1,2,3] and erlotinib (Tarceva) [3].
ERBB signaling pathways include downstream GTPases encoded by RAS genes. Some 15%–30% of lung adenocarcinomas contain activating mutations in the RAS family member KRAS. These mutations are most frequently found in codons 12 and 13 in exon 2 [5,6], and may be associated with unfavorable outcomes [7]. Interestingly, EGFR and KRAS mutations are rarely found in the same tumors, suggesting that they have functionally equivalent roles in lung tumorigenesis ([8]; M. Meyerson, personal communication). Furthermore, EGFR mutations are common in tumors from patients who have smoked less than 100 cigarettes in their lifetimes (“never smokers”) [3], while KRAS mutations more commonly occur in individuals with a history of substantial cigarette use [9].
We sought to determine whether KRAS mutations could also be used to predict primary sensitivity or resistance to gefitinib or erlotinib. We systematically evaluated 60 lung adenocarcinomas from patients with known responses to either of these drugs for the presence of mutations in EGFR (exons 18 through 21) and KRAS2 (exon 2). Here, we show that mutations in KRAS are associated with primary resistance to single-agent gefitinib or erlotinib. Our results suggest that a determination of mutational status for both EGFR and KRAS may help define which patients are likely to benefit from receiving gefitinib or erlotinib.
Methods
Tissue Procurement
Tumor specimens were obtained through protocols approved by the institutional review board of Memorial Sloan-Kettering Cancer Center, as previously described [3] (see Protocols S1–S3). Tumor material, obtained from patients prior to kinase inhibitor treatment for lung cancer, was collected retrospectively for patients on gefitinib, who received 250 mg or 500 mg orally once daily (n = 24), and prospectively for patients on erlotinib, who received 150 mg orally once daily (n = 36). The latter cohort of patients was part of a clinical trial of erlotinib for patients with bronchioloalveolar carcinoma. The analysis presented here includes specimens we previously reported on (n = 17 for gefitinib and n = 17 for erlotinib) [3].
All specimens were reviewed by a single reference pathologist (M. F. Z.). Imaging studies were assessed by a single reference radiologist (R. T. H.), who graded responses according to Response Evaluation Criteria in Solid Tumors (RECIST) [10]. Both observers were blinded to patient outcomes.
Eight of nine patients with tumors sensitive to gefitinib had objective partial responses as defined by RECIST, i.e., at least a 30% decrease in the sum of the longest diameters of target lesions, taking as reference the sum measured at baseline. The ninth patient had marked clinical improvement, as ascertained by two independent reviewing physicians and manifested by lessened dyspnea and cancer-related pain. However, this individual had radiographic lesions (pleural and bone metastases) that were deemed nonmeasurable by RECIST criteria. As erlotinib-treated patients were all in a clinical trial, all had disease measurable using RECIST guidelines. For both drugs in this study, tumors were considered refractory if they did not undergo sufficient shrinkage to qualify for partial response. This definition includes patients whose “best overall response” was either progression of disease (n = 26) or stable disease (n = 12) as defined by RECIST. No patients had a complete response.
Mutational Analyses of EGFR and KRAS in Lung Tumors
Genomic DNA was extracted from tumors embedded in paraffin blocks, except for tumor 109T, which was a fresh-frozen tumor specimen. Primers for EGFR analyses (exons 18–21) were as published [3]. For KRAS analyses, the following nested primer sets for exon 2 were used: huKRAS2 ex2F, 5′-
GAATGGTCCTGCACCAGTAA-3′; huKRAS2 ex2R, 5′-
GTGTGACATGTTCTAATATAGTCA-3′; huKRAS2 ex2Fint, 5′-
GTCCTGCACCAGTAATATGC-3′; and huKRAS2 ex2Rint, 5′-
ATGTTCTAATATAGTCACATTTTC-3′.
For both EGFR and KRAS, PCR was performed using the HotStarTaq Master Mix Kit (Qiagen, Valencia, California, United States), as per manufacturer's instructions. Use of this method often obviated the need for nested PCR sets. All sequencing reactions were performed in both forward and reverse directions, and all mutations were confirmed by PCR amplification of an independent DNA isolate.
In 12 cases, exon 19 deletions were also studied by length analysis of fluorescently labeled PCR products on a capillary electrophoresis device, using the following primers: EGFR-Ex19-FWD1, 5′-
GCACCATCTCACAATTGCCAGTTA-3′, and EGFR-Ex19-REV1, 5′-Fam-
AAAAGGTGGGCCTGAGGTTCA-3′. Using serial dilutions of DNA from the H1650 non-small-cell lung cancer cell line (exon 19 deletion-positive [11]), this assay detects the mutant allele when H1650 DNA comprises 6% or more of the total DNA tested, compared to a sensitivity of 12% for direct sequencing. These same cases were also screened for the exon 21 L858R mutation by a PCR–restriction fragment length polymorphism assay, based on a new Sau96I restriction site created by the L858R mutation (2,573T→G). The Sau96I-digested fluorescently labeled PCR products were analyzed by capillary electrophoresis, and the following primers were used: EGFR-Ex21-FWD1, 5′-
CCTCACAGCAGGGTCTTCTCTGT-3′, and EGFR-Ex21-REV1, 5′-Fam-
TCAGGAAAATGCTGGCTGACCTA-3′. Using serial dilutions of DNA from the H1975 cell line (L858R-positive [11]), this assay detects the mutant allele when H1975 DNA comprises 3% or more of the total DNA tested, compared to a sensitivity of 6% for direct sequencing (Q. Pan, W. Pao, and M. Ladanyi, unpublished data).
Statistics
Fisher's Exact Test was used to calculate p-values, and confidence intervals were calculated using Statistics with Confidence software [12].
Results
We identified 60 lung adenocarcinomas from individual patients with tumors shown to be sensitive or refractory to single-agent gefitinib or erlotinib and evaluated these tumors for mutations in EGFR and KRAS. Collectively, nine of 38 (24%) tumors refractory to either kinase inhibitor had KRAS mutations, while zero of 21 (0%) drug-sensitive tumors had such mutations (p = 0.02) (Table 1). The 95% confidence intervals (CIs) for these observations are 13%–39% and 0%–16%, respectively. Conversely, 17 of 22 (77%) tumors sensitive to either kinase inhibitor had EGFR mutations, in contrast to zero of 38 (0%) drug-resistant tumors (p = 6.8 × 10−11). The 95% CIs for these observed response rates are 57%–90% and 0%–9%, respectively. All 17 tumors with EGFR mutations responded to gefitinib or erlotinib, while all nine tumors with KRAS mutations did not (p = 3.2 × 10−7).
Table 1
EGFR and KRAS Mutation Status in Lung Adenocarcinomas Sensitive or Refractory to Gefitinib or Erlotinib
Lung tumors were examined for mutations in EGFR (exons 18–21) and KRAS (exon 2). In gefitinib-treated patients, six EGFR mutations involved exon 19 deletions that lacked the amino acids Leu-Arg-Glu-Ala, and two were exon 21 amino acid substitutions (L858R). A seventh case with an exon 19 deletion (involving 12 nucleotides) was detected by fluorescent capillary electrophoresis only, so exact sequence deletion information was unavailable (see Methods). In erlotinib-treated patients, three EGFR mutations were exon 19 deletions that lacked amino acids Leu-Arg-Glu-Ala, and five were exon 21 amino acid substitutions (L858R)
a One erlotinib-sensitive tumor was unavailable for KRAS examination
b The incidence of KRAS mutations in this cohort was low, probably because only cases involving bronchioloalveolar carcinoma were tested (see text)
Correlation of EGFR and KRAS mutational status with drug and treatment response is detailed in Table 1. The spectrum of KRAS mutations is shown in Figure 1 and Table 2. Results with gefitinib and erlotinib were similar overall. However, the incidence of KRAS mutations in the patients treated with erlotinib was low, probably because of the fact that all patients treated with this drug had bronchioloalveolar carcinoma, which rarely has RAS mutations [13]. Alternatively, our analyses involving only exon 2 of KRAS2 may have missed some RAS mutations. However, in our analysis of the exonic regions encoding the first 100 amino acids of KRAS in 110 surgically resected early-stage non-small-cell lung cancers, we have found 18 mutations, and all were in either codon 12 or codon 13, encoded by exon 2 (W. Pao, R. Wilson, H. Varmus, unpublished data). Another possibility is that the erlotinib-treated tumors have mutations in other RAS genes, since a minority of RAS mutations in lung cancer have been reported to occur in N- or HRAS [5,6].
Figure 1 Sequence Chromatograms Displaying the Types of KRAS Mutations Found in This Study
Table 2
KRAS Exon 2 Mutations Found in Non-Small-Cell Lung Cancers Refractory to Treatment with Gefitinib or Erlotinib
a Bold indicates mutations
Discussion
These results have important clinical implications. First, they extend previous data from our group and others showing that lung adenocarcinomas containing EGFR mutations are associated with sensitivity to gefitinib or erlotinib (17 of 17 in this series; 100% observed response rate; 95% CI, 82%–100%). Second, these data show that tumors with KRAS exon 2 mutations (n = 9) are associated with a lack of response to these kinase inhibitors (0% observed response rate; 95% CI, 0%–30%). Third, no drug-sensitive tumors had KRAS exon 2 mutations (n = 21). Whether KRAS mutational status can be used to predict responses to gefitinib or erlotinib in patients whose tumors have wild-type EGFR sequence is still under investigation: our analysis comparing response rates for tumors with neither EGFR nor KRAS mutations versus tumors with wild-type EGFR but mutated KRAS does not reach statistical significance (five of 22 versus zero of nine; p = 0.29). Nevertheless, these findings suggest that patients whose lung adenocarcinomas have KRAS mutations will not experience significant tumor regression with either drug.
The incidence of EGFR mutations in tumors responsive to EGFR kinase inhibitors has varied from 71% to 100% ([1,2,3] and this paper). Thus, at this point, patients whose tumors test negative for EGFR mutations should not necessarily be precluded from treatment with either gefitinib or erlotinib. Data presented here suggest that clinical decisions regarding the use of these agents in patients with lung adenocarcinomas might be improved in the future by pre-treatment mutational profiling of both EGFR and KRAS. These findings warrant validation in large prospective trials using standardized mutation detection techniques.
Supporting Information
Protocol S1 Preclinical Studies of Blood, Urine, Bone Marrow, and Tissues Collected from Patients with Thoracic Malignancies
(32 KB PDF).
Click here for additional data file.
Protocol S2 Multicenter Phase II Trial of OSI-774 (Erlotinib, Tarceva) in Patients with Advanced Bronchioloalveolar Cell Lung Cancer
(1.9 MB PDF).
Click here for additional data file.
Protocol S3 Protocol Approval Letters
(60 KB PDF).
Click here for additional data file.
Accession Numbers
The LocusLink (http://www.ncbi.nlm.nih.gov/LocusLink/) accession number for the KRAS2 sequence discussed in this paper is 3845; the GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) accession number for the KRAS2 sequence discussed in this paper is NT_009714.16.
Patient Summary
Background
Two drugs, gefitinib (Iressa) and erlotinib (Tarceva), have been developed that can make lung cancers smaller in some patients. The drugs work by blocking the effect of a molecule called the epidermal growth factor receptor (EGFR), which relays instructions to cells to grow and divide. Recently, researchers found that these drugs most effectively shrink tumors that have acquired abnormal variations (mutations) in the EGFR gene. These mutations somehow allow tumor cells to escape normal safety mechanisms that keep cells from growing out of control. Some lung cancers also have mutations in another gene called KRAS. Interestingly, KRAS mutations and EGFR mutations are rarely ever found in the same tumor.
Why Was This Study Done?
Unfortunately, EGFR mutations are only found in a minority of patients with lung cancer. This means that gefitinib or erlotinib might be given to a lot of patients who may not benefit from this treatment. Ideally, the drugs would be given only to patients who we know will benefit from them. This study examined whether studying the KRAS gene (to see if it had a mutation) could help predict which patients had tumors that would respond well to the drugs.
What Did the Researchers Do?
They took 60 lung cancer samples from patients who had been treated with one of the drugs and either responded (that is, their tumors shrunk in size) or not, and tested whether the tumors had normal or abnormal KRAS.
What Did They Find?
Tumors that got significantly smaller while treated with gefitinib or erlotinib (a total of 22) had a normal KRAS gene. Most of these tumors had EGFR mutations. Conversely, tumors that had abnormal KRAS (a total of nine) did not shrink while treated with gefitinib or erlotinib.
What Does This Mean?
Both gefitinib and erlotinib are expensive and have side effects. Testing for EGFR and KRAS mutations is relatively straightforward, and one could test for abnormalities in both genes first and then decide which patients should be treated with either of the two drugs.
What Next?
Before doing EGFR and KRAS tests on a routine basis and taking the results into account when making a decision about who should be treated with gefitinib or erlotinib, larger studies need to be done to see whether the results reported here hold up.
More Information Online
US Food and Drug Administration information page on Iressa: http://www.fda.gov/cder/drug/infopage/iressa/iressaQ&A.htm
Cancer Research UK information page about erlotinib: http://www.cancerhelp.org.uk/help/default.asp?page=10296
We acknowledge J. Doherty for help with PCR, T. McDonough for clinical data assembly, J. Somar and T. Zheng for technical assistance with DNA extraction from the paraffin blocks, D. Wong and D. Tabarini from the Memorial Sloan-Kettering Cancer Center sequencing core facility for sequencing, E. Venkatraman for statistical calculations, K. Politi for critical reading of the manuscript, and M. McClellan and R. Wilson from the Genome Sequencing Center at Washington University in St. Louis for sequencing sample 109T. TYW received research grant support from the Johns Hopkins University School of Medicine (Henry Strong Denison Award for Medical Research). WP received grant support from the National Institutes of Health (NIH CA009512) and the CHEST Foundation of the American College of Chest Physicians and the LUNGevity Foundation. This work was funded in part by an anonymous donor. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Citation: Pao W, Wang TY, Riely GJ, Miller VA, Pan Q, et al. (2005) KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib. PLoS Med 2(1): e17.
Abbreviations
CIconfidence interval
EGFRepidermal growth factor receptor
RECISTResponse Evaluation Criteria in Solid Tumors
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References
Lynch TJ Bell DW Sordella R Gurubhagavatula S Okimoto RA Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib N Engl J Med 2004 350 2129 2139 15118073
Paez JG Janne PA Lee JC Tracy S Greulich H
EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy Science 2004 304 1497 1500 15118125
Pao W Miller V Zakowski M Doherty J Politi K EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib Proc Natl Acad Sci U S A 2004 101 13306 13311 15329413
Stephens P Hunter C Bignell G Edkins S Davies H Lung cancer: Intragenic ERBB2 kinase mutations in tumours Nature 2004 431 525 526
Rodenhuis S Slebos RJC Boot AJM Evers SG Mooi WJ Incidence and possible clinical significance of K-ras oncogene activation in adenocarcinoma of the human lung Cancer Res 1988 48 5738 5741 3048648
Suzuki Y Orita M Shiraishi M Hayashi K Sekiya T Detection of ras gene mutations in human lung cancers by single-strand conformation polymorphism analysis of polymerase chain reaction products Oncogene 1990 5 1037 1043 2197591
Rodenhuis S Slebos RJ The ras oncogenes in human lung cancer Amer Rev Resp Dis 1990 142 S27 S30 2252272
Gazdar A Shigematsu H Herz J Minna J Mutations and addiction to EGFR: The Achilles ‘heal' of lung cancers? Trends Mol Med 2004 10 481 486 15464447
Ahrendt SA Decker PA Alawi EA Zhu YR Sanchez-Cespedes M Cigarette smoking is strongly associated with mutation of the K-ras gene in patients with primary adenocarcinoma of the lung Cancer 2001 92 1525 1530 11745231
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Sordella R Bell DW Haber DA Settleman J Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways Science 2004 305 1163 1167 15284455
Altman DG Machin D Bryant TN Gardner MJ editors Statistics with confidence, 2nd ed 2000 London BMJ Books 240
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Ras oncogene point mutation: An infrequent event in bronchioloalveolar cancer J Thor Cardiovasc Surg 1992 104 1465 1469
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1569620610.1371/journal.pmed.0020019Case ReportEmergency MedicineNeurology/NeurosurgeryOpthalmologyPrimary CareNeurologyStrokeOphthalmologyEmergency MedicinePainful Horner Syndrome as a Harbinger of Silent Carotid Dissection Case ReportNautiyal Amit
1
Singh Sonal
1
*DiSalle Michael
1
O'Sullivan John
2
1Case report fromDepartment of Medicine, Unity Health SystemRochester, New YorkUnited States of America2Department of Neurology, Unity Health SystemRochester, New YorkUnited States of America
Competing Interests: The authors have declared that no competing interests exist.
Author Contributions: AN and JO cared for the patient; AN, SS, and MD drafted the manuscript; and JO obtained the neuroradiology. All authors approved the final manuscript.
*To whom correspondence should be addressed. E-mail: [email protected] 2005 25 1 2005 2 1 e1919 11 2004 2 12 2004 Copyright: © 2005 Nautiyal et al.2005This 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 author and source are properly credited.A painful Horner's syndrome should alert clinicians to the possibilty of a silent carotid dissection
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PRESENTATION of CASE
A 43-y-old white female presented to the hospital in July 2004 with pain in the left eye and left upper lid ptosis. She did not perceive any difference in perspiration between the two halves of her face. She was a nonsmoker and denied any history of head or neck trauma, or ocular, cardiac, vascular, or neurologic disease. Neuro-ophthalmological examination was normal except for 1 mm of left upper eyelid ptosis (drooping of the eyelid), miosis (constriction of the pupil), and mild enophthalmos (recession of the eyeball into the orbit) consistent with classic left-sided Horner syndrome (Figure 1). There was no carotidynia (a neck pain syndrome associated with tenderness to palpation over the carotid bifurcation) or carotid bruit. A chest radiograph obtained to rule out an underlying left apical superior sulcus tumor was normal. Magnetic resonance imaging/magnetic resonance angiography of the brain with cross-sectional imaging of the neck was obtained, which revealed extracranial left internal carotid artery dissection (Figures 2 and 3). The patient was treated with unfractionated heparin and coumadin and made an uneventful recovery. The patient was seen in the clinic a few months later and did not have any complications at follow-up.
Figure 1 Photograph of Patient Showing Left-Sided Horner Syndrome
Figure 2 Magnetic Resonance Imaging/Magnetic Resonance Angiography of the Neck Showing Left Internal Carotid Artery Dissection
Figure 3 T2-Weighted Magnetic Resonance Imaging Showing Blood in the Arterial Wall and Narrowing of the Lumen of the Left Internal Carotid Artery
This is also known as the “crescent sign,” a hallmark of internal carotid artery dissection.
DISCUSSION
Horner syndrome—characterized by the constellation of miosis, ptosis, anhidrosis (lack of sweating), enophthalmos, and anisocoria (unequal pupil size)—is present in up to 58% of internal carotid artery dissections [1]. Most patients experience neck, facial, and head pain ipsilateral to the lesion because of ischemia or stretching of the trigeminal pain fibers surrounding the carotid arteries [2]. Ophthalmic manifestations have been reported to occur in up to 62% of patients with internal carotid artery dissection [2]. Common findings in descending order of frequency are painful partial Horner syndrome (due to disruption of the third-order neuron oculosympathetic fibers) as seen in our patient, transient monocular vision loss, and permanent visual loss [2].
De Bray et al. studied the prognosis of 90 cases of isolated Horner syndrome due to internal carotid artery dissection [3]. They found that 91% of cases of Horner syndrome due to internal carotid artery dissection were painful. The risk of an early ischemic stroke within the first 2 wk was high (around 17%) without initial antithrombotic treatment [3].
Internal carotid artery dissection is a potentially life-threatening condition and carries a substantial risk of disabling stroke [4]. Carotid dissection is under-recognized as a cause of Horner syndrome and can be missed [5]. It is important to diagnose dissection because anticoagulation can prevent carotid thrombosis and embolism [5]. The investigation of choice is magnetic resonance imaging and angiography scan of the head and neck [5].The treatment advocated for dissection is anticoagulation for 3–6 mo [5].
Learning Points
Painful Horner syndrome should alert clinicians to the possibility of a silent carotid dissection until proven otherwise [6].
Magnetic resonance imaging and angiography scan of the head and neck is the imaging modality of choice to look for dissection [5].
For patients with carotid dissection, anticoagulation with warfarin and coumadin is recommended for 3–6 mo to prevent carotid thrombosis and embolism [5].
Citation: Nautiyal A, Singh S, DiSalle M, O'Sullivan J (2005) Painful Horner syndrome as a harbinger of silent carotid dissection. PLoS Med 2(1): e19.
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de Bray JM Baumgartner RB Guillon B Dziewas R Ringelstein E Prognosis of dissections with an initially isolated Horner's syndrome [abstract]. 13th European Stroke Conference; Mannheim-Heidelberg, Germany; 2004 May 14. Available: http://www.esc-mannheim-2004.org/Mannheim/m_S4_oral.asp
2004 Accessed 6 December 2004
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| 15696206 | PMC545208 | CC BY | 2021-01-05 11:13:38 | no | PLoS Med. 2005 Jan 25; 2(1):e19 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020019 | oa_comm |
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